Citation
Studies of the Adeno-Associated Virus Capsid

Material Information

Title:
Studies of the Adeno-Associated Virus Capsid
Creator:
Vanvliet, Kim
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (193 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Genetics (IDP)
Committee Chair:
Snyder, Richard O.
Committee Members:
Bungert, Jorg
Muzyczka, Nicholas
Agbandje-McKenna, Mavis
Graduation Date:
8/11/2007

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Antibodies ( jstor )
Capsid ( jstor )
Capsid proteins ( jstor )
Dependovirus ( jstor )
Gene therapy ( jstor )
Isols ( jstor )
Parvovirus ( jstor )
Symmetry ( jstor )
Virion ( jstor )
Genetics (IDP) -- Dissertations, Academic -- UF
adeno, associated, capsid, dynamics, mass, protease, spectrometry, virus
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Medical Sciences thesis, Ph.D.

Notes

Abstract:
Adeno-associated virus (AAV) is a promising gene transfer vector that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The AAV capsid plays an essential role in cell binding, internalization, and trafficking, which are critical processes for gene delivery to specific cells. The structural proteins of the capsid spontaneously self-assemble into preformed shells, and then DNA packaging occurs. Although the viral capsid proteins self-assemble, little is known about this process. Potential subunits of assembly have been isolated by other investigators and this data was utilized to develop the two general models for Parvovirus assembly discussed here. Specific interactions that comprise the AAV-2 capsid subunit interfaces are analyzed with respect to several capsid mutants and their impact on the formation of a macromolecular structure. A better understanding of specific interactions required for AAV capsid assembly will allow for the development of customized targeting AAV vectors in the future. The AAV capsid is remarkably stable, which is a desirable characteristic for gene therapy vectors; however, the work presented here demonstrates that AAV serotypes exhibit differential susceptibility to proteases. The capsid fragmentation pattern upon limited proteolysis, as well as the susceptibility of the serotypes to a series of proteases, provides a unique fingerprint for each serotype that can be used for capsid identity validation. In addition, protease susceptibility is utilized to study dynamic structural changes of intact capsids in solution. A high-throughput method for AAV capsid serotype identification (AAV-CSI) utilizing denatured capsid proteins was also developed. This will be useful to verify customized gene therapy vectors that will be produced in the future, which may contain components of more than one AAV serotype. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2007.
Local:
Adviser: Snyder, Richard O.
Statement of Responsibility:
by Kim Vanvliet.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Vanvliet, Kim. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Classification:
LD1780 2007 ( lcc )

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This item has the following downloads:


Full Text







VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate rh49 AAS99247
isolate rh50 AAS99248
isolate rh51 AAS99249
isolate rh52 AAS99250
isolate rh64 AAS99259
isolate rh53 AAS99251
isolate rh61 AAS99257
isolate rh58 AAS99255
isolate rh57 AAS99254
AAV8 VP1 AANO3857
AAV8 YP 077180


YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YNGFSTPWGY
YNGFSTPWGY
YNGFSTPWGY
FNGFSTPWGY
FNGFSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGFSTPWGY
FFGFSTPWGY
FFGFSTPWGY
FFGFSTPWGY
FFGFSTPWGY


FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHSHW
FDFNRFHSHW
FDFNRFHSHW
FDFNRFHSHW
FDFNRFHCHF
FDFNRFHCHF
FDYNRFHCHF
FDYNRFHCHF
FDYNRFHCHF
FDYNRFHCHF


SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRPIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDRQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLVN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN


NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKKL
NNWGFRPKKL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKKL
NNWGFRPKKL
SNWGFRPKKL
NNWGFRPKKL
NNWGFRPKKL
NNWGFRPKKL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGLRPKAM
NNWGMRPKAM
NNWGMRPKAM
NHWGLRPKSM
NHWGLRPKSM
NYWGFRPRSL
NYWGFRPRSL
NYWGFRPRSL
NYWGFRPRSL
NHWGFRPKRL
NNWGLRPKSL
NNWGIRPKAM
NNWGIRPKAM
NNWGIRPKAM
NNWGIRPKAM


SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
RFKLFNIQVK
RFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
SFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
RVKI FN IQVK
RVKI FN IQVK
RVKI FN IQVK
QVRIFNIQVK
QVRIFNIQVK
RVKI FN IQVK
RVKI FN IQVK
RVKI FN IQVK
RVKI FN IQVK
RVKL FN IQVK
RFKIFNIQVK
RFRLFNIQVK
RFRLFNIQVK
RFRLFNIQVK
RFRLFNIQVK


VP1 isolate rh43 AAS99245
VP1 isolate pil AAS99238
VP1 isolate pi3 AAS99240
VP1 isolate pi2 AAS99239
VP1 isolate rh1 AAS99241
AAV7 VP1 AANO3855
AAV7 YP 077178
VP1 isolate rh48 AAS99246
VP1 isolate rh62 AAS99258
VP1 isolate rh55 AAS99253
VP1 isolate rh54 AAS99252
VP1 isolate rh60 AAS99256
AAV9 VP1 AAS99264
VP1 isolate hu31 AAS99281
VP1 isolate hu32 AAS99282
AAV11 AAT46339
AAV4 NP 044927
AAV4 VP1 AAC58045
BOVINE AAV AAR26465
BOVINE AAV YP 024971
AAV5 VP1 AAD13756
AAV5 YP 068409
CAPRINE AAV1 AAU84890
GOAT AAV VP1 ABC69726
RAT AAV1 AAZ79676
MOUSE AAV1 AAZ79672
AVIAN AAV ATCC VR865 AAO32087
AVIAN AAV ATCC VR865 AAT48613
AVIAN AAV ATCC VR865 NP 852781
AVIAN AAV Strain DA1 AAT48615












VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate hu52
isolate hu47


AAS 992 9 9
AAS 992 9 5
AAS 992 9 3
AAS9 93 0 5
AAS 992 9 7
AAS9 93 0 3
AAS9 93 0 4
AAS 992 7 8
AAS 992 7 9
AAU 05364
AAS 992 63
AAS9 93 0 9
AAS 9 9310
AAU 053 62
AAU 05371
AAU 05358
AAU 05372
AAU 05366
AAU 05368
AAU 05360
AAS 992 7 7
AAS 992 6 9
AAS 992 71
AAS 992 7 2
AAS 992 7 5
AAS 992 7 3
AAS 992 7 4


.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN
.VDFTVDTN


GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG
GVYSEPRPIG


TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
ARYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL


isolate
isolate
isolate
isolate
isolate
isolate
isolate


hu45
hu58
hu49
hu56
hu57
hu28
hu29


VP1 isolate huT70
VP1 isolate hul3
VP1 isolate hu63
VP1 isolate hu64
VP1 isolate huT40
VP1 isolate huLG15
VP1 isolate huT17
VP1 isolate huT41
VP1 isolate huT71
VP1 isolate huT88
VP1 isolate huT32
VP1 isolate hu27
VP1 isolate hul9
VP1 isolate hu20
VP1 isolate hu21
VP1 isolate hu24
VP1 isolate hu22
VP1 isolate hu23































Figure 3-5. Intermolecular residues are shown between the yellow chain, and the Asp231.
Residues that also may play a role in the phenotype of Mutl9 include Tyr397 and
Pro3 99. This figure was generated using the molecular modeling software Deep
View.


Figure 3-6. Asp231 has been changed to Ala231, as in Mutl9. Note that the primary interaction
that is disrupted is the intermolecular interaction with Lys692; however, an
intermolecular interaction with Pro399 is also disrupted. One or both interactions may
result in the inability to form intact capsids. This figure was generated using the
molecular modeling software Deep View.












AVIAN AAV ATCC VR865 AAO32087
AVIAN AAV ATCC VR865 AAT48613
AVIAN AAV ATCC VR865 NP 852781
AVIAN AAV Strain DA1 AAT48615
AVIAN AAV Strain DA1 YP 077183
DUCK AAV Strain FM AAA83225
MUSCOVY DUCK PARVOVIRUS YP 068412
MUSCOVY DUCK VP1 YP 068411
MUSCOVY DUCK PARVOVIRUS YP 068413
Goose AAV VP1 AAA83230
GOOSE AAV VP1 NP 043515
AAV3 NP 043941
AAV3 Strain H AAC55049
AAV3B VP1 AAB95452
VP1 isolate hul AAS99260
VP1 isolate hu4 AAS99287
VP1 isolate hu2 AAS99270
VP1 isolate hu3 AAS99280
VP1 isolate hu60 AAS99307
VP1 isolate hu61 AAS99308
VP1 isolate hu25 AAS99276
VP1 isolate hul5 AAS99265
VP1 isolate hul6 AAS99266
VP1 isolate hul8 AAS99268
VP1 isolate hu7 AAS99313
VP1 isolate hul0 AAS99261
VP1 isolate hull AAS99262
VP1 isolate hu9 AAS99314
VP1 isolate hu53 AAS99300
VP1 isolate hu55 AAS99302
VP1 isolate hu54 AAS99301
VP1 isolate huS17 AAU05370
AAV2 VP1 AACO3780
VP1 isolate hu34 AAS99283
VP1 isolate hu35 AAS99284
VP1 islolate hu51 AAS99298
VP1 isolate hu52 AAS99299
VP1 isolate hu47 AAS99295
VP1 isolate hu45 AAS99293
VP1 isolate hu58 AAS99305
VP1 isolate hu49 AAS99297


TNMAAQYRNW
TNMAAQYRNW
TNMAAQYRNW
TNMATQYRNW
TNMATQYRNW
GAFGAMGRNW
GAFGAMGRNW
GAFGAMGRNW
GAFGAMGRNW
GAYGTMGRNW
GAYGTMGRNW
QSMSLQARNW
QSMSLQARNW
QSMSLQARNW
TNMSLQAKNW
TNMSLQAKNW
TNMSLQAKNW
TNMSLQAKNW
TNMSLQAKNW
TNMSLQAKNR
TNMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
TSMSLQAKNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW


LPGPFFRDQQ
LPGPFFRDQQ
LPGPFFRDQQ
LPGPFVRDQQ
LPGPFVRDQQ
LPGPKLLDQR
LPGPKLLDQR
LPGPKLLDQR
LPGPKLLDQR
LPGPKFLDQR
LPGPKFLDQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR


IFTGASNITK
IFTGASNITK
IFTGASNITK
IFTGASNITQ
IFTGASNITQ
VRAYSGGTDN
VRAYSGGTDN
VRAYSGGTDN
VRAYSGGTDN
VRAYTGGTDN
VRAYTGGTDN
LSKTANDNNN
LSKTANDNNN
LSKTANDNNN
LSKQANGNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANDNNN
LSKQANENNN
VSKTSADNNN
VSKTSADNNN
VSKTSADNNN
VSKTSADNNN
VSKTSADNNN
VSKTSADNNN
VSKTSADNNN
VSKTSADNNN
VSKTAADNNN


..NVFSVW
..NVFSVW
..NVFSVW
..NVFNVW
..NVFNVW
..ANWSIW
..ANWSIW
..ANWSIW
..ANWSIW
..ANWNIW
..ANWNIW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....NFPW
....EYSW
....EYSW
....EYSW
....EYSW
....EYSW
....EYSW
....EYSW
....EYSW
....EYSW


EKGKQWELDN
EKGKQWELDN
EKGKQWELDN
DKGKQWVIDN
DKGKQWVIDN
SKGNKVFLKD
SKGNKVFLKD
SKGNKVFLKD
SKGNKVFLKD
SNGNKVNLKD
SNGNKVNLKD
TAASKYHLNG
TAASKYHLNG
TAASKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYHLNG
TAATKYRLNG
TAATKYHLNG
TGATKYYLNG
TGATKYHLNG
TGATKYHLNG
TAATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
IGATKYHLNG
TGATKYHLNG










time-of-flight mass spectroscopy (MALDI-TOF) analysis, which is close to the theoretical mass

of the C-terminal peptide that results from cleavage at R588 (Table 4-1). The R588 tryptic site

was confirmed by N-terminal sequencing, which mapped residueS 589QAATADVNTQGV600 as

the terminal peptide for the VP1,2,3T fragment.

Table 4-1. Predicted AAV VP I tryptic fragment mass from cleavage in the G-H loop. Trypsin
cleavage sites of AAV-2 VP I were determined using the program Peptide Cutter
found on the ExPASy server (www.expasy.org). Based on the sites where trypsin
cleaves, these sequences were entered into the program Peptide Mass on the ExPASy
server and the mass of each tysin frament was determined.
Fragment Averag~e Mass Peptide

VPI 81944.65 All Cys in reduced form

1 549 61112.40 VPI start to 549
137 549 45757.24 VP2 start to 549
203 549 39231.12 VP3 start to 549
550 -735 20850.26 C terminal end
1 556 61912.28 VPI start to 556
137 556 46557.12 VP2 start to 556
203 556 40031.00 VP3 start to 556
557 -735 20050.38 C terminal end
1 566 63128.65 VPI start to 566
137 566 47773.49 VP2 start to 566
203 566 41247.37 VP3 start to 566
567 -735 18834.01 C terminal end
1 585 65176.85 VPI start to 585
137 585 49821.69 VP2 start to 585
203 585 43295.57 VP3 start to 585
586 -735 16785.81 C terminal end
1 588 65504.19 VPI start to 588 (VPT)
137 588 50149.03 VP2 start to 588 (VP2T)
203 588 43 622.91 VP3 start to 588 (VP3T)
589 -735 16458.47 C terminal end (VP1,2,3T)
1 609 67743.68 VPI start to 609
137 609 52388.52 VP2 start to 609
203 609 45862.40 VP3 start to 609
610 -735 14218.98 C terminal end










LIST OF ABBREVIATIONS

Acidic amino acid interaction

Angstrom

Adeno-Associated Virus

Adeno-Associated Virus-Capsid Serotype Identification

Basic amino acid interaction

Canine Parvovirus

Cryo-Electron Microscopy

cold-sensitive

Defective Interfering Particle

Deoxyribonucleic acid

Feline Parvovirus

Hydrophobic amino acid integration

heparin negative, virus is unable to bind heparin

heparin positive, virus binds heparin

heat-sensitive

Low infectious particle phenotype

Mass Spectrometry

Mutant

Minute Virus of Mice

non-infectious

Polar, uncharged amino acid interaction

partially defective

Pentamers of Trimers

Porcine Parvovirus


A

a

AAV

AAV-CSI

B

CPV

Cryo-EM

cs

DI particle

DNA

FPV

H

hep-

hep+

hs

lip mutant

MS

Mut

MVM

ni

P

pd

POTs

PPV























I _~Rep68





Figure 1-1. The single-stranded DNA genome of AAV-2. The inverted terminal repeats (ITRs)
flank the two open reading frames rep and cap. The rep gene encodes four nonstructural
proteins, Rep78, Rep68, Rep52 and Rep40. The cap gene encodes three structural proteins
VP1, VP2 and VP3. The location of the promoters, p5, pl9, and p40 are depicted by arrows.
Vectors

Three features of AAV-2 which may limit its use as a gene therapy vector include: 1) its

small size, with an ability to package approximately 4.7 kb, 2) the difficulty and expense of

production of large quantities of high titer virus for clinical trials, and 3) the wide range of tissue

tropism of AAV-2, as a result of using heparin sulfate proteoglycan as a receptor, which could

result in expression of the transgene in non-target tissues. Studies have addressed the small size

of the AAV capsid and an evaluation of the packaging limits of AAV has shown that the AAV

capsid is capable of packaging and protecting DNA up to 6.0 kb; however, post-entry, capsids

with DNA larger than the AAV genome are preferentially degraded by the proteosome. It has

been shown that proteosome inhibitors can be used to overcome this barrier [35]. The


ITR
p5


LI


I ~1 78'

7:

~VP376:

I ~Re 78


ITR
PolyA

Cap-C~


7 U
2 kDa

3 kDa


pl9 p40


Rep























STable 5-1. AAV Serotype Amino Acid Identity Table for AAV Serotypes 1 -11. AAV Seroty es were aisne ad4aon ildeen it table
e ~was constructed using Vector NTI (Invitrogen). Numeric values represent percent dniy AAV- is 65% identical to AV1 4
AAV-8, and 83% identical to AAV-2, representing highly homologous serotypes. oA- s6%ie tia e. AAV-5, is
identical to AAV-8, 61% identical to AAV-2, 54% identical to AAhV-.5 represserino ya le Aes oolongo smsb rs fprproteins
54 59% identical to the other serotypes, also representin95a le d homm 7oou 7,o e whichaesidentcl AAV-2 VorPrtin
used in the alignment are. AAV-1 VPI sequences AAV-354 StainH AAC275504 which are identical, AAV-3B VPI
AACO3780, AAV-3 VPI sequences NP 044927 and AAC58045 which are identical, AAV-5 VPI sequences YP 068408
a AD35 hc r dnia AV V AAB95452, AAV-4 VPI sequencesNPa AANO3855 and YP 077178 which
aedide 1ial A6AwV 8c r s ATee 6P 7780 And A4N50 8A Vwh ch alre ide t al AAV-9 VPI AAS99264, AAV-10
AAT46337, AAV-11 AAT46339.












VP1 isolate huLG15
VP1 isolate huT17
VP1 isolate huT41
VP1 isolate huT71
VP1 isolate huT88
VP1 isolate huT32


AAU 05371
AAU 05358
AAU 05372
AAU 05366
AAU 05368
AAU 05360
AAS 992 7 7
AAS 992 6 9
AAS 992 71
AAS 992 7 2
AAS 992 7 5
AAS 992 7 3
AAS 992 7 4


.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYRY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY


LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG



RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTPFGG
RLKEDTSFGG
RLKEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG


EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA



NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK


LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!

150
KRVLEPFG.L
KRVLEPFG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPFG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.P
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L


VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate
isolate
isolate
isolate


hu27
hul9
hu20
hu21


isolate hu24
isolate hu22
isolate hu23


101
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYPR
LDSGDNPYLK
LDSGDNPYLK
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPHLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR


AAV VR195 ABA71699
AAV VR355 ABA71701
AAV1 NP 049542


YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAELQE
YNHADAEFQE


VP1 isol

VP1 isol
VP1 isol
VP1 isol

VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol


AAV1 VP1 AAD27757
ate hu48 AAS99296
AAV6 VP1 AAB95450
ate hu43 AAS99291
ate hu44 AAS99292
ate hu46 AAS99294
AAV10 AAT46337
ate rh40 AAS99244
ate hu37 AAS99285
ate hu42 AAS99290
ate hu40 AAS99288
ate hu67 AAS99312
ate rh38 AAS99243
ate hu41 AAS99289
ate hu66 AAS99311
ate hul7 AAS99267
,late hu6 AAS99306
ate rh25 AAS99242
ate hu39 AAS99286
ate rh49 AAS99247
ate rh50 AAS99248
ate rh51 AAS99249
ate rh52 AAS99250


VP1 iso
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol





Proabii~Leondover 95% 80% to 94%b~~~ i~~ ~ o19


1007~0J21 kV vi~ru:.i; DI:-plaing3 f urnbe~i :r Un;que Peptidej~
Protegin narni- Iurn ArCse:.:-ionPiotgin rniCAAA BBB
AAV5 1ao~ gia53384;? u
cAAV8 1 gllf1199499 o1739 6 8
AAV4 1 gll9E29E.3 Y0622.5
AAV4~ unnamed p 1 91~122923 E.<><
AAV2 VP3 1 5'1221426 56698 1

HSP70[IAcanthan-1gl 69trua9'7-1
E2A; DNA binding 1 gll9626179 5906; 2
Thioredoxin 1 g1l230;11 1165d.0. 2
AAV2 VP1 1 qll16.16.1 j's 167 6
Goose VP3 4gl422 191dP 4
Goose Parvovirus L JI1737E.2L 1-1.


CCC EEE GGG
1 1 11


HHH KKK LLL MMM PPP QQ QQQ RR
6 5 9 9 8

1 14
10 14


17 1
1 1


2 2
3


1 3
5 4


2 1


1 2
1


Figure 5-4. Mass spec data analyzed using the software program Scaffold enabled unambiguous serotype identification for 13/17
samples (76%) and the correct serotype assignment was made for 17/17 samples (100%) based on the serotype with the
largest number of unique fragments. The viral proteins, accession numbers, and protein mass are the result of the database
search. The unknown samples are listed as their alphabetic code, and the values in the table are the number of unique
trypsin fragments. This value was used to correctly assign the serotype. Codes: AAA AAV-1; BBB AAV-8; CCC -
AAV-8; EEE AAV-4; GGG AAV-5; HHH AAV-2; KKK AAV-4; LLL AAV-4; IV1V1V AAV-4; PPP AAV-1;
QQ AAV-5; QQQ AAV-5; RR AAV-5; RRR AAV-5; SSS AAV-8; XXX AAV-8; ZZZ AAV-5


RRR SSS XXX Z.ZZ
7 10
7E 10rr









LIST OF REFERENCES


1. Allander T, Tammi MT, Eriksson M, Bj erkner A, Tiveljung-Lindell A, Andersson B:
Cloning of a human parvovirus by molecular screening of respiratory tract samples.
Proc NatlAcadSci USA 2005, 102: 12891-12896.


2. Jones MS, Kapoor A, Lukashov VV, Simmonds P, Hecht F, Delwart E: New DNA viruses
identified in patients with acute viral infection syndrome. J Virol 2005, 79:8230-8236.


3. Berns KI, Bohenzky RA: Adeno-associated viruses: an update. Adv Virus Res 1987,
32:243-306.


4. Bankiewicz KS, Forsayeth J, Eberling JL, Sanchez-Pernaute R, Pivirotto P, Bringas J,
Herscovitch P, Carson RE, Eckelman W, Reutter B, Cunningham J: Long-term clinical
improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC. Mol1
Ther 2006, 14:564-570.


5. Crystal RG, Sondhi D, Hackett NR, Kaminsky SM, Worgall S, Stieg P, Souweidane M,
Hosain S, Heier L, Ballon D, Dinner M, Wisniewski K, Kaplitt M, Greenwald BM, Howell
JD, Strybing K, Dyke J, Voss H: Clinical protocol. Administration of a replication-
deficient adeno-associated virus gene transfer vector expressing the human CLN2
cDNA to the brain of children with late infantile neuronal ceroid lipofuscinosis. Hum
Gene Ther 2004, 15: 1131-1154.


6. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ, Ozelo MC, Hoots K,
Blatt P, Konkle B, Dake M, Kaye R, Razavi M, Zajko A, Zehnder J, Rustagi PK, Nakai H,
Chew A, Leonard D, Wright JF, Lessard RR, Sommer JM, Tigges M, Sabatino D, Luk A,
Jiang H, Mingozzi F, Couto L, Ertl HC, High KA, Kay MA: Successful transduction of
liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune
response. Nat M~ed 2006, 12:342-347.


7. During MJ, Kaplitt MG, Stern MB, Eidelberg D: Subthalamic GAD gene transfer in
Parkinson disease patients who are candidates for deep brain stimulation. Hum Gene
Ther 2001, 12:1589-1591.


8. Stedman H, Wilson JM, Finke R, Kleckner AL, Mendell J: Phase I clinical trial utilizing
gene therapy for limb girdle muscular dystrophy: alpha-, beta-, gamma-, or delta-
sarcoglycan gene delivered with intramuscular instillations of adeno-associated
vectors. Hum Gene Ther 2000, 11:777-790.









for other parvoviruses such as MVM that suggest that trimers are the intermediate, and that VP I

and VP2 are both capable of translocating into the nucleolus where virus assembly occurs [79]

when singly expressed in transfected cells; however, only VP2 is able to assemble into capsid by

itself. Recent data for MVM suggests that trimers are actually transported through the nuclear

pore complex, not intact capsids [80]. For MVM, there are 2 proteins, VPI and VP2, present in a

ratio of 1:5 in the assembled capsid. Because trimers are more stable than pentamers and dimers,

it is thought that this energetically expensive subunit forms first and is transported through the

nuclear pore. How the stoichiometry of the viral structural proteins (1 VPl: 5 VP2) is maintained

in the assembled MVM capsid is unclear. If pentamers are the structural intermediate, as in

OX174, then the stoichiometry of 1 VPl: 4 VP2 would approximate the observed 1:5 ratio;

however, recent data does not support the hypothesis that MVM assembles via a pentameric















Mlonomeric Subunits
Pentameric Trimers of Pentamers Mlacromolecular
Intermediate Assembly
Figure 2-3. Model of Parvovirus Assembly through a Pentameric Intermediate. In this model,
monomeric subunits come together to form pentamers, and then 3 of these pentamers
come together to form trimers of pentamers (TOPs), and then pentamers add on to
form the capsid. This is the favored model for Adeno-Associated Virus (AAV)
assembly.











AVIAN AAV Strain DA1 YP 077183
DUCK AAV Strain FM AAA83225
MUSCOVY DUCK PARVOVIRUS YP 068412
MUSCOVY DUCK VP1 YP 068411
MUSCOVY DUCK PARVOVIRUS YP 068413
Goose AAV VP1 AAA83230
GOOSE AAV VP1 NP 043515
AAV3 NP 043941


FFGFSTPWGY
YAGYSTPWGY
YAGYSTPWGY
YAGYSTPWGY
YAGYSTPWGY
YAGYSTPWGY
YAGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGCSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY


FDYNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCRF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF


SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLVN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDRQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN


NNWGIRPKAM
NHWGIRPKAL
NHWGIRPKAL
NHWGIRPKAL
NHWGIRPKAL
NHWGIRPKSL
NHWGIRPKSL
NNWGFRPKKL
NNWGFRPKKL
NNWGFRPKKL
NNWGFRPKRL
NNRGFRPKRL
NNWGFRPKRL
SNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NSWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL


RFRLFNIQVK
KFKIFNVQVK
KFKIFNVQVK
KFKIFNVQVK
KFKIFNVQVK
KFKIFNVQVK
KFKIFNVQVK
SFKLFNIQVR
SFKLFNIQVR
SFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVR
NFKLFNIQVK
NFKLFNIQVK
NLKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK


AAV3 Strain H
AAV3B VP1
VP1 isolate hul
VP1 isolate hu4
VP1 isolate hu2
VP1 isolate hu3
VP1 isolate hu60
VP1 isolate hu61
VP1 isolate hu25
VP1 isolate hul5
VP1 isolate hul6
VP1 isolate hul8
VP1 isolate hu7
VP1 isolate hul0
VP1 isolate hull
VP1 isolate hu9
VP1 isolate hu53
VP1 isolate hu55
VP1 isolate hu54
VP1 isolate huS17
AAV2 VP1


AAC55049
AAB 9 54 52
AAS 992 60
AAS 992 8 7
AAS 992 70
AAS 992 80
AAS9 93 0 7
AAS9 93 0 8
AAS 992 7 6
AAS 992 65
AAS 992 6 6
AAS 992 6 8
AAS 9 9313
AAS 992 61
AAS 992 62
AAS 9 9314
AAS 9 9 30
AAS9 9 302
AAS9 9 301
AAU 05370
AAC O37 80
AAS 992 8 3
AAS 992 8 4
AAS 992 9 8
AAS 992 9 9
AAS 992 9 5
AAS 992 9 3
AAS9 93 0 5
AAS 992 9 7
AAS9 93 0 3
AAS9 93 0 4
AAS 992 7 8
AAS 992 7 9


VP1 isolate
VP1 isolate
VP1 islolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate


hu34
hu35
hu51
hu52
hu47
hu45
hu58
hu49
hu56
hu57
hu28
hu29









CHAPTER 4
STUDIES OF THE AAV CAPSID IN SOLUTION

Introduction

Proteolytic structural mapping. The three-dimensional structure of several autonomous

parvoviruses [53, 93, 94], plus those of AAV-2 [48], AAV-4 [50], and AAV-5 [51], have been

determined by X-ray crystallography or cryoelectron microscopy (cryo-EM). Several features of

the virus cannot be determined based on the crystal structure. For example, the unique N-

terminus of VP 1, as well as the unique N-terminus of VP2, are not present in the crystal

structures of AAV. This is likely due to the fact that these monomers are present in the AAV

capsid in low abundance, (~5 copies each), and icosahedral averaging is used to solve the crystal

structure. In addition to being present in low abundance, as discussed in Chapter 2, the N-

terminus of VPI and VP2 may be disordered in assembled capsids which would preclude

crystallographic structure determination of these regions.

In the near future, customized AAV gene therapy vectors may consist of modified capsids

that allow for specific targeting to treat patients with various diseases. The 3D structures of the

AAV capsids will provide a basis for rational vector design, however, the 3D structures available

for autonomous parvoviruses and dependoviruses only provide a "snapshot" of the capsid

topology in a low energy conformation. Our knowledge about the AAV viral capsid structure in

solution is limited; however, this structure must be dynamic to carry out the various functions

required for viral attachment and entry, as well as trafficking within the cell. AAV-2 has been

shown to utilize heparin sulfate proteoglycan as a cell surface receptor, and the specific amino

acid residues involved in this interaction have been mapped to basic amino acids at the three-fold

axis of symmetry on the capsid surface, including R585 and R588 [95, 96]. Studies of the AAV

capsid proteins have shown that the unique N-terminus of VP I is required for infectivity [69].











3-20 The location of residues that are mutated in Mut46. ............__....._ ................74

3-21 Intramolecular interactions with the residues that are changed in Mut46,. .......................75

3 -22 Intramolecular interactions after the residues in Mut46 have been changed ................... ..76

3-23 Transfection of Mut 33. ........._._ ........... ...............82.....

3-24 Green cell assay of transfections at 320 C. ............. ...............83.....

3-25 Transfections at 37 OC............... ...............84...

3-26 Green cell assay after transfection at 39.5 OC.. ............ ...............85.....

4-1 Western blot of several AAV-2 preps............... ...............89.

4-2 Tryptic mapping of full AAV-2 capsids ..........._ ..... ._ ....__ ............. ...90

4-3 Trypsinized AAV-2 virions remain intact .............. ...............93....

4-4 Proteolysis distinguishes full and empty AAV-2 particles ................. .. ....._ ..........97

4-5 AAV-2, AAV-1 and AAV-5 capsids can be distinguished proteolytically ................... ....99

4-6 AAV-2, AAV-1, and AAV-5 have different susceptibility to Chymotrypsin. ................101

4-7 Capsid structure .............. ...............102....

4-8 AAV-1, AAV-2 and AAV-5 Homology models. ............ .....__. .........._.. ....0

5-1 Phylogenetic Tree of AAV Serotypes 1 1 1................ .......... ........ .............1 15

5-2 Coomassie stained samples for mass spectrometry ................. ............................116

5-3 Coomassie stained samples for mass spectrometry identification ................. ...............118

5-4 Mass spec data analyzed using the software program Scaffold............... .................1

5-5 Mass spectrometry data for K544E AAV-4 mutant and wt AAV-4 ............... .... .........._.122









stained. To determine if these additional bands were degradation products or smaller proteins

that co-purified with the AAV structural proteins, a western blot was performed and probed with

polyclonal antibody to AAV-2. This is shown in Figure 4-1. This assay verifies that the

fragments are degradation products of AAV-2 that co-purified with the intact virus. The

classical method for purifying AAV-2 vectors included using trypsin, deoxycholate and the

performing cesium chloride gradients followed by heparin chromatography. To determine if

trypsin was responsible for the additional bands, AAV2-GFP vector virions were produced as

described in the materials and methods and digested with trypsin. For proteolyic mapping of the

AAV-2 capsid, several antibodies are available that detect various regions of the AAV capsid

proteins [65, 100]. The B1 antibody epitope is on the C-terminal end of the capsid protein, and is

primarily internal at the 2-fold axis of symmetry in assembled capsids. This antibody is useful in

detecting denatured AAV proteins and the epitope is highly conserved among the AAV

serotypes. Al antibody recognizes the unique N terminal region of VP 1, while the A69 epitope is

in the N-terminal region of VP2 for AAV-2. Polyclonal antibodies have been produced to AAV-

2 capsids, as well as other serotypes. Figure 4-2 is a western blot of AAV2-GFP digested with

trypsin as described in the materials and methods. Odd lanes (lane 1, 3, 5 and 7) are undigested

AAV2-GFP. Even lanes (lane 2, 4, 6 and 8) have been digested with trypsin. Lanes 1 and 2 are

probed with polyclonal antisera to AAV-2 resulting in the detection of two digestions products at

~40 kDa, and a smaller 15 to 20 kDa fragment. Lanes 3 and 4 are probed with B l. This shows

that the smaller 15 to 20 kDa fragment is derived from the C-terminal region that is common to

all three of the structural proteins, VP 1, VP2, and VP3. This fragment was named VP 1,2,3T. The

~40 kDa N-terminal fragment is not detectable since the N-terminal fragment no longer harbors

the B1 epitope. This fragment was designated VP3T. Lanes 5 and 6 are probed with A69











VP1
VP1
VP1
VP1
VP1



VP1
VP1
VP1


isolate rh64 AAS99259
isolate rh53 AAS99251
isolate rh61 AAS99257
isolate rh58 AAS99255
isolate rh57 AAS99254
AAV8 VP1 AANO3857
AAV8 YP 077180
isolate rh43 AAS99245
isolate pil AAS99238
isolate pi3 AAS99240


LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LQAGDNPYLR
LQAGDNPYLR
LQAGDNPYLR
LEAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LQAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLR
LKAGDNPYLK
LKAGDNPYLK
LKAGDNPYLK
LKAGDNPYLR
LKAGDNPYLK
LKAGDNPYLK
LEAGDNPYLK
LEAGDNPYLK
LEAGDNPYLK
LEAGDNPYLK
LEAGDNPYLK
LEAGDNPYLK
LESGENPYLT
LEAGDNPYLK
IKDGHNPYFE
IKDGHNPYFE
IKDGHNPYFE
LKDGHNPYFE
LKDGHNPYFE
LKAGDNPYIK


YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQQ
YNHADAEFQQ
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
YNHADAEFQE
FNHADRQLQK
YNHADREFQE
YNEADRRFQE
YNEADRRFQE
YNEADRRFQE
YNEADRRFQE
YNEADRRFQE
FKHADQEFID


RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLQEDTSFGG
RLKEDTSFGG
RLKEDTSFGG
RLKEDTSFGG
RLQEDTSFGG
RLQGDTSFGG
RLQGDTSFGG
KLASDTSFGG
KLASDTSFGG
KLADDTSFGG
KLADDTSFGG
KLADDTSFGG
KLADDTSFGG
DLAEDQSFEG
ALKDDTSFEG
RLKDDTSFGG
RLKDDTSFGG
RLKDDTSFGG
RLKDDTSFGG
RLKDDTSFGG
NLQGDTSFGG


NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGRAVFQAK
NLGKAVFQAK
NLGKAVFQAK
NLGKAVFQAK
NLGKAVFQAK
NLGKAVFQAK
NLGKAVFQAK
NLARGLFEAK
NLARGLFEAK
NLGKAIFQAK
NLGKAIFQAK
NLGKAIFQAK
NLGKAIFQAK
NLGKAIFQAK
NLGKAVFQAK


KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRLLEPLG.L
KRLLEPLG.L
KRLLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRVLEPLG.L
KRILEPLG.L
KRILEPLG.L
KRVLEPFG.L
KRVLEPFG.L
KRVLEPFG.L
KRVLEPFG.L
KLAAQVVG.V
KLVAEPLG.L
KRVLEPFG.L
KRVLEPFG.L
KRVLEPFG.L
KRVLEPFG.L
KRVLEPFG.L
KRILEPLG.L


VP1 isolate pi2 AAS99239
VP1 isolate rh1 AAS99241
AAV7 VP1 AANO3855
AAV7 YP 077178
VP1 isolate rh48 AAS99246
VP1 isolate rh62 AAS99258
VP1 isolate rh55 AAS99253
VP1 isolate rh54 AAS99252
VP1 isolate rh60 AAS99256
AAV9 VP1 AAS99264
VP1 isolate hu31 AAS99281
VP1 isolate hu32 AAS99282
AAV11 AAT46339
AAV4 NP 044927
AAV4 VP1 AAC58045
BOVINE AAV AAR26465
BOVINE AAV YP 024971
AAV5 VP1 AAD13756
AAV5 YP 068409
CAPRINE AAV1 AAU84890
GOAT AAV VP1 ABC69726
RAT AAV1 AAZ79676
MOUSE AAV1 AAZ79672
AVIAN AAV ATCC VR865 AAO32087
AVIAN AAV ATCC VR865 AAT48613
AVIAN AAV ATCC VR865 NP 852781
AVIAN AAV Strain DA1 AAT48615
AVIAN AAV Strain DA1 YP 077183
DUCK AAV Strain FM AAA83225
MUSCOVY DUCK PARVOVIRUS YP 068412
MUSCOVY DUCK VP1 YP 068411


LKAGDNPYIK FKHADQEFID NLQGDTSFGG NLGKAVFQAK KRILEPLG.L











GOAT AAV VP1 ABC69726
RAT AAV1 AAZ79676
MOUSE AAV1 AAZ79672
AVIAN AAV ATCC VR865 AAO32087
AVIAN AAV ATCC VR865 AAT48613
AVIAN AAV ATCC VR865 NP 852781
AVIAN AAV Strain DA1 AAT48615
AVIAN AAV Strain DA1 YP 077183
DUCK AAV Strain FM AAA83225
MUSCOVY DUCK PARVOVIRUS YP 068412
MUSCOVY DUCK VP1 YP 068411
MUSCOVY DUCK PARVOVIRUS YP 068413
Goose AAV VP1 AAA83230
GOOSE AAV VP1 NP 043515
AAV3 NP 043941
AAV3 Strain H AAC55049
AAV3B VP1 AAB95452
VP1 isolate hul AAS99260
VP1 isolate hu4 AAS99287
VP1 isolate hu2 AAS99270
VP1 isolate hu3 AAS99280
VP1 isolate hu60 AAS99307
VP1 isolate hu61 AAS99308
VP1 isolate hu25 AAS99276
VP1 isolate hul5 AAS99265
VP1 isolate hul6 AAS99266
VP1 isolate hul8 AAS99268
VP1 isolate hu7 AAS99313
VP1 isolate hul0 AAS99261
VP1 isolate hull AAS99262
VP1 isolate hu9 AAS99314
VP1 isolate hu53 AAS99300
VP1 isolate hu55 AAS99302
VP1 isolate hu54 AAS99301
VP1 isolate huS17 AAU05370
AAV2 VP1 AACO3780
VP1 isolate hu34 AAS99283
VP1 isolate hu35 AAS99284
VP1 islolate hu51 AAS99298
VP1 isolate hu52 AAS99299
VP1 isolate hu47 AAS99295


DVYLQGPIWA KIPETGAHFH PSPAMGGFGL KHPPPMMLIK NTPVPGN.IT
DVYLQGPIWA KIPHTGAHFH PSPMMGGFGL RNPPPMMLIK NTPVPGN.VT
DIYLQGQIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPADPPT
DIYPTGTHLA KIPDTDNHFH PSPLIGRFGC KHPPPQIFIK NTPVPANPSE
DIYPTGTHLA KIPDTDNHFH PSPLIGRFGC KHPPPQIFIK NTPVPANPSE
DIYPTGTHLA KIPDTDNHFH PSPLIGRFGC KHPPPQIFIK NTPVPANPSE
DIYLQGPIWA KIPDTDNHFH PSPLIGGFGC KHPPPQIFIK NTPVPANPSE
DIYLQGPIWA KIPDTDNHFH PSPLIGGFGC KHPPPQIFIK NTPVPANPSE
DIYLQGPIWA KIPKTDGKPH PSPNLGGFGL HNPPPQVFIK NTPVPADPPL
DIYLQGPIWA KIPKTDGKPH PSPNLGGFGL HNPPPQVFIK NTPVPADPPL
DIYLQGPIWA KIPKTDGKPH PSPNLGGFGL HNPPPQVFIK NTPVPADPPL
DIYLQGPIWA KIPKTDGKPH PSPNLGGFGL HNPPPQVFIK NTPVPADPPL
DIYLQGPIGA KIPKTDGKFH PSPNLGGFGL HNPPPQVFIK NTPVPADPPV
DIYLQGPIGA KIPKTDGKFH PSPNLGGFGL HNPPPQVFIK NTPVPADPPV
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLTGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK STPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLVGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLRGPIWA KIPHADGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGP KHPPPQILIK NTPVPANPST
DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST











AVIAN PArVATCC VR865 NP 852781
AVIAN PArV Strain DA1 AAT48615
AVIAN PArV Strain DA1 YP 077183
DUCK AAV Strain FM AAA83225
MUSCOVY DUCK PARVOVIRUS YP 068412
MUSCOVY DUCK VP1 YP 068411
MUSCOVY DUCK PARVOVIRUS YP 068413
Goose AAV VP1 AAA83230
GOOSE AAV VP1 NP 043515
AAV3 NP 043941
AAV3 Strain H AAC55049
AAV3B VP1 AAB95452
VP1 isolate hul AAS99260
VP1 isolate hu4 AAS99287
VP1 isolate hu2 AAS99270
VP1 isolate hu3 AAS99280
VP1 isolate hu60 AAS99307
VP1 isolate hu61 AAS99308
VP1 isolate hu25 AAS99276
VP1 isolate hul5 AAS99265
VP1 isolate hul6 AAS99266
VP1 isolate hul8 AAS99268
VP1 isolate hu7 AAS99313
VP1 isolate hul0 AAS99261
VP1 isolate hull AAS99262
VP1 isolate hu9 AAS99314
VP1 isolate hu53 AAS99300
VP1 isolate hu55 AAS99302
VP1 isolate hu54 AAS99301
VP1 isolate huS17 AAU05370
AAV2 VP1 AACO3780
VP1 isolate hu34 AAS99283
VP1 isolate hu35 AAS99284
VP1 islolate hu51 AAS99298
VP1 isolate hu52 AAS99299
VP1 isolate hu47 AAS99295
VP1 isolate hu45 AAS99293
VP1 isolate hu58 AAS99305
VP1 isolate hu49 AAS99297
VP1 isolate hu56 AAS99303
VP1 isolate hu57 AAS99304


IPQYGYCTLN
IPQYGYCTLN
IPQYGYCTLN
LPQYGYCTMH
LPQYGYCTMH
LPQYGYCTMH
LPQYGYCTMH
LPQYGYCTMH
LPQYGYCTMH
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLS
VPQYGYLTLN
VPQYGYPTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN
VPQYGYLTLN


YN.N..EAVD
YN.N..EAVD
YN.N..EAVD
TNQSGARFND
TNQSGARFND
TNQSGARFND
TNQSGARFND
TNQNGARFND
TNQNGARFND
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NE...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG


RSAFYCLDYF
RSAFYCLDYF
RSAFYCLDYF
RSAFYCLEYF
RSAFYCLEYF
RSAFYCLEYF
RSAFYCLEYF
RSAFYCLEYF
RSAFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSPFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLECF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCPEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF


PSDMLRTGNN
PSDMLRTGNN
PSDMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN


FEFTYTFEDV
FEFTYTFEDV
FEFTYTFEDV
FEFSFEFEEV
FEFSFEFEEV
FEFSFEFEEV
FEFSFEFEEV
FEFTFDFEEV
FEFTFDFEEV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
LTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FQFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV









Table 3-9. Parvovirus structural capsid protein alignment for Mutl9 residues. A structural
alignment was performed in DALI for other parvoviruses whose structures have been
solved and the amino acids were evaluated to determine if the region is highly
conserved. The amino acids are color coded based on general properties, pink are
aromatic amino acids, green are neutral amino acids, grey are aliphatic amino acids,
red are acidic amino acids and aqua are basic amino acids.
Parvovirus Sequence
AAV2 (PDB#11p3) W CDS
Porcine Parvovirus (PDB#1k3v) FNNQT
MVM (PDB#1mvm) YDNQT
CPV (PDB#4dpy) FNNQT

Table 3-10. Parvovirus structural capsid protein alignment for Mut24 residues. A structural
alignment was performed in DALI for other parvoviruses whose structures have been
solved and the amino acids were evaluated to determine if the region is highly
conserved. The amino acids are color coded based on general properties, pink are
aromatic amino acids, green are neutral amino acids, grey are aliphatic amino acids,
red are acidic amino acids and aqua are basic amino acids.
Parvovirus Sequence
AAV2 (PDB#11p3) FSP D
Porcine Parvovirus (PDB#1k3v) FNPAD
MVM (PDB#1mvm) LQPSD
CPV (PDB#4dpy) FNPGD

Table 3-11. Parvovirus structural capsid protein alignment for Mut26 residues. A structural
alignment was performed in DALI for other parvoviruses whose structures have been
solved and the amino acids were evaluated to determine if the region is highly
conserved. The amino acids are color coded based on general properties, pink are
aromatic amino acids, green are neutral amino acids, grey are aliphatic amino acids,
red are acidic amino acids and aqua are basic amino acids.
Parvovirus Sequence
AAV2 (PDB#11p3) V EVT
Porcine Parvovirus (PDB#1k3v) L T IT
MVM (PDB#1mvm) L TVT
CPV (PDB#4dpy) L TVS
Table 3-12. Parvovirus structural capsid protein alignment for Mut33 residues. A structural
alignment was performed in DALI for other parvoviruses whose structures have been
solved and the amino acids were evaluated to determine if the region is highly
conserved. The amino acids are color coded based on general properties, pink are
aromatic amino acids, green are neutral amino acids, grey are aliphatic amino acids,
red are acidic amino acids and aqua are basic amino acids.
Parvovirus Sequence
AAV2 (PDB#11p3) DI D
Porcine Parvovirus (PDB#1k3v) T EAT
MVM (PDB#1mvm) S EA I
CPV (PDB#4dpy) T EAT










88. Hauck B, and Xiao, W.: Characterization of Tissue Tropism Determinants of Adeno-
Associated Virus Type 1. Journal of Virology 2003, 77:2768 2774.


89. Wu P, Xiao W, Conlon T, Hughes J, Agbandj e-McKenna M, Ferkol T, Flotte T, Muzyczka
N: Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and
construction of AAV2 vectors with altered tropism. J Virol 2000, 74:8635-8647.


90. Tullis GE, Burger LR, Pintel DJ: The minor capsid protein VP1 of the autonomous
parvovirus minute virus of mice is dispensable for encapsidation of progeny single-
stranded DNA but is required for infectivity. J Virol 1993, 67:131-141.


91. Lombardo E, Ramirez JC, Agbandj e-McKenna M, Almendral JM: A beta-stranded motif
drives capsid protein oligomers of the parvovirus minute virus of mice into the
nucleus for viral assembly. J Virol 2000, 74:3 804-3 814.


92. Clark KR, Voulgaropoulou F, Johnson PR: A stable cell line carrying adenovirus-
inducible rep and cap genes allows for infectivity titration of adeno-associated virus
vectors. Gene 7lher 1996, 3:1124-1132.


93. Agbandj e M, McKenna R, Rossmann MG, Strassheim ML, Parrish CR: Structure
determination of feline panleukopenia virus empty particles. Proteins 1993, 16: 155-
171.


94. Tsao J, Chapman MS, Agbandj e M, Keller W, Smith K, Wu H, Luo M, Smith TJ,
Rossmann MG, Compans RW, et al.: The three-dimensional structure of canine
parvovirus and its functional implications. Science 1991, 251:1456-1464.


95. Opie SR, Warrington KH, Jr., Agbandj e-McKenna M, Zolotukhin S, Muzyczka N:
Identification of amino acid residues in the capsid proteins of adeno-associated virus
type 2 that contribute to heparan sulfate proteoglycan binding. J Virol 2003, 77:6995-
7006.


96. Kern A, Schmidt K, Leder C, Muller OJ, Wobus CE, Bettinger K, Von der Lieth CW, King
JA, Kleinschmidt JA: Identification of a heparin-binding motif on adeno-associated
virus type 2 capsids. J Virol 2003, 77: 11072-11081.


97. Kronenberg S, Kleinschmidt JA, Bottcher B: Electron cryo-microscopy and image
reconstruction of adeno-associated virus type 2 empty capsids. EM~BO Rep 2001,
2:997-1002.









centrifugation (3000g for 10 minutes), and DOC was added to the supernatant to a Einal

concentration of 0.5%. The supernatant was digested with 50U/ml benzonase for 30 minutes at

37oC, filtered (Acrodisc 25mm PF, Pall), and purified by heparin affinity chromatography.

Virions and empty capsid preparations were dialyzed into 50mM Tris-Cl pH 8 containing

100mM NaCl using a 10,000 MWCO membrane (Pierce), aliquoted, and stored frozen at -20oC

or -80oC.


Proteolytic digestion. 0.8Clg of virions (equivalent to ~1.2X1011 capsids) were digested

with 5 Cg (0.02% Einal concentration) of trypsin (Gibco) or 80ug of co-chymotrypsin (Sigma) in a

25 Cl reaction at 37oC for up to 24 hours. The products of proteolysis were denatured at 100oC

using Laemmli sample buffer at final concentrations of 1% SDS and 655mM P-mercaptoethanol,

and separated by SDS-PAGE. After transferring the proteins to nitrocellulose (25mM

Tris/192mM Glycine/0. 1% (w/v) SDS/20%MeOH for 2 hours at 0.5 Amp in a Criterion-transblot

apparatus (Bio Rad) that reached 45oC), they were probed with antisera to AAV-2 polyclonall,

Al, Bl, A69 [65, 100]) and AAV-5 from Progen at dilutions of 1:250 polyclonall) to 1:2000

(monoclonal). The AAV-2 polyclonal antiserum cross-reacts with other AAV serotypes, but

cross-reacts weakly with AAV-5 under our conditions. Based on the peptides used to generate

the monoclonal antisera, B1 (antibody epitope IGTRYLTR) recognizes AAV1-3, and AAV5-10;

A69 (antibody epitope LNFGQTGDADSV) is specific for AAV-2; and Al (antibody epitope

KRVLEPLGL) recognizes AAV-1, AAV-2, AAV-4, AAV-7, AAV-8, AAV-10, and AAV-11.

Bands were visualized by chemiluminescence using HRP-conjugated secondary antibodies

(Amersham) and captured on X-ray film.

Mass spectroscopy and N-terminal sequencing. For mass spectroscopy and N-terminal

sequencing, DOC/CsCl/heparin purified rAAV2-GFP virions were digested with trypsin (0.02%




































O 2007 Kim Marie Van Vliet










Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

STUDIES OF THE ADENO-ASSOCIATED VIRUS CAPSID

By

Kim Marie Van Vliet

August 2007

Chair: Richard O. Snyder
Maj or: Medical Sciences Molecular Genetics and Microbiology

Adeno-associated virus (AAV) is a promising gene transfer vector that has been shown to

effect long-term gene expression and disease correction with low toxicity in animal models, and

is well tolerated in human clinical trials. The AAV capsid plays an essential role in cell binding,

internalization, and trafficking, which are critical processes for gene delivery to specific cells.

The structural proteins of the capsid spontaneously self-assemble into preformed shells, and then

DNA packaging occurs. Although the viral capsid proteins self-assemble, little is known about

this process. Potential subunits of assembly have been isolated by other investigators and this

data was utilized to develop the two general models for Parvovirus assembly discussed here.

Specific interactions that comprise the AAV-2 capsid subunit interfaces are analyzed with

respect to several capsid mutants and their impact on the formation of a macromolecular

structure. A better understanding of specific interactions required for AAV capsid assembly will

allow for the development of customized targeting AAV vectors in the future.

The AAV capsid is remarkably stable, which is a desirable characteristic for gene therapy

vectors; however, the work presented here demonstrates that AAV serotypes exhibit differential

susceptibility to proteases. The capsid fragmentation pattern upon limited proteolysis, as well as

the susceptibility of the serotypes to a series of proteases, provides a unique fingerprint for each











VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate rh25 AAS99242
isolate hu39 AAS99286
isolate rh49 AAS99247
isolate rh50 AAS99248
isolate rh51 AAS99249
isolate rh52 AAS99250
isolate rh64 AAS99259
isolate rh53 AAS99251
isolate rh61 AAS99257
isolate rh58 AAS99255
isolate rh57 AAS99254
AAV8 VP1 AANO3857


IPQYGYLTLN
IPQYGYLTLN
IPQYGNLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTPN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
VPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
IPQYGYLTLN
VPQYGYCGIV
VPQYGYCGLV
VPQYGYCGLV
VPQYGYCGLV
VPQYGYCGLV
LPQYGYATLN
LPQYGYATLN
LPQYGYATLN
LPQYGYATLN
LPQYGYATLN
LPQYGYCTRQ
IPQYGYCTLN
IPQYGYCTLN


NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQAVG
NG...SQALG
NG...SQSVG
NG...SQSVG
NG...SQSVG
ND...SQSVG
NG...SQSVG
NG...SQSVG
NG...SQAVG
DG...SQAVG
DG...GQAVG
DG...SQAVG
TG.ENQNQTD
TGNTSQQQTD
TGNTSQQQTD
TGGSSQNQTD
TGGSSQNQTD
RD.NTENPTE
RD.NTENPTE
RD.NGDNPTE
RD.NGDNPTE
DS.NNGNPTN
DG.NSNNPTP
YN.N..EAVD
YN.N..EAVD


RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RSSFYCLEYF
RNAFYCLEYF
RNAFYCLEYF
RNAFYCLEYF
RNAFYCLEYF
RNAFYCLEYF
RSSFFCLEYF
RSSFFCLEYF
RSSFFCLEYF
RSSFFCLEYF
RSSFFCLEYF
RSAFYCLEYF
RSAFYCLDYF
RSAFYCLDYF


PSQMRRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQVLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSQMLRTGNN
PSKMLRTGNN
PSKMLRTGNN
PSKMLRTGNN
PSKMLRTGNN
PSKMLRTGNN
PSKMLRTGNS
PSDMLRTGNN
PSDMLRTGNN


FEFSYQFEDV
FSFSYTFEDV
FSFSYTFEDV
FSFSYTFEDV
FSFSYTFEDV
FSFSYTFEDV
FSFSYTFEDV
FSFSYTFEDV
FSFSYPFEDV
FSFSYTFEDV
FSFSYTFEDV
FQFTYTFEDV
FQFTYTFEDV
FQFTYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FQFSYTFEDV
FEFSYSFEDV
FEFSYSFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FTFSYTFEDV
FSFSYTFEDV
FQFSYEFENV
FQFSYEFENV
FQFSYEFENV
FEMAYNFEKV
FEITYSFEKV
FEITYSFEKV
FEMVYKFENV
FEMVYKFENV
FEFTYNFEEV
FEFTYNFEEV
FEFTYSFEEV
FEFTYSFEEV
FEFTFEFEDV
FEFTYNFEKV
FEFTYTFEDV
FEFTYTFEDV


AAV8 YP 077180
VP1 isolate rh43 AAS99245
VP1 isolate pil AAS99238
VP1 isolate pi3 AAS99240
VP1 isolate pi2 AAS99239
VP1 isolate rh1 AAS99241
AAV7 VP1 AANO3855
AAV7 YP 077178


VP1
VP1
VP1
VP1
VP1

VP1
VP1


isolate rh48
isolate rh62
isolate rh55
isolate rh54
isolate rh60
AAV9 VP1
isolate hu31
isolate hu32
AAV11


AAS 992 4 6
AAS 992 5 8
AAS 992 5 3
AAS 992 52
AAS 992 5 6
AAS 992 64
AAS 992 81
AAS 992 8 2
AAT4 63 3 9


AAV4 NP 044927
AAV4 VP1 AAC58045
BOVINE AAV AAR26465
BOVINE AAV YP 024971
AAV5 VP1 AAD13756
AAV5 YP 068409
CAPRINE AAV1 AAU84890
GOAT AAV VP1 ABC69726
RAT AAV1 AAZ79676
MOUSE AAV1 AAZ79672
AVIAN PArVATCC VR865 AAO32087
AVIAN PArVATCC VR865 AAT48613









subject to cesium chloride gradients, AAV fractions were identified based on refractive index on

cesium chloride. These fractions were dialyzed and analyzed by green cell assay on C12 cells

[92].

Additional methods of production were explored by co-transfecting plM45 or the plM45

based mutant plasmid and UF5, followed by infecting the cells with adenovirus to provide the

helper function. The temperature sensitive phenotype was still present when using adenovirus to

supply the helper function. This production method was evaluated because other investigators

[64] have used this method for their temperature sensitive mutant studies.

Briefly, flasks were transfected for 5-7 hours and then infected with adenovirus (MOI=2)

and incubated at 32 OC, or 37 OC or 39.5 oC. Plates were incubated for 48 to 60 hours and cells

were harvested by aspirating them off the cell surface. Cells were pelleted by centrifugation.

Pellets were resuspended in 30 mls Lysis buffer. The cell lysate was prepared by repeated freeze

thaw cycles of the cell pellet.

The lysate was brought to 25% with Ammonium sulfate in an oakridge tube and a 25%

ammonium sulfate precipation was performed. This was incubated on ice for 1 hour, then

centrifuged 10 minutes at 5000 x g (8000 rpm). The supernatant was transferred to new oakridge

tubes and brought to 50% saturation with ammonium sulfate to precipitate rAAV and

adenovirus. These were incubated on ice for 1 hour and then centrifuged for 20 minutes at

12,000 X g (12,000 rpm). After removing the supernatant, pellets were centrifuged one more

time for 5 minutes at 5000 X g (8,000 rpm) and residual liquid was removed. This pellet

contained AAV and Ad and was dissolved in 1.37 g/ml CsC1. Cesium gradients were setup and

underlayed with 1.5 g/ml CsC1. Centrifugation was performed for 36 hours at 288,000 X g

(41,000 rpm). The CsCl gradient was dripped and fractions were collected. Refractometry was



















Polyclonal


AAV1 A~AV2 AAV1
0 1 5 12 24 0 1 5 12 24 0 1 5 12 24
100 kDa-



25 kDa, J- -
20 kDa -


AAV2 AAV5
0 1 5 12 24 0 1 5 12 24
-100



25
--20
Polyclonal


1.00E+11


1.00E+10



1.00E+09


1.00E+08


1.00E+07



1.00E+06


1.00E+05


5 10 15


Hours


Figure 4-6. AAV-2, AAV-1, and AAV-5 have different susceptibility to Chymotrypsin. A.
Chymotrypsin time course. Purified AAV-1, AAV-2, and AAV-5 were incubated at
37oC without chymotrypsin (0) or digested with chymotrypsin at 37oC for the time
(hours) indicated above each lane. The capsid proteins of AAV-1 and AAV-2 were
probed with anti-AAV2 polyclonal or B1 antisera, and AAV-5 was probed with anti-
AAV5 polyclonal sera. The asterisk indicates an additional AAV-2 band observed
with chymotrypsin that was not seen following trypsin digestion in Fig. 6A. B.
Infectivity. Infectious titering of rAAV1-GFP, rAAV2-GFP, and rAAV5-GFP
virions was performed following digestion with trypsin or chymotrypsin by infecting
C12 cells in the presence of Adenovirus (reported as the average + SDEV from three
separate infections). AAV-1 and AAV-5 infect C12 cells less efficiently than AAV-2
[103].









verifying the capsid serotype involves partial proteolysis of the capsid and analyzing the

fragments [46] as described in Chapter 4, or alternatively, other investigators have developed a

serotype specific ELISA utilizing antibodies that react to intact capsid proteins [114]. Several

monoclonal antibodies for AAV-2 capsids have been described, such as A20 which recognizes

intact AAV-2 or AAV-3 capsids [72], mAb C37 and C24 which selectively react with AAV-2

[100], and mAb D3 which shows broad reactivity with different serotypes [100]. Antibody B4 is

a monoclonal antibody that recognizes intact AAV-5 capsids; however, it has not been tested for

serotype specificity [115]. Other monoclonal antibodies have recently been generated that

recognize AAV1/6 (ADKla and b), AAV-4 (ADK4) and AAV-5 (ADK~a and b) [114] for the

purpose of serotype identity testing. ADKla and b, ADK4 or ADK~a and b were each shown to

recognize a serotype specific conformational epitope and do not cross-react with serotypes

AAV1-6, 8 and 9, with the exception of ADKla and b which also reacts with AAV-6, whose

primary sequence differs from AAV-1 by six amino acids. In 1999, Grimm, et al. developed a

capture ELISA for AAV-2 using the monoclonal antibody A20 [116]. Recently, newly generated

monoclonal antibodies have been utilized to develop a capture ELISA assay for serotypes 1/6, 4

and 5. However, with the potential of over a hundred AAV serotypes, it will be difficult to

generate unique conformational antibodies that do not cross-react with other serotypes. In

addition to using different serotypes, capsids may be specifically altered for improved targeting

to transduce specific cells and capsids have been modified to express peptide fragments on the

AAV surface.

Methods for determining AAV serotype identity utilizing intact capsids were described in

Chapter 4. The focus of this chapter includes techniques to determine the AAV capsid serotype

identity using denatured viral capsid proteins. The western blot assay for evaluating proteolytic









the hypothesis that it is the improper folding of the monomeric subunits that result in the

phenotype of Mut46, which is the inability to produce intact capsids. Figure 3-21 shows the

intramolecular interactions that occur in the wild-type capsid monomers. It should be noted

however that when Glu681, Ile682, and Glu683 were changed to alanine, that many of these

intramolecular interactions are still maintained. In Figure 3-22, these residues have been mutated

to alanine and intramolecular distances have been calculated. Based on this model, the primary

interaction that is disrupted in this mutant involves interactions with Glu683. Upon changing this

residue to Ala683, there is a 2.81 A+ interaction with Arg23 8 that is disrupted. After the mutation

this distance is approximately 7 A+. There is also an interaction between Arg683 and Val239 that

is disrupted as a result of the mutation. Interestingly, in Mut20 [89] Arg23 8 is mutated and the























Figure 3-21. Intramolecular interactions with the residues that are changed in Mut46, Glu681,
Ile682, and Glu683. Glu681 is pink, Ile682 is blue, and Glu683 is aqua. This figure
was generated using the molecular modeling software Deep View.












Name:
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9 am:
SName:
Nae:
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Name:
Name:
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Name:
Name:
Name:
Name:
Name:
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Name:


AAV3B VP1 AAB95452
VP1 isolate hul AAS99260
VP1 isolate hu4 AAS99287
VP1 isolate hu2 AAS99270
VP1 isolate hu3 AAS99280
VP1 isolate hu60 AAS99307
VP1 isolate hu61 AAS99308
VP1 isolate hu25 AAS99276
VP1 isolate hul5 AAS99265
VP1 isolate hul6 AAS99266
VP1 isolate hul8 AAS99268
VP1 isolate hu7 AAS99313
VP1 isolate hul0 AAS99261
VP1 isolate hull AAS99262
VP1 isolate hu9 AAS99314
VP1 isolate hu53 AAS99300
VP1 isolate hu55 AAS99302
VP1 isolate hu54 AAS99301
VP1 isolate huS17 AAU05370
AAV2 VP1 AACO3780
VP1 isolate hu34 AAS99283
VP1 isolate hu35 AAS99284
VP1 islolate hu51 AAS99298
VP1 isolate hu52 AAS99299
VP1 isolate hu47 AAS99295
VP1 isolate hu45 AAS99293
VP1 isolate hu58 AAS99305
VP1 isolate hu49 AAS99297
VP1 isolate hu56 AAS99303
VP1 isolate hu57 AAS99304
VP1 isolate hu28 AAS99278
VP1 isolate hu29 AAS99279
VP1 isolate huT70 AAU05364
VP1 isolate hul3 AAS99263
VP1 isolate hu63 AAS99309
VP1 isolate hu64 AAS99310
VP1 isolate huT40 AAU05362
VP1 isolate huLG15 AAU05371
VP1 isolate huT17 AAU05358
VP1 isolate huT41 AAU05372
VP1 isolate huT71 AAU05366


Len:
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Len:
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Len:
Len:
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Len:
Len:
Len:
Len:
Len:
Len:
Len:
Len:
Len:
Len:


778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778
778


Check:
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1343 Weight:
1391 Weight:
1303 Weight:
1141 Weight:
2673 Weight:
1875 Weight:
1690 Weight:
772 Weight:
1483 Weight:
2044 Weight:
2981 Weight:
996 Weight:
2978 Weight:
2837 Weight:
1894 Weight:
1757 Weight:
57 Weight:
9885 Weight:
1727 Weight:
3108 Weight:
2718 Weight:
2744 Weight:
3098 Weight:
4413 Weight:
4162 Weight:
4008 Weight:
2430 Weight:
3468 Weight:
4209 Weight:
1345 Weight:
3099 Weight:
2731 Weight:
3586 Weight:
1295 Weight:
2301 Weight:
2688 Weight:
1971 Weight:
590 Weight:
1851 Weight:
2922 Weight:
2922 Weight:









intramolecular interactions that are involved in proper protein folding. Mutl9 occurs at the

beginning of the first beta-sheet structure in VP3. If an intramolecular interaction was

responsible for the lack of capsids, the prediction would be that monomers would be formed that

are unable to associate into a capsid structure. The intermolecular interactions that may be

disrupted in Mutl9 are shown in Figure 3-5. In addition to Lys692, the interaction with Pro399

may also be disrupted as a result of Asp23 1 being changed to alanine.


Figure 3-2. Residues at the two fold symmetry axis that are changed in Mutl9. The aqua residue
is His229. The pink residue is Asp231. The residue shown in yellow is Lys692. The
residues that are mutated in Mutl9 are colored differently than the main chain,
usually white, except where they would not be visible, in which case they are aqua.
Yellow arrows point to the region of the mutation. This figure was generated using
the molecular modeling software Deep View.









disrupts these interactions, as the side chain in alanine is much smaller and is now not in close

enough proximity to interact. In addition, this charge:charge interaction is also disrupted and

may be required to hold the monomers together.


Figure 3-7. Residues at the 5-fold symmetry axis that are changed in Mut24. The view is from
the inside of the capsid. The aqua residue is Arg294. The pink residue is Asp295.
This figure was generated using the molecular modeling software Deep View.

When Glu689 was changed to an alanine in Mut47 [89], the phenotype is a non-infectious

capsid that is A20 negative. The lack of infectivity provides further evidence for the importance

of the interaction between Arg294 and Glu689. When Asp295 is changed to an alanine, the

intermolecular interaction between it and Tyr397 is disrupted. This residue is from the light pink

chain that is part of the 3-fold axis of symmetry shown in Figure 3-11i. Based on this, the

predicted subunits formed by Mut24 are monomers, assuming that the mutation at the 5-fold axis

of symmetry prevents pentameric interactions. To further evaluate the mutations that were made









TOPs Trimers of Pentamers

VIPERdb Virus Particle Explorer Database

wt wild-type


































Figure 3-18. Interactions in the region of Mut33. The yellow residue is Asp469, the aqua residue
is Arg471, and the white residue is Asp472. This figure was generated using the
molecular modeling software Deep View.


Figure 3-19. Model of residues mutated in Mu
modeling software Deep View.


.is figure was generated using the molecular









relies on for cleavage (K or R), this would not substantially change the number of amino acids in

the fragment that would result in a fragment too large to detect. The software Mascot which was

utilized to analyze the mass spec data can potentially detect this mutation. This experiment will

be repeated utilizing a different protease that doesn't rely on basic residues and the software

Mascot will be used determine if the fragment containing the mutation can be identified.

A database has been built that includes all of the viral proteins for all of the known

serotypes. For the studies presented in this chapter, the MS data was searched thru the NCBI

database, which was limited to ALL viral proteins. We have built a database that includes only

the AAV sequences, as well as the mutant sequence for AAV-4. This is currently being further

developed and this will be useful for customized gene therapy vectors in the future that have

been modified. These sequences can be deposited into the database, and clinical grade preps can

be tested and verified. Several investigators have shown that single amino acid mutations in the

AAV capsid can effect tropism [128]. For AAV-2, mutation of several amino acids has been

shown to result in vector vector targeting to cardiac tissue. A vector like this could potentially be

utilized in clinical trials and AAV Capsid Serotype Identifieation (AAV-CSI) would be useful

for validating that the therapeutic gene has been packaged into the correct capsid.

Materials and Methods

Viruses and cell lines. rAAV2-GFP vector virions were produced by transient transfection

of HEK293 cells, cells were harvested, then lysed with 0.5% deoxycholate (DOC), incubated for

30 minutes at 370C with 50 U/ml ofbenzonase, and purified on sequential CsCl density

gradients. Cesium fractions were dialyzed and heparin chromatography was performed as a

concentration step [111] rAAV1-GFP, rAAV4-GFP, rAAV5-GFP and rAAV8-GFP vector

virions were purified by freeze/thaw and CsCl gradients followed by Q-sepharose

chromatography. Infectivity of the AAV vectors were assayed on C12 cells in the presence of

123











VP1 isolate rh38 AAS99243
VP1 isolate hu41 AAS99289
VP1 isolate hu66 AAS99311
VP1 isolate hul7 AAS99267
VP1 isolate hu6 AAS99306
VP1 isolate rh25 AAS99242
VP1 isolate hu39 AAS99286
VP1 isolate rh49 AAS99247
VP1 isolate rh50 AAS99248
VP1 isolate rh51 AAS99249
VP1 isolate rh52 AAS99250
VP1 isolate rh64 AAS99259
VP1 isolate rh53 AAS99251
VP1 isolate rh61 AAS99257
VP1 isolate rh58 AAS99255
VP1 isolate rh57 AAS99254
AAV8 VP1 AANO3857
AAV8 YP 077180
VP1 isolate rh43 AAS99245
VP1 isolate pil AAS99238
VP1 isolate pi3 AAS99240
VP1 isolate pi2 AAS99239
VP1 isolate rh1 AAS99241
AAV7 VP1 AANO3855
AAV7 YP 077178
VP1 isolate rh48 AAS99246
VP1 isolate rh62 AAS99258
VP1 isolate rh55 AAS99253
VP1 isolate rh54 AAS99252
VP1 isolate rh60 AAS99256
AAV9 VP1 AAS99264
VP1 isolate hu31 AAS99281
VP1 isolate hu32 AAS99282
AAV11 AAT46339
AAV4 NP 044927
AAV4 VP1 AAC58045
BOVINE AAV AAR26465
BOVINE PAY YP 024971
AAV5 VP1 AAD13756
AAV5 YP 068409
CAPRINE AAV1 AAU84890


DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQVLIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK STPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPV
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPV
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPV
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPL
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHP..QILIK NTPVPANPPE
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT
DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT
DIYYQGPIWA KIPHADGHFH PSPLIGGFGL KHPPPQIFIK NTPVPANPAT
DIYYQGPIWA KIPHTDGHFH PSPLIGGFGL KHPPPQIFIK NTPVPANPAT
DIYYQGPIWA KIPHTDGHFH PSPLIGGFGL KHPPPQIFIK NTPVPANPAT
DIYYQGPIWA KIPHTDGHFH PSPLIGGFGL KSPPPQIFIK NTPVPANPAT
DIYYQGPIWA KIPHTDGHFH PSPLIGGFGL KSPPPQIFIK NTPVPANPAT
DVYLQGPIWA KIPETGAHFH PSPAMGGFGL KHPPPMMLIK NTPVPGN.IT
DVYLQGPIWA KIPETGAHFH PSPAMGGFGL KHPPPMMLIK NTPVPGN.IT
DVYLQGPIWA KIPETGAHFH PSPAMGGFGL KHPPPMMLIK NTPVPGN.IT










Table 3-4 Continued
H-H
H-H
H-H
H--FI
H-P
H-B
H-P
H H
H H
H P
H H
H H
H H
H-P
H H
H H
H H
H-P
H-P
B-B
B-P
P-P
P-P
P-P
P P
B-P
B P
A P
A P
P--B
P-P
P--H
P H
P H
P H
P-P
P-P
P--A
P-P
P-P
P-P
P--H
P--B
P-P
P--H


L442:A357
L442:L540
L442:1F542
L442:M634
I43 8:Y281
I43 8:H3 58
I43 8:4Q359
I438:P373
P436:I260
P436:Y272
P436:G376
P436:L378
L601:P481
L601:Y483
L601:P521
L601:G599
L445:V488
L445:S501
L445:Q536
R566:R389
R566:N511
Q584:4Q486
IQ584:N495
IQ584:N496
Q584:Y500
R475:Y508
R475:S515
D528:N382
D528:N511
S580:R484
S580:4Q486
N476:A619
N476:P632
N476:L633
N476:M634
S458:N495
S458:N497
T448:E499
T448:Y500
T448:S501
N43 5:Y281
N435:V353
N43 5:H3 58
N43 5:Y375
N435:G376


Mut22 unchanged











Matt29 --wt








Mut3 6 unchanged


Mut37 ni, full
particle









This interaction also involves the 2-fold axis of symmetry. If this interaction is responsible for

the phenotype of Mutl9, the expected subunit would also be a trimer. Another possibility is that

both Lys592 and Pro399 are needed to interact with Asp231. Using the methods established here,

it is not possible to determine if both interactions are required for intact capsid formation.

However, Pro399 is 3.60 A+ away from Asp231, while Lys692 is only 2.78 A+ away. The model of

Mutl9 after the amino acids are changed to alanine is shown in Figure 3-6.

Another approach to evaluating the effect of the alanine mutation is to do a structural

alignment of several parvovirus capsid proteins and look at whether or not the residues that are

changed in Mutl9 are conserved. The capsid proteins of several parvoviruses were aligned, and

then evaluated to determine if the residues that were mutated in Mutl9 are conserved among

other parvoviruses. These are shown in Table 3-9. The table includes AAV, Porcine Parvovirus,

MVM, and CPV. Based on the table, the residues in Mutl9 appear to be unique to AAV. AAV

residue in this position. Based on the structural alignment, the prediction would be that this

residue might be able to be changed without affecting the overall structure, since there is such a

wide range of amino acids in this position for other parvoviruses. AAV is also the only

parvovirus in the table that has a negatively charged acidic residue at Asp231. Based on this, the

prediction would be that changing Asp231 to alanine, should be tolerated by AAV, as the other

parvoviruses listed have neutral amino acids in this position. However, the model indicates that

the negatively charged Asp231 interacts with the positively charged Lys692. In the capsid

protein alignment, amino acid 692 in porcine parvovirus and MVM are neutral residues, N and T

respectively, while CPV, like AAV-2 also has a positively charged basic residue (H) in this

position. Based on the data, the charge-charge interaction between Asp231 and Lys692 is

probably necessary for AAV capsid stability.










Table 3-1. The Hyve strongest interactions at the icosahedral 5-fold axis of symmetry (VIPERdb).
The amino acids that are the strongest at the icosahedral 5-fold axis of symmetry are
listed, using VPI numbering. This refers to interactions between subunits Al and A2
in the icosahedral virus particle in Figure 2-2. The type of interactions are indicated,
B = basic, H = hydrophobic, P= Polar, uncharged. The phenotype of AAV mutants
generated by Wu, et al. [84] are listed, wt = wild type.

Amino VP I Energy
Acid numbering Type (kcal/mol) Wu, et al. mutant phenotype [84]
ARG 404 B -3.76
LYS 665 B -3.57 Mut45, wt
PHE 661 H -3.11
TYR 397 P -3.07
MET 402 H -2.14

Table 3-2. Specific amino acid interactions at the icosahedral 5-fold axis of symmetry
(VIPERdb). Specific amino acids at the icosahedral 5-fold axis of symmetry are
listed, using VPI numbering, with the strongest interactions listed first, and the
weakest interactions listed last. This list includes all interactions between subunits Al
and A2 in the icosahedral virus particle depicted in Figure 2-2. The type of
interactions are indicated, A = acidic, B = basic, H = hydrophobic, P = Polar,
uncharged. The phenotype of AAV mutants generated by Wu, et al. are listed, either
in the column to the left of the amino acid list if the mutation was made in the 1st
amino acid of the pair listed, or in the column to the right of the amino acid list if the
mutation was made in the 2nd amino acid of the pair listed. cs cold-sensitive, hep+
= heparin positive, hs = heat-sensitive, ni = non-infectious, pd = partially defective,
wt = wild-type. Amino acids that were not mutated by Wu, et al. [84], but flank the
mutated amino acids are designated unchanged.
Type of Mutant Phenotype VP I Mutant Phenotype
Interaction (Wu, et al.[84]) numbering (Wu, et al.[84])
B -H R404:V221
B -H R404: G222
B -P R404: S224
B -P R404: S225
B -P R404:N317
B -P R404:Q319
B -P R404:T405
B A Mut45 wt K665:D368
H-P F661:Y252
H H F661:G360
H H F661:M371
H -H F661:P373










R588


B1 epitope


Figure 4-7. Capsid structure. A. Capsid monomer. Ribbon representation of an AAV-2 VP3
monomer (amino acids 217-73 5) rotated 900 from standard icosahedral orientation.
P-strands and co-helices are represented as arrows and coils, respectively. Small
arrows indicate the location of R588 (blue spheres) and the B 1 epitope at the extreme
C-terminus (green). The 2-fold (oval), 3-fold (triangle) and 5-fold (pentamer) axes
are indicated. B. Side view of an AAV-2 VP3 trimer. Amino acids R585 and R588
in the three G-H loops from three VP3 monomers (gray, salmon, wheat) are
represented as blue spheres and indicated by arrows.









nucleus. Functional NLM' s depend on a correct three-dimensional protein conformation. For

AAV2, the sequence KGKLTMRAKLR is not present in the C-terminal region of VP3;

however, an alignment of the MVM capsid protein with the AAV2 capsid protein sequence

reveals that this is the location of the mutations introduced in Mut46. This further supports the

suggestion that the phenotype of Mut46 is the result of improper protein folding which disrupts

the intrastrand hydrogen bonding interactions of the P-sheet, which also may result in a defect in

protein trafficking through the nuclear pore complex. Other mutations introduced into Mut46 by

Wu et al. [89], such as a serine substitution at amino acid position 682 (Mut46subserl5), and a

FLAG substitution at amino acid position 682 (Mut46subfigll1) also resulted in a noninfectious,

no capsid phenotype.

Methods for Mutant Studies

The capsid mutants were evaluated biochemically in an effort to determine their

properties with respect to capsid assembly and packaging. DNA for the mutant AAV capsids was

transfected and analyzed in an attempt to isolate and identify an assembly intermediate. For

AAV, isolating an intermediate of assembly is complicated due to the requirement of a helper

virus such as adenovirus for production of AAV. Adenovirus assembly is known to proceed via

pentameric intermediates and hexon and penton proteins isolated from AAV preps can easily be

mistaken for pentameric AAV intermediates by electron microscopy. Baculovirus produced

AAV vectors do not require a helper virus; however, the baculovirus proteins that are providing

the helper function for AAV production have not yet been elucidated and few antibodies are

available for baculovirus proteins (anti-gp64). The method developed and described in Chapter 5

for AAV Capsid Serotype Identification (AAV-CSI) will be useful for verifying and validating

that the assembly intermediate isolated is an actual AAV intermediate and not a contaminating

subunit from the helper virus used to produce the AAV. Of the capsid mutants described here,










18. Strauss BE, Costanzi-Strauss E: Combating oncogene activation associated with
retrovirus-mediated gene therapy of X-linked severe combined immunodeficiency.
Braz J2~ed Biol Res 2007, 40:601-613.


19. Chen J, Wu Q, Yang P, Hsu HC, Mountz JD: Determination of specific CD4 and CD8 T
cell epitopes after AAV2- and AAV8-hF.IX gene therapy. Mol1 Ther 2006, 13:260-269.


20. Jooss K, Yang Y, Fisher KJ, Wilson JM: Transduction of dendritic cells by DNA viral
vectors directs the immune response to transgene products in muscle fibers. J Virol
1998, 72:4212-4223.


21. Zaiss AK, Muruve DA: Immune responses to adeno-associated virus vectors. Curr
Gene Ther 2005, 5:323-331.


22. Wang L, Figueredo J, Calcedo R, Lin J, Wilson JM: Cross-presentation of adeno-
associated virus serotype 2 capsids activates cytotoxic T cells but does not render
hepatocytes effective cytolytic targets. Hum Gene Ther 2007, 18:185-194.


23. Gao G, Vandenberghe LH, Alvira MR, Lu Y, Calcedo R, Zhou X, Wilson JM: Clades of
Adeno-associated viruses are widely disseminated in human tissues. J Virol 2004,
78:6381-6388.


24. Gao G, Vandenberghe LH, Wilson JM: New recombinant serotypes of AAV vectors.
Curr Gene 7Jher 2005, 5:285-297.


25. Schmidt M, Grot E, Cervenka P, Wainer S, Buck C, Chiorini JA: Identification and
characterization of novel adeno-associated virus isolates in ATCC virus stocks. J Virol
2006, 80:5082-5085.


26. Chen CL, Jensen RL, Schnepp BC, Connell MJ, Shell R, Sferra TJ, Bartlett JS, Clark KR,
Johnson PR: Molecular characterization of adeno-associated viruses infecting
children. J Virol 2005, 79:14781-14792.


27. Rutledge EA, Halbert CL, Russell DW: Infectious clones and vectors derived from
adeno-associated virus (AAV) serotypes other than AAV type 2. J Virol 1998, 72:309-
319.











Poly
ABCD


B1
ABCD


A69 Al
ABCD ABCD



*- 4
I.p


150
100
75


kDa
kDa
kDa -


-VP1
- VP2, VP1T

TVP2T
-VP3T


50 kDa

37 kDa


25 kDa
2 0 kDka


15 kDa
1 2 54


1M


-VP1,2,3 T


9 10 11 12 15 14 15 1C


Full Empy
0 1 5 12 24 0 1 5 12 24
100 kDa -I
-~ VP1
75ik~aa-3 ....-- --- --- VP2,VP1T~
L C~~~_ I ~f_ C~ VP3
5~~~ k~ VP3T
37rkDa -


Fu~ll....... Empty
0 0.65 2 0 0.5 2


- 100 k~a
- 75 kD~a
- 50 kMa

-- 37 krDa


25 kDa


-- 25 kcDa


Trypsin


Chymotrypsin


Figure 4-4. Proteolysis distinguishes full and empty AAV-2 particles. A. Trypsin. Purified full
(F-T/iodixanol/heparin) and empty AAV-2 capsids were treated with 0.02% trypsin,
and the capsid proteins were probed with the indicated antibodies (AAV-2 polyclonal,
Al, Bl, A69). Lanes A, AAV-2 full capsids; lanes B, AAV-2 full capsids digested
with trypsin for 24 hours; lanes C, AAV-2 empty capsids digested with trypsin for 24
hours; lanes D, AAV-2 empty capsids. B. Trypsin time course. Purified full (F-
T/iodixanol/heparin) and empty AAV-2 virions were treated with trypsin for the time
(hours) indicated above each lane and probed with anti-AAV2 polyclonal antisera. C.
Chymotrypsin time course. Purified full (F-T/iodixanol/heparin) and empty AAV-2
capsids were treated with chymotrypsin for the time (hours) indicated above each lane
and the capsid proteins were probed with anti-AAV2 polyclonal antisera.


* b- -h I *
... Ce .


\~ r










The potential intermediates of assembly for AAV could include pentameric subunits, 12 of

which must come together to make an intact capsid, trimeric subunits, 20 of which comprise an

intact capsid, or dimeric subunits, 30 of which must come together to produce an intact capsid.

The two favored hypotheses for intermediates in assembly are: 1. Assembly via a pentameric

intermediate or 2. Assembly via a trimeric intermediate.

Assembly via a Pentameric Intermediate

If a pentameric intermediate is the assembly intermediate, pentamers form first

(intrapentameric interactions), and then interpentameric interactions form and subsequent

pentamers are added. This model requires a trimer of pentamers or TOPs, to produce the 5-fold,

2-fold and 3-fold interactions. In (DX174, a ssDNA bacteriophage, the first detectable

intermediates in assembly are pentamers of the F protein [74]. If the mechanism of assembly for

ssDNA viruses is conserved, the hypothesis regarding AAV assembly is that pentamers form






lb 10X


5 1








A7.7









AAV viral proteins which would not be distinguishable in an SDS page gel to assess purity. In

Chapter 4, we showed that proteolysis of AAV-2 VP I results in a degradation product, VP IT,

that is similar in size to VP2, which were detected using available antibodies for AAV-2. Fewer

antibodies are available for other serotypes, and the A69 antibody that recognizes the N-terminus

VP2 is unique for AAV serotype 2. For AAV-2, the site of proteolytic cleavage mapped to a

critical region in the capsid required for heparin binding and as a result, capsids with a cleavage

in the GH loop are removed during heparin chromatography; however, if the cleavage had not

been in a critical region, or for other serotypes such as AAV-8 that may be less stable, this

technique will be useful for detecting degradation products of VP I that are similar in size to

VP2 .

Another goal of this work was to determine if a capsid mutation has occurred, if it could be

identified using this method. Because the raw data utilized for mass spec is based on the

fragmentation pattern of a known protein, this method will not be able to identify if a capsid

mutation has occurred during production. For example, a mutation in AAV-4 that resulted in the

change of a basic amino acid at amino acid 544 to an acidic amino acid would no longer have a

trypsin cleavage site at this position and the resulting fragment would be much larger than

expected. However, in the database search, this larger fragment would not be present in the

database. Therefore, for this region of the AAV capsid, there is a lack of coverage in the MS data

for these amino acids. Figure 5-5 is LC/MS/MS data for wt AAV-4 and AAV-4 R544E mutant

which shows partial coverage for wt AAV-4 in the region of the mutation which allows

verification that R544 is a basic residue in the wild type virus, and a lack of coverage in R544E,

which is due to a known mutation in the starting material used in this assay. Coverage or the

number of amino acids detected after mass spec analysis is rarely 100%, so the lack of coverage











VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate hu47


AAS 992 9 5
AAS 992 9 3
AAS9 93 0 5
AAS 992 9 7
AAS9 93 0 3
AAS9 93 0 4
AAS 992 7 8
AAS 992 7 9
AAU 05364
AAS 992 63
AAS9 93 0 9
AAS 9 9310
AAU 053 62
AAU 05371
AAU 05358
AAU 05372
AAU 05366
AAU 05368
AAU 05360
AAS 992 7 7
AAS 992 6 9
AAS 992 71
AAS 992 7 2
AAS 992 7 5
AAS 992 7 3
AAS 992 7 4


TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKFVSF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF
TFSAAKEASF

751
..VDFTVDNN
..VDFTVDNN
..VDFTVDNN
..VDFTVDNN
..VDFTVDNN
..VDFTVDNN
..VDFTVDNN
..VDFTVDNN
..VDFTVDNN
..VDFAVNTE
..VDFAVNSE
..VDFAVNTE
..VDFAVNTE


ITQYSTGQVS
ITQYSTGQVS
ITQYSTGRVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS


GLYTEPRPIG
GLYTEPRPIG
GLYTEPRPIG
GLYTEPRPIG
GLYTEPRPIG
GLYTEPRPIG
GLYTEPRPIG
GLYTEPRPIG
GLYTEPRPIG
GTYSEPRPIG
GTYSEPRPIG
GTYSEPRPIG
GTYSEPRPIG


VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE

778
TRYLTRPL
TRYLTRPL
TRYLTRPL
TRYLTRPL
TRYLTRPL
TRYLTRPL
TRYLTRPL
TRYLTRPL
TRYLTRPL
TRYLTRNL
TRYLTRNL
TRYLTRNL
TRYLTRNL


NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ


YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN
YTSNYNKSVN


isolate
isolate
isolate
isolate
isolate
isolate
isolate


hu45
hu58
hu49
hu56
hu57
hu28
hu29


VP1 isolate huT70
VP1 isolate hul3
VP1 isolate hu63
VP1 isolate hu64
VP1 isolate huT40
VP1 isolate huLG15
VP1 isolate huT17
VP1 isolate huT41
VP1 isolate huT71
VP1 isolate huT88
VP1 isolate huT32
VP1 isolate hu27
VP1 isolate hul9
VP1 isolate hu20
VP1 isolate hu21
VP1 isolate hu24
VP1 isolate hu22
VP1 isolate hu23


AAV VR195 ABA71699
AAV VR355 ABA71701
AAV1 NP 049542


AAV1 VP1 AAD27757
ate hu48 AAS99296
AAV6 VP1 AAB95450
ate hu43 AAS99291
ate hu44 AAS99292
ate hu46 AAS99294
AAV10 AAT46337
ate rh40 AAS99244
ate hu37 AAS99285
ate hu42 AAS99290


VP1 isol

VP1 isol
VP1 isol
VP1 isol

VP1 isol
VP1 isol
VP1 isol












VP1 isolate hul3
VP1 isolate hu63
VP1 isolate hu64
VP1 isolate huT40
VP1 isolate huLG15
VP1 isolate huT17
VP1 isolate huT41
VP1 isolate huT71
VP1 isolate huT88
VP1 isolate huT32


AAS 992 63
AAS9 93 0 9
AAS 9 9310
AAU 053 62
AAU 05371
AAU 05358
AAU 05372
AAU 05366
AAU 05368
AAU 05360
AAS 992 7 7
AAS 992 6 9
AAS 992 71
AAS 992 7 2
AAS 992 7 5
AAS 992 7 3
AAS 992 7 4



ABA71699
ABA71701


SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWYCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST

301
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYGTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY


WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGDRVITTS
WMGGRVITTS
WMGDRVITTS



FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
LDFNRFHCHF


TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTCN



SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN


NHLYKQISSQ
NHLYKQISSQ
NHLYRQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ



NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL


S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH
S..GASNDNH

350
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVE
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKPFNIQVK
SFKLFNIQVK
SFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
SFKLFNIQVK


VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate
isolate
isolate
isolate


hu27
hul9
hu20
hu21


isolate hu24
isolate hu22
isolate hu23


AAV VR195
AAV VR355


AAV1 NP 049542
AAV1 VP1 AAD27757
ate hu48 AAS99296
AAV6 VP1 AAB95450
ate hu43 AAS99291
ate hu44 AAS99292
ate hu46 AAS99294
AAV10 AAT46337
ate rh40 AAS99244
ate hu37 AAS99285
ate hu42 AAS99290
ate hu40 AAS99288
ate hu67 AAS99312
ate rh38 AAS99243
ate hu41 AAS99289
ate hu66 AAS99311
ate hul7 AAS99267
late hu6 AAS99306
ate rh25 AAS99242
ate hu39 AAS99286


VP1 isol


VP1
VP1
VP1

VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1


isol
isol
isol

isol
isol
isol
isol
isol
isol
isol
isol
isol


VP1 iso
VP1 isol
VP1 isol






































the AAV capsid, and disassembly experiments to date suggest it is not possible to gently disrupt

the capsid and isolate an intermediate of assembly. The energy required to disrupt the 3-fold

interactions results in the capsids being broken down completely into monomers when heated

and treated with SDS (data not shown). For MVM, one study evaluated critical residues

involved in inter-trimeric contacts and showed that both hydrophobic interactions and hydrogen

bonding between interfacial side chains could potentially make maj or contributions toward

holding trimeric subassemblies together. These inter-trimeric contacts were nearly identical for

both strains of MVM, MVMi which is pathogenic and MVMp which is nonpathogenic [86].

The five strongest interactions at the icosahedral 2-fold axis of symmetry are listed in

Table 3-5. The specific residues and types of interactions that occur at the icosahedral 2-fold axis

of symmetry are listed in Table 3-6. While the strongest interaction at the 2-fold axis is a

hydrophobic interaction, electrostatic interactions contribute 13.63 kcal/mol per subunit at the 2-


V


Mut47 unchanged
Mut42 ni, no capsid

Mut42 ni, no capsid


Table 3-4 Continued
H B
P A
B H
P B
B -P
H P
H P
P P
A A
P-P
P P
P A
H P
H H
H B
P B
P P
P P
B A


A425:R389
S422:D625
H629: G626
Y424 :H623
H426:T624
V595:Y483
V595:T597
S691:Y393
D608:D625
T732:S391
Q607:T624
Q607:D625
L430:Y508
L430:L510
G466:K549
Q473 :H3 58
N582:Q486
T568:T624
R729:D625


Mut29
Mut43
Mut43

Mut43


wt
wt
unchanged

unchanged


Mut43

Mut43


Mut3 8 wt


Mut43
Mut43


unchanged
wt











250 kDa
150 kDa
-- 100 kDa

-75 kDa

-50 kDa

S -37 kDa


-25 kDa
e 1 2 3 4 20 kDa
Figure 4-1. Western blot of several AAV-2 preps. Lane 1 AAV-2 standard. Lane 2 AAV-2
sample. Lane 3 AAV-2 sample. Lane 4 AAV-2 sample. Using polyclonal
antibody, AAV fragments are detectable in the sample lanes.

antibody. This detects the unique N-terminal region that is common in VPI and VP2. Lane 6

shows that upon cleavage with trypsin there is a unique fragment that runs just smaller than VP3.

A69 does not recognize VP3, so this fragment is the result of a C-terminal trypsin cleavage event

that results in the N-terminal fragment shifting in size after proteolysis to approximately 50 kDa.

This fragment was designated VP2T. Lanes 7 and 8 are probed with Al antibody. Lane 8 shows

a shift after proteolysis in the size of VP l. Because it is detectable by Al and the epitope for the

antibody is on the N-terminus of VP 1, this fragment was designated VP 1T. Figure 4-2 also

shows a diagram of the approximate locations of the epitopes for the various antibodies to the

AAV structural proteins, as well as and arrow which indicates the trypsin cleavage site.

Fine mapping of the trypsin cleavage site. Trypsin cleavage sites of AAV-2 VP I were

determined using the program Peptide Cutter on the ExPASy server (www.expasy.org). Based

on the potential basic residues where trypsin can cleave, these sequences were entered into the

program Peptide Mass on the ExPASy server and the mass of each trypsin fragment was

determined. This is shown in Table 4-1. Theoretical prediction of trypsin sites in the capsid G-H

loop [48], comprising amino acid residues 416 to 645, and fragment masses for AAV-2











VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate hu45
isolate hu58


AAS 992 9 3
AAS9 93 0 5
AAS 992 9 7
AAS9 93 0 3
AAS9 93 0 4
AAS 992 7 8
AAS 992 7 9
AAU 05364
AAS 992 63
AAS9 93 0 9
AAS 9 9310
AAU 053 62
AAU 05371
AAU 05358
AAU 05372
AAU 05366
AAU 05368
AAU 05360
AAS 992 7 7
AAS 992 6 9
AAS 992 71
AAS 992 7 2
AAS 992 7 5
AAS 992 7 3
AAS 992 7 4


DVYLQGPIWA
DVYLQGPIWA
DVHLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPTWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLRGPIWA

701
EFSATKEASF
EFSATKEASF
EFSATKEASF
EFSATKEASF
EFSATKEASF
EFSATKEASF
EFSATKEASF
EFSATKEASF
EFSATKEASF
TFSQAKLASF
TFSQAKLASF
TFSQAKLASF
TFSQAKLASF
TFSQAKLASF


KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFY
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH



ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSAGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS
ITQYSTGQVS


PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLVGGFGL
PSPPMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL



VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE
VEIEWELQKE


KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK



NSKRWNPEVQ
NSKRWNPEVQ
NSKRWNPEVQ
NSKRWNPEVQ
NSKRWNPEVQ
NSKRWNPEVQ
NSKRWNPEVQ
NSKRWNPEVQ
NSKRWNPEVQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ
NSKRWNPEIQ


NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST
NTPVPANPST

750
YTSNYAKSAN
YTSNYAKSAN
YTSNYAKSAN
YTSNYAKSAN
YTSNYAKSAN
YTSNYAKSAN
YTSNYAKSAS
YTSNYAKSAN
YTSNYAKSAN
YTSNYYKSTN
YTSNYYKSTN
YTSNYYKSTN
YTSNYYKSTN
YTSNYYKSTN


isolate
isolate
isolate
isolate
isolate


hu49
hu56
hu57
hu28
hu29


VP1 isolate huT70
VP1 isolate hul3
VP1 isolate hu63
VP1 isolate hu64
VP1 isolate huT40
VP1 isolate huLG15
VP1 isolate huT17
VP1 isolate huT41
VP1 isolate huT71
VP1 isolate huT88
VP1 isolate huT32
VP1 isolate hu27
VP1 isolate hul9
VP1 isolate hu20
VP1 isolate hu21
VP1 isolate hu24
VP1 isolate hu22
VP1 isolate hu23


AAV VR195 ABA71699
AAV VR355 ABA71701
AAV1 NP 049542
AAV1 VP1 AAD27757
VP1 isolate hu48 AAS99296
AAV6 VP1 AAB95450
VP1 isolate hu43 AAS99291
VP1 isolate hu44 AAS99292
VP1 isolate hu46 AAS99294
AAV10 AAT46337
VP1 isolate rh40 AAS99244
VP1 isolate hu37 AAS99285
VP1 isolate hu42 AAS99290
VP1 isolate hu40 AAS99288









CHAPTER 3
AAV-2 ASSE1VBLY AND SUBUNIT INTERACTIONS

Introduction

Understanding the capsid subunit interactions that are critical for AAV virion assembly

will allow for the production of customized vectors in the future. An analysis of the types of

residues located at the 5-fold axis of symmetry, the 3-fold axis of symmetry, as well as the 2-fold

axis of symmetry provides a starting point for evaluating residues that are important for virus

assembly. The interactions at the 5-fold axis of symmetry are shown in Table 3-1, with the

strongest interactions listed first. The specific amino acids involved in the strongest

intrapentermic interactions at the 5-fold axis of symmetry are shown in Table 3-2. For the

mutants produced by Wu, et al. [84], an analysis of the phenotype when that amino acid has been

mutated is also included.

The strongest interaction energies for the 3-fold axis of symmetry are listed in Table 3-3.

The specific residues involved in these interactions, as well as the phenotype of capsids with

mutations in these residues are listed in Table 3-4. The mutations at the 3-fold axis may provide

information regarding assembly and packaging since mutants have been constructed in which the

strongest interactions at the 3-fold axis have been disrupted. One of these mutants (Mut3 1)

results in the formation of empty particles but is unable to package DNA. This would be

consistent with a conserved mechanism for packaging in small DNA viruses, like OX174 where

DNA is inserted at the 3-fold, and exits the virion thru the 5-fold pore. However, due to other

mutant studies, the most likely site for DNA packaging for AAV is through the 5-fold pore [85].

The interactions at the 3-fold axis of symmetry are very strong and would represent a significant

barrier to packaging. These interactions are probably responsible for the remarkable stability of









127. Becerra SP, Rose JA, Hardy M, Baroudy BM, Anderson CW: Direct mapping of adeno-
associated virus capsid proteins B and C: a possible ACG initiation codon. Proc Natl
Acad'Sci USA 1985, 82:7919-7923.


128. Wu Z, Asokan A, Grieger JC, Govindasamy L, Agbandj e-McKenna M, Samulski RJ:
Single amino Acid changes can influence titer, heparin binding, and tissue tropism in
different adeno-associated virus serotypes. J Virol 2006, 80: 11393-11397.


129. Laemmli UK: Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 1970, 227:680-685.


130. Keller A, Nesvizhskii AI, Kolker E, Aebersold R: Empirical statistical model to estimate
the accuracy of peptide identifications made by MS/MS and database search. Anal
Chem 2002, 74:5383-5392.


131. Nesvizhskii AI, Keller A, Kolker E, Aebersold R: A statistical model for identifying
proteins by tandem mass spectrometry. Anal Chem 2003, 75:4646-4658.































Figure 3-1. Residues at the five fold symmetry axis that are changed in Mutl9. The aqua residue
is His229. The pink residue is Asp231i. Yellow arrows point to the location of the
mutation. This figure was generated using the molecular modeling software
DeepView.

using the conformational antibody A20; however, capsid proteins were subsequently detected

using B1 antibody and the buoyant density in cesium chloride for these capsid proteins was

consistent with the production of intact capsids (Hartladge, Warrington and Muzyczka,

unpublished data). Figure 3-3 shows that the 3-fold interactions are still intact as a result of the

amino acid changes in Mutl9. Based on this, the predicted subunit of assembly that should be

produced from Mutl9 is a trimer. To determine what interactions were disrupted by changing

His229 and Asp231 to alanine, the amino acids WHCDS were selected in the model.Amino acids

were added to the view within 4 A+ of the selected residues. Based on this, intermolecular

distances and intramolecular distances were determined. The model shows several

intramolecular interactions between His229 and Thr242, Thr243 and Thr244, as seen in Figure

3-4; however, when His229 was changed to an alanine, these interactions are still maintained

(data not shown). This suggests that the lack of capsids is probably not due to disrupting the









Trypsin-Treated Virons Remain Intact. The presence of tryptic fragments in the AAV2-

GFP vector preps indicated that the cleaved products remain tightly associated with the capsid,

as these fragments are still present after virions are subj ected to cesium chloride gradients. The

buoyant density of these capsids are ~1.40 g/cm3, indicating a protein and DNA composition that

is similar to other vector preps. The value is slightly less than the density of wt AAV-2 (1.41 -

1.45 g/cm3) because the rAAV2-GFP vector is smaller (4331 nucleotides) than the wt AAV

genome (Genbank AFO43303). Figure 4-3A shows Negative-stain EM analysis of the trypsin-

treated and untreated purified rAAV2-GFP samples. This confirmed the intact nature of the

treated capsids (Figure 4-3A). However, the negative staining pattern suggested a difference

between the samples in their permeability to uranyl acetate, indicating a possible structural

rearrangement or flexibility due to the cleavage event.

Antibodies, such as A20, are available that recognize conformational epitopes that are

present only on assembled capsids for AAV-2. Native immuno-dot blot analysis with the A20

anti-capsid antibody showed that virions remain intact following 24 hours of trypsin digestion.

Figure 4-3B is a native immuno-dot blot of AAV2-GFP virions that have been digested with

trypsin in a time course experiment for the various times indicated. Control samples were heated

at 650C or 750C for 30 minutes. Previously, it had been shown that treating AAV-2 650C,

capsids were still intact and recognized by A20, but that at 75 OC, capsids are no longer intact as

shown by a loss of signal when probed with A20. An immunodot bot was also performed after

exposing AAV2-GFP to SDS and Methanol (MeOH). This is shown in Figure 4-3C. AAV-2

virions were digested with trypsin for 5 hours or 12 hours (TS and T12) and treated with 0.1%

SDS and 20% MeOH for 2 hours at 37 or 45 OC, transferred to nitrocellulose and probed with B1

antibody. The B1 epitope was not recognized until the virions were trpsinized and treated with









minutes at room temperature. The buffer was removed. The gel was washed 3 times with 100 Cl1

50% ACN/50 mM ABC with agitation each for 15 minutes. The gel wash washed until it was

colorless. The gel pieces were dried in a speed vac until they were completely dry, 10 15

minutes. Next, the proteins were digested with trypsin. Promega Trypsin was prepared at a

concentration of 12.5 ng/Cl~ reconstituded in 50 mM ABC, pH 8.4, with 5 mM CaCl2. Enzyme

and buffer were kept cold in an ice bucket and the ratio of enzyme to protein was approximately

1:20. The dried gel piece was chilled on ice, and the gel pieces were covered with ice cold

trypsin digestion buffer. The tube was kept on ice for 45 minutes. The enzyme solution was

removed and replaced with 5 to 20 Cl1 of 50 mM ABC and 5 mM CaCl2 without enzyme. Tubes

were incubated overnight at 370C in a water bath. The supernatant was pipetted into a clean tube.

Stop the reaction with 5.0% glacial acetic acid to a final concentration of 0.5% acetic acid.

Mass spec protocol

Capillary rpHPLC separation of protein digests was performed on a 15 cm x 75 um i.d.

PepMap C18 column (LC Packings, San Francisco, CA) in combination with an Ultimate

Capillary HPLC System (LC Packings, San Francisco, CA) operated at a flow rate of 200

nL/min. Inline mass spectrometric analysis of the column eluate was accomplished by a hybrid

quadrupole time-of-flight instrument (QSTAR, Applied Biosystems, Foster City, CA) equipped

with a nanoelectrospray source.

Fragment ion data generated by Information Dependent Acquisition (IDA) via the QSTAR

were searched against the NCBI nr sequence database using the Mascot (Matrix Science,

London, UK) database search engine. Probability-based MOWSE scores above the default

significant value were considered for protein identification in addition to validation by manual

interpretation of the tandem MS data.










78. Zlotnick A, Aldrich R, Johnson JM, Ceres P, Young MJ: Mechanism of capsid assembly
for an icosahedral plant virus. Virology 2000, 277:450-456.


79. Lombardo E, Ramirez JC, Garcia J, Almendral JM: Complementary roles of multiple
nuclear targeting signals in the capsid proteins of the parvovirus minute virus of mice
during assembly and onset of infection. J Virol 2002, 76:7049-7059.


80. Riolobos L, Reguera J, Mateu MG, Almendral JM: Nuclear transport of trimeric
assembly intermediates exerts a morphogenetic control on the icosahedral parvovirus
capsid. J2~ol Biol 2006, 357: 1026-103 8.


81. Zadori Z, Szelei J, Lacoste MC, Li Y, Gariepy S, Raymond P, Allaire M, Nabi IR, Tij ssen
P: A viral phospholipase A2 is required for parvovirus infectivity. Dev Cell 2001,
1:291-302.


82. Grieger JC, Johnson JS, Gurda-Whitaker B, Agbandj e-McKenna M, Samulski RJ: Surface
exposed Adeno-associated virus Vpl-NLS capsid fusion protein rescues infectivity of
non-infectious wild-type Vp2/Vp3 and Vp3-only capsids, but not 5-fold pore mutant
virions. J Virol 2007.


83. Sitharam M, Agbandj e-McKenna M: Modeling virus self-assembly pathways: avoiding
dynamics using geometric constraint decomposition. J Comput Biol 2006, 13: 1232-
1265.


84. Wu P, Xiao, W., Conlon, T., Hughes, J., Agbandj e-McKenna, M., Ferkol, T., Flotte, T.,
and Muzyczka, N.: Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2)
Capsid Gene and Construction of AAV2 Vectors with Altered Tropism. Journal of
Virology 2000, 74:8635 8647.


85. Bleker S, Sonntag, F., and Kleinschmidt, J.A.: Mutational Analysis of Narrow Pores at
the Fivefold Symmetry Axes of Adeno-Associated Virus Type 2 Capsids Reveals a
Dual Role in Genome Packaging and Activation of Phospholipase A2 Activity. Journal
of Virology 2005, 79:2528 2540.


86. Reguera J, Carreira A, Riolobos L, Almendral JM, Mateu MG: Role of interfacial amino
acid residues in assembly, stability, and conformation of a spherical virus capsid. Proc
NatlAcad'Sci USA 2004, 101:2724-2729.


87. Chiorini JA, Kim F, Yang L, Kotin RM: Cloning and characterization of adeno-
associated virus type 5. J Virol 1999, 73: 1309-1319.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ...._ ................ ...............11......


LIST OF FIGURES .............. ...............12....


AB S TRAC T ............._. .......... ..............._ 16...


CHAPTER


1 INTRODUCTION ................. ...............18.......... ......


The Family Parvoviridae .............. ...............18....
The AAV Genome ................. ...............20........... ....
Vectors ................. ...............21.................


2 MODELS OF AAV CAPSID ASSEMBLY................ ...............2


Capsid Structure.................. .............2
Capsid Assembly and Packaging ................. ...............25................
Virus Assembly for ssDNA Viruses ........._...... ...............27._.___.....
Assembly via a Pentameric Intermediate .............. ...............28....
Assembly via a Trimeric Intermediate .............. ...............29....


3 AAV-2 ASSEMBLY AND SUBUNIT INTERACTIONS ................ .........................35


Introducti on ................. ...............35.................
AAV-2 Mutant Studies ........._.__....... .__. ...............48...
Amino Acid Alignment .............. ...............49....
M odeling ........._.__....... .__ ...............51....
The Mutants ........._..... ............_.. ............._. .............5
Mut 19 228 232 WHCDS WACAS ................. ...............52........... .
Mut24 291 -295 FSPRD FSPAA .............. ...............58....
Mut26 320 324 VKEVT VAAVT ............. .....__ ...............64
Mut33 469 472 DIRD AIAA............... ...............69..
Mut46 681 683 EIE AAA .............. ...............73....
Methods for Mutant Studies .............. ...............78....


4 STUDIES OF THE AAV CAPSID INT SOLUTION .............. ...............86....


Introducti on .................. ......... ...............86.......
Proteolytic structural mapping .............. ...............86....
Trypsin digestion of AAV-2 ............. ...... ._ ...............87...









CHAPTER 5
MASS SPECTROMETRY FOR AAV CAPSID SEROTYPE IDENTIFICATION

AAV-CSI Introduction

There are 11 serotypes of AAV, each with unique properties. AAV-2 was the first to be

discovered, is the most studied and the maj ority of clinical trial data has come from studies with

AAV-2. Currently, there are two Phase 1 clinical trials with AAV-2, one to deliver RPE65 to eye

in patients, which was shown to restore vision in animal models, and one to provide aromatic L-

amino acid decarobxylase to patients with Parkinson's disease. However, serotypes other than

AAV-2 have been shown to have unique cellular receptors and different transduction

efficiencies. AAV-4 has also been utilized to deliver transgenes to the retina of the eye. In

addition, AAV-8 has been shown to have higher transduction efficiency than AAV-2. AAV-1

has been shown to be superior for transducing skeletal muscle and is currently in Phase 1 clinical

trials to investigate its use in the treatment of limb girdle muscular dystrophy type 2D

(LGMD2D), as well as for the treatment of alphal-antitrypsin (AAT) deficiency. Recently, over

100 AAV genotypic variants have been discovered [23, 26, 104], which provide the potential for

an expanding number of AAV serotypes that could be developed into gene therapy vectors in the

future.

A PCR-based assay using unique primers to the cap genes of different serotypes has been

developed to distinguish the serotype of wt virus for AAV-2, AAV-3B or AAV-6 [1 13];

however, in gene therapy vectors, the rep and cap genes have been removed and replaced with a

therapeutic gene, so this assay cannot be used to determine the capsid serotype. For patient

safety, the therapeutic transgene is sequenced from viral DNA to demonstrate that the gene

therapy vector harbors the correct transgene. However, there are very few methods available to

verify the capsid serotype of the therapeutic AAV vector. One recently developed method for






































Figure 3-25.Transfections at 37 oC. Green cell assay of transfections at 37 oC. Lysates were
prepared from cells transfected at 37 OC and a green cell assay for infectivity was
performed at 32 OC, or 37 oC. A. Green cell assay at 32 OC of Mut33 produced at 37
OC was not performed. B. Green cell assay at 37 OC of Mut33 produced at 37 oC. C.
Green cell assay at 32 OC of plM45 produced at 37 oC. D. Green cell assay at 37 OC
of plM45 produced at 37 oC.


Green Cell Assay Infectivity Data
Transfections at 37 oC

320C Inf 370C Inf




Mut33
37 oC
Tnf


plM45
37 oC
Tnf









Table 3-15. Evaluation of the effect of the mutations introduced in Mut26 on amino acid
interactions. The amino acids that interact with Lys321 and Glu322 were modeled
using SPDB Viewer and the distances before the mutations were introduced were
determined. The type of interaction, either intermolecular or intramolecular, was
evaluated. The amino acids were mutated using the molecular modeling software
Deep View for visualization. The interactions were evaluated after the mutations were
introduced to determine which interactions were disrupted as a result of the mutation.
Figure 3-14 and Figure 3-15 show the interactions before and after the mutation
respectively, and the colors in parenthesis above refer to the residues in these figures.
Amino Acid Distance (A+) Interacting Was interaction Type of
that is changed residue di srup~ted? interaction
Lys321 3.16 AAsn3 3 5(yellow) Yes Intermolecular
Lys321 4.30 A Asn3 35(yellow) Yes Intermolecular
Lys321 3.41 A Ala333 No Intramolecular
Lys321 2.38 1 Val320 No Intramolecular
Lys321 3.39 A Val320 No Intramolecular
Lys321 2.96 1 Tyr734 No Intramolecular
Lys321 31 Tyr734 No Intramolecular
Glu322 3.90 a l~e332 (green) Yes Intermolecular
Glu3 22 2.78 A~ Asn3 35 Yes Intramolecular
Glu3 22 2.98 a Asn3 35 Yes Intramolecular
Glu3 22 3.65 a Asn3 35 Yes Intramolecular
Glu3 22 3.82 a Asn3 35 Yes Intramolecular
Glu322 3.38 1 Thr732 No Intramolecular
Glu322 2.59 a Ala333 No Intramolecular
Glu322 3.56 a Ala333 No Intramolecular
Glu322 3.18 1 Ala333 No Intramolecular
Glu322 3.60 a Ala333 No Intramolecular

Mut33 469 472 DIRD AIAA

Mut33 is an alanine substitution mutation, where the charged residue Asp469, Arg471, and

Asp472 have been changed to alanine. Utilizing the same methods described for Mutl9, Mut24

and Mut26, the residues that are mutated in Mut33 were evaluated. Mut33 is not involved in the

5-fold axis of symmetry or the 2-fold axis of symmetry. The lack of an effect at the 5-fold axis of

symmetry is shown in Figure 3-16. The mutations are located at the 3-fold axis of symmetry in

Mut33 as shown in Figure 3-17. There are two intramolecular interactions that are disrupted in

Asp469 as a result of the mutation, as shown in Figure 3-18. However, as shown in Figure 3-19,

intermolecular interactions, as well as intramolecular interactions are disrupted as a result of the










Not treated Trypsin treated













-~t -S', ++++D
~~~ -~ + ++MO



'r1 *r
37~~~~rrr 37 3 3 7 745 4 40

Figure ~ ~ ~ ~ lf 4-3 Trysinze AA- viron reai itc.AEltrnmroop.Ngivstai
eletro mcroraps f utrete rA V2-FPcapid (FTidxnlHp ri)o
trypsi-trea ed rAAV2~R-GFP virons Th samples wer viwe n itch -70
trnsisinelcro icocoeat3,00 mg ifain Eac hihpw red
insert~C is 7000.B m uodtbo.rA 2GPvr in wer di ese wit
trpi n smlswre ae t th nictdtmepit (hus. An imun dot





codiiosAA -2viios TO) wer diese with t rysi forC 75 ad12hur T

an T2 rseciel),ad ratdwih0.1 D n 0 eHfr2husa


37oC~ or 45C trnfre to nircluoe an rbe ih atioy









infectious virus can be produced that lack VP2 entirely [71]. Additionally, the N-terminus of

VP2 has been replaced with green fluorescent protein and these capsids still assemble and

maintained infectivity. This demonstrates that VP2 can tolerate peptide insertions and may be

useful for incorporating peptides into the capsid for cell-specific targeting of AAV.

Virus Assembly for ssDNA Viruses

Spontaneous assembly of macromolecular structures such as a virus capsid is a process

that is not clearly understood. For a T=1 icosahedral capsid, such as AAV, 60 subunits must

come together in the correct orientation to provide a stable capsid, and AAV assembles with high

fidelity. There are features on the surface of the AAV capsid that distinguish the icosahedral 5-

fold, 3-fold or 2-fold axis of symmetry, for example, the 5-fold pore, the 3-fold mounds or the 2-

fold dimple. These regions on the capsid are important for various steps in the virus life cycle.

The 3-fold mounds are implicated in receptor binding, and the 5-fold pore has been implicated in

both externalization of the unique N-terminus of VP I as well as DNA packaging. In vitro studies

have identified a number of potential assembly intermediates [60, 72].

Studies of CPV structure estimating the free energy of association have revealed that the

formation of 3-fold and 5-fold contacts likely takes precedence over 2-fold interactions [52, 53].

For AAV-2, the crystal structure is known, and the individual amino acid interactions at the

interface between subunits have been analyzed and will be discussed further in Chapter 3. The

strongest interactions for AAV-2 are at the 3-fold axis of symmetry, followed by the 5-fold axis

of symmetry, and the weakest interactions are at the 2-fold axis of symmetry. The image in

Figure 2-2 depicts an icosahedral virus particle (taken from VIPERdb) [73]. The subunit

association energies for AAV-2 and other parvoviruses are shown in Table 2-1. This data verifies

that the subunit association energies for other parvoviruses for the 5-fold, 3-fold and 2-fold

interactions are comparable (data from VIPERdb) [73].









random integration into the host genome, and have the potential to activate proto-oncogenes [16-

18]. 4) Low immunogenicity in animal models recently, it was reported that AAV2 vectors can

activate functional cytotoxic CD8+ T cells specific to the AAV2 capsid in mice after

intramuscular inj section [19, 20]. Recent clinical trial data suggests that T cell mediated immunity

to AAV capsid antigens caused destruction of AAV-2 transduced hepatocytes in humans [6, 21].

Studies in mice have shown that AAV-2 capsids are able to activate cytotoxic T cells but did not

render the transduced hepatocytes as effective cytolytic targets [22]. This suggests that currently

available animal models may not be suffcient for studying the immune response to the AAV

capsid that has been shown to occur in human patients. In addition, humoral immune responses

are generated to the AAV capsid that may result in viral neutralization [21]. There are 11 known

serotypes of AAV with nearly 100 recently discovered genotypic variants [23-27]. The use of

other serotypes of AAV may circumvent the humoral response and allow for repeated treatments

if necessary [28]. However, immune suppressive therapy may be needed during treatment with

AAV to mitigate the host immune response in human patients, as well as animals[29]. Recent

data in a canine model of Duchenne Muscular Dystrophy demonstrated that a brief course of an

immunosuppressive regimen was well tolerated and sumfcient to permit sustained AAV-

mediated dystrophin expression [30]. A recent study evaluating the immune response to AAV-8

expressing F.IX in rhesus macaques, the natural host for AAV-8, evaluated preexisting

neutralizing antibodies to AAV-8, a standard T-cell immunosuppressive regimen, as well as

effcacy and safety of administration. This study showed that low titers of preexisting antibody

abrogate transduction, and the use of an immunosuppressive regimen did not induce toxicity, or

impair AAV transduction or F.IX synthesis [29]. AAV vectors may also be useful for providing

long term gene expression in immunocompromised hosts, which is an advantage over other











































Mutl9 -unchanged








Mutl9 -unchanged


Table 3-2 Continued
P-P
P-P
P-H
P-H
P-H
H-P
H H
H H
B-H


T651:T246
T651:Q677
Y257:F365
Y257:A367
Y257:V714
P652:T246
P652:V369
L336:V221
K258:G718

A386:V710
V387:Y704
L256:43718
V221:'V221
N382:K706
N326:T329
N326:T331
Y275:'V710
Y275:T713
Y275:'V714
Q401:N227
N335:K321
N3 3 5 N334
S338:4Q319
A218:N223
C394:F365
C394:P366
S400:W228
S662:M371
N407:N223
F669:V369
S390:V710
T339:S224
T339:N317
A655:I332
Q341:W228
F273: V710


Mut21 unchanged






Mut21 -pd, unstable
cap sid


Mut21 unchanged


Matt48 -pd, hep+







Matt26 -hs


Mkitl8 -unchanged












VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate rh50 AAS99248
isolate rh51 AAS99249
isolate rh52 AAS99250
isolate rh64 AAS99259
isolate rh53 AAS99251
isolate rh61 AAS99257
isolate rh58 AAS99255
isolate rh57 AAS99254
AAV8 VP1 AANO3857
AAV8 YP 077180


SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
SSGNWHCDST
VSGNWHCDST
VSGNWHCDST
ASGNWHCDST
SSGNWHCDST
ASGNWHCDST
ASGNWHCDST
ASGNWHCDST
ASGNWHCDST
SSGNWHCDST
ASGNWHCDST
SSGNWHCDST
SSGNWHCDSQ
SSGNWHCDSQ
SSGNWHCDSQ
ASGDWHCDST
ASGDWHCDST
ASGDWHCDST
ASGDWHCDST
ASGDWHCDST
ASGDWHCDST
ASGDWHCDST
ASGDWHCDST
ASGDWHCDST
ASGDWHCDSK
ASGDWHCDST
SSGNWHCDSQ
SSGNWHCDSQ
SSGNWHCDSQ
SSGNWHCDSQ
SSGNWHCDSQ


WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
RLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WLGDRVITTS
WSEGKVTTTS
WSEGHVTTTS
WSEGHVTTTS
WSESHVTTTS
WSESHVTTTS
WMGDRVVTKS
WMGDRVVTKS
WMGDRVVTKS
WMGDRVVTKS
WLGNRVLTRS
WLDNCVITRT
WLENGVVTRT
WLENGVVTRT
WLENGVVTRT
WLDNGVVTRT
WLDNGVVTRT


TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWALPTHN
TRTWALPTYN
TRTWALPTYN
TRTWALPTYN
TRTWVLPTYN
TRTWVLPTYN
TRTWVLPTYN
TRTWVLPTYN
TRTWVLPTYN
TRTWVLPSYN
TRTWVLPSYN
TRTWVLPSYN
TRTWVLPSYN
TRTWVLPSYN
TRTWNLPTYN
TRTWVLPSYN
TRTWVLPSYN
TRTWVLPSYN
TRTWVLPSYN
TRTWVLPSYN


NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQTSNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISNG
NHLYKQISSE
NHLYKQISSE
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISSQ
NHLYKQISNG
NHLYKQISNS
NHLYKQISNS
NHLYKQISNS
NHLYLRLGTT
NHLYKRLGES
NHLYKRLGES
NHLYLRLGSS
NHLYLRLGSS
NHQYREIKSG
NHQYREIKSG
NHQYREIKSG
NHQYREIKSG
NHLYKQISDA
NHIYKRLNGT
NHLYKRIQGP
NHLYKRIQGP
NHLYKRIQGP
NHLYKRIQGP
NHLYKRIQGP


TSGGSTNDNT
TSGGSTNDNT
TSGGSTNDNT
TSGGSTNDNT
TSGGSTNDNT
TSGGSTNDNT
TSGGSTNDNT
TSGGSTNDNT
TSGGATNDNT
TSGGATNDNT
TSGGATNDNT
TSGGSSNDNT
TSGGSSNDNT
TSGGSSNDNT
TSGGSTNDNT
T.AGSTNDNT
T.AGSTNDNT
S.AGSTNDNV
S.AGSTNDNV
S.AGSTNDNV
S.AGSTNDNV
TSGGSTNDNV
TSGGSSNDNA
TSGGSSNDNA
TSGGSSNDNA
S.....SSNT
L.....QSNT
L.....QSNT
N.....ASDT
N.....ASDT
S.VDGSNANA
S.VDGSNANA
S.VDGSNANA
S.VDGSNANA
SGVHSLPGSR
T....SGDQS
S..GGDNNNK
S..GGDNNNK
S..GGDNNNK
G..GTDPNNK
G..GTDPNNK


VP1 i
VP1
VP1
VP1
VP1



VP1 i
VP1 i
VP1 i


solate rh43 AAS99245
isolate pil AAS99238
isolate pi3 AAS99240
isolate pi2 AAS99239
isolate rh1 AAS99241
AAV7 VP1 AANO3855
AAV7 YP 077178
solate rh48 AAS99246
solate rh62 AAS99258
solate rh55 AAS99253


VP1 isolate rh54 AAS99252
VP1 isolate rh60 AAS99256
AAV9 VP1 AAS99264
VP1 isolate hu31 AAS99281
VP1 isolate hu32 AAS99282
AAV11 AAT46339
AAV4 NP 044927
AAV4 VP1 AAC58045
BOVINE AAV AAR26465
BOVINE AAV YP 024971
AAV5 VP1 AAD13756
AAV5 YP 068409
CAPRINE AAV1 AAU84890
GOAT AAV VP1 ABC69726
RAT AAV1 AAZ79676
MOUSE AAV1 AAZ79672
AVIAN AAV ATCC VR865 AAO32087
AVIAN AAV ATCC VR865 AAT48613
AVIAN AAV ATCC VR865 NP 852781
AVIAN AAV Strain DA1 AAT48615
AVIAN AAV Strain DA1 YP 077183












VP1 isolate rh61 AAS99257
VP1 isolate rh58 AAS99255
VP1 isolate rh57 AAS99254
AAV8 VP1 AANO3857
AAV8 YP 077180
VP1 isolate rh43 AAS99245
VP1 isolate pil AAS99238
VP1 isolate pi3 AAS99240
VP1 isolate pi2 AAS99239
VP1 isolate rh1 AAS99241
AAV7 VP1 AANO3855
AAV7 YP 077178


...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MAADGY
...MTDGY
...MTDGY
...MSFVDH
...MSFVDH
.. .MS
.. .MS
.. .MS
.. .MS
.. .MS
.. .MS
MSLISDAIPD
MSLISDAIPD
MSLISDAIPD
MSLISDAIPD
MSLISDAIPD
.MST


LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
LPDWLEDT..
LPDWLEDT..
LPDWLEDN..
LPDWLEDN..
LPDWLEDN..
PPDWLES...
PPDWLES...
FVDHPPDWLE
FVDHPPDWLE
FVDHPPDWLE
FVDHPPDWLE
FFDWIG...R
FFDWLGKQ..
WLERLVKK..
WLERLVKK..
WLERLVKK..
WLERLVKK..
WLERLVKK..
FLEKFEDW. .


.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIREWW
.LSEGIHEWW
.LSEGIREWW
.LSEGIRQWW
.LSEGIRQWW
.LSEGIREWW
.LSEGVREWW
.LSEGVREWW
.IGDGFREFL
.IGDGFREFL
EVGEGLREFL
EVGEGLREFL
EVGEGLREFL
EVGEGLREFL
KYAN GAAE FW
.YAQGAAEFW
.GVNAAADFY
.GVNAAADFY
.GVNAAADFY
.GVNAAADFY
.GVNAAADFY
.YETAAASWR


DLKPGAPKPK
DLKPGAPKPK
ALKPGAPKPK
ALKPGAPKPK
ALKPGAPKPK
DLKPGAPKPK
ALKPGAPQPK
ALKPGAPQPK
ALKPGAPQPK
ALKPGAPKPK
DLKPGAPKPK
DLKPGAPKPK
DLKPGAPKPK
DLKPGAPKPK
DLKPGAPKPK
DLKPGAPKPK
DPKPGAPKPK
ALKPGAPQPK
KLKPGPPPPK
KLKPGPPPPK
DLKPGAPKPK
ALQPGAPKPK
ALQPGAPKPK
GLEAGPPKPK
GLEAGPPKPK
GLEAGPPKPK
GLEAGPPKPK
GLEAGPPKPK
GLEAGPPKPK
DLEPGPPKPK
DLKSGPPAPK
HLESGPPRPK
HLESGPPRPK
HLESGPPRPK
HLESGPPHPK
HLESGPPHPK
HLKAGAPKPK


ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQKQ!....
ANQQHQ ....
PAERHK....
PAERHK....
ANQQKQ!....
ANQQHQ ....
ANQQHQ ....
ANQQKQ!....
ANQQKQ!....
PNQQ.......
PNQQ.......
PNQQ.......
PNQQ.......
..KA......
KARKDG ....
ANQQTQ ....
ANQQTQ ....
ANQQTQ ....
ANQQTQ ....
ANQQTQ ....
SNQQSQSVST


VP1
VP1
VP1
VP1


isolate rh48
isolate rh62
isolate rh55
isolate rh54


AAS 992 4 6
AAS 992 5 8
AAS 992 5 3
AAS 992 52
AAS 992 5 6
AAS 992 64
AAS 992 81
AAS 992 8 2


VP1 isolate rh60
AAV9 VP1
VP1 isolate hu31
VP1 isolate hu32


AAV11 AAT46339
AAV4 NP 044927
AAV4 VP1 AAC58045
BOVINE AAV AAR26465
BOVINE AAV YP 024971
AAV5 VP1 AAD13756
AAV5 YP 068409
CAPRINE AAV1 AAU84890
GOAT PAY VP1 ABC69726
RAT AAV1 AAZ79676
MOUSE AAV1 AAZ79672
AVIAN AAV ATCC VR865 AAO32087
AVIAN AAV ATCC VR865 AAT48613
AVIAN AAV ATCC VR865 NP 852781
AVIAN AAV Strain DA1 AAT48615
AVIAN AAV Strain DA1 YP 077183
DUCK AAV Strain FM AAA83225
MUSCOVY DUCK PARVOVIRUS YP 068412
MUSCOVY DUCK VP1 YP 068411
MUSCOVY DUCK PARVOVIRUS YP 068413
Goose AAV VP1 AAA83230


.........MS .......... .......... .......... ..........

.......MST FLEKFEDW.. .YETAAASWR HLKAGAPKPK SNQQSQSVST









In terms of building a macromolecular structure, both of the models, either pentamers of

trimers (POTs), or trimers of pentamers (TOPs) demonstrate that the important interactions for

achieving an intact capsid are the two-fold interactions. Although they are the weakest

interactions, there are many of them. In addition, when either the pentamer or the trimer is added,

these weak two-fold interactions would enable a new subunit to attach, detach, reattach, in effect

allowing it to be sampled so that it is added in the correct orientation (like a puzzle piece). The

assembly model favored for AAV based on all of the data is trimers of pentamers (TOPs). This

model allows for two pentamers to come together, so intrapentameric interactions form first, then

interpentameric interactions second, which are stabilized by the two-fold interactions at the

pentameric interfaces, and as the trimer of pentamers come together, these interactions allow for

the energentically expensive three-fold intratrimeric interactions. This model of AAV assembly

incorporates what is currently known about assembly, capsid stability and disassembly. The

AAV capsid is stable over a wide range of pH and temperature. It has been shown that upon

treating the AAV capsid at 65 OC for 30 minutes that it' s possible to expose the unique N-

terminus of VP 1, and VP2 [70]. However, upon heat treatment of the AAV capsid, experiments

to date to isolate an assembly intermediate have been unsuccessful. This suggests that this is an

all-or-none phenomenon. The energy required to break the strong interactions at the 3-fold axis

of symmetry results in complete disruption of the AAV capsid. This is consistent with a

pentameric intermediate, where the strongest interaction at the 3-fold axis of symmetry would

form last, and for capsid disassembly to occur, would need to be disrupted first. If trimers are the

assembly intermediate, and they form pentamers of trimers, and then the weak 2-fold interactions

form last, in a disassembly model, these should be relatively easy to disrupt giving either a











AAS9 93 0 5
AAS 992 9 7
AAS9 93 0 3
AAS9 93 0 4
AAS 992 7 8
AAS 992 7 9
AAU 05364
AAS 992 63
AAS9 93 0 9
AAS 9 9310
AAU 053 62
AAU 05371
AAU 05358
AAU 05372
AAU 05366
AAU 05368
AAU 05360
AAS 992 7 7
AAS 992 6 9
AAS 992 71
AAS 992 7 2
AAS 992 7 5
AAS 992 7 3
AAS 992 7 4


TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN
TDEEEIRTTN

651
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA
DVYLQGPIWA


PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGTV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGAV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PAATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV
PVATEQYGSV


KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGHFH
KIPHTDGNFH
KIPHTDGNFH
KIPHTDGNFH
KIPHTDGNFH
KIPHTDGNFH
KIPHTDGNFH


STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTR
STNLQSGNTQ
STNLQSGNTQ
STNLQSSNTQ
STNLQGGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STNLQSGNTQ
STYLQSGNTQ


PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGFGL
PSPLMGGLGL
PSPLMGGFGL
PSPLMGGFGL


ASTADVNTQG
AATADVNTQG
AAT SDVNTQG
AAT SDVNTQG
AATADVNTQG
AATADVNTQG
AAT SDVNTQG
AATADVNTQG
AAT SDVNTQG
AAT SDVNTQG
AAT SDVNTQG
AAT SDVNTQG
AAT SDVNTQG
AATADVNTQG
AATADVNTQG
AATADVNTQG
AAT SDVNTQG
AAT SDVNTQG
AAT SDVNTQG
AAT SDVNTQG
AAT SDVNTQG
AAT SDVNTQG
AAT SDVNTQG
AAT SDVNTQG



KHPPPQILIK
KHPPPQILIK
KNPPPQILIK
KNPPPQILIK
KNPPPQILIK
KHPPPQILIK
KNPPPQILIK
KNPPPQILIK
KNPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK
KHPPPQILIK


VLPGMVWQDR
VLPGMVWQDR
VLPERVWQDR
VLPERVWQDR
VLPGMVGQDR
VLPGMVWQDR
VLPGMVWQDR
VLPGMVWQDR
VLPERVWQDR
VLPERVWQDR
VLPGMVWQDR
VLPGMVWQDR
VLPGMVWQDR
VLPGMVWQDR
VLPGMVWQDR
VLPGMVWQDR
VLPGMVWQDR
VLPERVWQDR
VLPERVWQDR
VLPERVWQDR
VLPERVWQDR
VLPERVWQDR
VLPERVWQDR
VLPERVWQDR

700
NTPVPANPPA
NTPVPANPPA
NTPVPANPPA
NTPVP.ANPPA
NTPVP.ANPPA
NTPVP.ANPPA
NTPVP.ANPPA
NTPVP.ANPPA
NTPVP.ANPPA
NTPVPADPPT
NTPVPADPPT
NTPVPADPPT
NTPVPADPPT
NTPVPADPPT
NTPVPADPPT


VP1 isolate hu58
VP1 isolate hu49
VP1 isolate hu56
VP1 isolate hu57
VP1 isolate hu28
VP1 isolate hu29
VP1 isolate huT70
VP1 isolate hul3
VP1 isolate hu63
VP1 isolate hu64
VP1 isolate huT40
VP1 isolate huLG15
VP1 isolate huT17
VP1 isolate huT41
VP1 isolate huT71
VP1 isolate huT88
VP1 isolate huT32


VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate
isolate
isolate


hu27
hul9
hu20


isolate hu21
isolate hu24
isolate hu22
isolate hu23


PArVVR195 ABA71699
PArVVR355 ABA71701
AAV1 NP 049542


AAV1 VP1
VP1 isolate hu48
AAV6 VP1
VP1 isolate hu43
VP1 isolate hu44
VP1 isolate hu46
AAV10
VP1 isolate rh40
VP1 isolate hu37
VP1 isolate hu42
VP1 isolate hu40
VP1 isolate hu67


AAD27757
AAS 992 9 6
AAB9 54 50
AAS 992 91
AAS 992 92
AAS 992 9 4
AAT4 63 3 7
AAS 992 4 4
AAS 992 8 5
AAS 992 90
AAS 992 8 8
AAS 9 9312











VP1 isolate hu6 AAS99306
VP1 isolate rh25 AAS99242
VP1 isolate hu39 AAS99286
VP1 isolate rh49 AAS99247
VP1 isolate rh50 AAS99248
VP1 isolate rh51 AAS99249
VP1 isolate rh52 AAS99250
VP1 isolate rh64 AAS99259
VP1 isolate rh53 AAS99251
VP1 isolate rh61 AAS99257
VP1 isolate rh58 AAS99255
VP1 isolate rh57 AAS99254
AAV8 VP1 AANO3857
AAV8 YP 077180
VP1 isolate rh43 AAS99245
VP1 isolate pil AAS99238
VP1 isolate pi3 AAS99240
VP1 isolate pi2 AAS99239
VP1 isolate rh1 AAS99241
AAV7 VP1 AANO3855
AAV7 YP 077178
VP1 isolate rh48 AAS99246
VP1 isolate rh62 AAS99258
VP1 isolate rh55 AAS99253
VP1 isolate rh54 AAS99252
VP1 isolate rh60 AAS99256
AAV9 VP1 AAS99264
VP1 isolate hu31 AAS99281
VP1 isolate hu32 AAS99282
AAV11 AAT46339
AAV4 NP 044927
AAV4 VP1 AAC58045
BOVINE AAV AAR26465
BOVINE AAV YP 024971
AAV5 VP1 AAD13756
AAV5 YP 068409
CAPRINE AAV1 AAU84890
GOAT AAV VP1 ABC69726
RAT AAV1 AAZ79676
MOUSE AAV1 AAZ79672
AVIAN AAV ATCC VR865 AAO32087


PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLV
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYVHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSSYAHSQ SLDRLMNPLI
PFHSMYAHSQ SLDRLMNPLL
PFHSMYAHSQ SLDRLMNPLI
PFHSMYAHSQ SLDRLMNPLI
PFHSMYAHSQ SLDRLMNPLL
PFHSMYAHSQ SLDRLMNPLL
PFHSSFAPSQ NLFKLANPLV
PFHSSFAPSQ NLFKLANPLV
PFHCSFAPSQ NLFKLANPLV
PFHCSFAPSQ NLFKLANPLV
PFHTGFAPCQ NLFKLSNPLV
PFHSMWAHNQ SLDRLMNPLI
PFHSMFAHNQ TLDRLMNPLV


DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSRAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLSSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP
DQYLYYLSRT QTTGG.TANT QTLGFSQGGP
DQYLYYLSRT QTTGG.TANT QTLGFSQGGP
DQYLYYLSRT QTTGG.TANT QTLGFSQGGP
DQYLYYLSRT QTNG..TNAT QTLLEAQAGP
DQYLYYLSRT QTNG..TNAT QTLLEAQAGP
DQYLYYLSRT QTNG..TNAT QTLLEAQAGP
DQYLYYLVRT QTTG..TGGT QTLAFSQAGP
DQYLYYLART QSNPGGTAGN RELQFYQGGP
DQYLYYLART QSNPGGTAGN RELQFYQGGP
DQYLYYLART QSNAGGTAGN RELQFYQGGP
DQYLYYLART QSNAGGTAGN RELQFYQGGP
DQYLYYLART QSNAGGTAGN RELQFYQGGP
DQYLYYLART QSNPGGTSGN RELQFYQGGP
DQYLYYLSRT QSTEG.TAGT QQLLFSQAGP
DQYLYYLSKT INGSG..QNQ QTLKFSVAGP
DQYLYYLSKT INGSG..QNQ QTLKFSVAGP
DQYLYYLSKT INGSG..QNQ QTLKFSVAGP
DQYLWHLQST TSGETLNQGN AATTFGKIRS
DQYLWGLQST TTGTTLNAGT ATTNFTKLRP
DQYLWGLQST TTGTTLNAGT ATTNFTKLRP
DQYLWELQST TSGGTLNQGN SATNFAKLTK
DQYLWELQST TSGGTLNQGN SATNFAKLTK
DQYLYRFVST NNT....... GGVQFNKNLA
DQYLYRFVST NNT....... GGVQFNKNLA
DQYLYRFVST SAT....... GAIQFQKNLA
DQYLYRFVST SAT....... GAIQFQKNLA
DQYLYRFQGT DNSSS..STP GVVKFEKCVA
DQYLYYLDVT SSTG...... ..FTYQKGVH
DQYLWAFSSV SQAGS...SG RALHYSRATK





Table 3-3. The Hyve strongest interactions at the icosahedral 3-fold axis of symmetry (VIPERdb).
The amino acids that are the strongest at the icosahedral 3-fold axis of symmetry are
listed, using VPI numbering. This refers to interactions between subunits Al and A7
in the icosahedral virus particle in Figure 2-2. The type of interactions are indicated,
B = basic, H = hydrophobic, P = Polar, uncharged. The phenotype of AAV mutants
generated by Wu, et al. [84]are listed, wt = wild type.


Amino


VPI


Energy
Type (kcal/mol)


Acid numbering


Wu, et al. [84] mutant phenotype
Mut47, ni, A20- (Hartladge, Warrington,
Muzyczka, unpublished data)
Mut31, ni, empty particle
Mut33, hs


LYS
ARG
ARG
TYR
PRO


-4.88
-3.92
-3.79
-3.44
-3.07


Table 3-4. Specific amino acid interactions at the icosahedral 3-fold axis of symmetry
(VIPERdb). Specific amino acids at the icosahedral 3-fold axis of symmetry are
listed, using VPI numbering, with the strongest interactions listed first, and the
weakest interactions listed last. This list includes all interactions between subunits Al
and A7 in the icosahedral virus particle depicted in Figure 2-2. The type of
interactions are indicated, A = acidic, B = basic, H = hydrophobic, P = Polar,
uncharged. The phenotype of AAV mutants generated by Wu, et al. [84] are listed,
either in the column to the left of the amino acid list if the mutation was made in the
1st amino acid of the pair listed, or in the column to the right of the amino acid list if
the mutation was made in the 2nd amino acid of the pair listed. ala ins = alanine
insertion, cs = cold-sensitive, hep- = heparin negative, hep+ = heparin positive, hs =
heat-sensitive, ni = non-infectious, pd = partially defective, wt = wild-type. Amino
acids that were not mutated by Wu, et al.[84], but flank the mutated amino acids are


designated unchanged.
Type of Mutant Phenotypes
Interaction (Wu, et al. [84])
B P Mut47 ni, A20
B P
B H
B P Mut31 ni, empty
B -H particle
B P
B B
B P
B -P Mut33 -hs
B A
B P
B H


VPI
numbering
K692:Q349
K692:Y397
K692:F398
R432:Y272
R432:L378
R432:N3 81
R432:R513
R432:S515
R471: S267
R471:D269
R471:N270
R471:W502


Mutant Phenotype
(Wu, et al. [84])



Mut22 unchanged


Mut3 6
Mut3 6

Mut22
Mut22


pd, hep+
unchanged

ni, full particle
unchanged









fold axis. Hydrophobic interactions contribute -12.79 kcal/mol per subunit at the 2-fold axis and

there are nearly twice as many interactions as compared with electrostatic interactions. This is

consistent with the suggestion that weak electrostatic interactions provide capsid stability. Polar

interactions contribute -8.75 kcal/mol per subunit and there are a similar number of polar

interactions as hydrophobic interactions. Based on this analysis, the maj ority of interactions at

the 2-fold axis of symmetry are hydrophobic interactions and polar hydrogen bonding

interactions; however, although fewer in number, the electrostatic interactions have a greater

energy contribution at the 2-fold axis of symmetry. In addition, for both Mutl9 and Mut24, when

charged residues are changed to alanine, the resulting phenotype is the absence of capsids, and

no infectious particles. Particularly with the residues involved in Mut24, the residues in Mut24

that are unchanged in this mutant (FSPRD FSPAA), S292 and P293 (VPI numbering),

Table 3-5. The five strongest interactions at the icosahedral 2-fold axis of symmetry (VIPERdb).
The amino acids that are the strongest at the icosahedral 2-fold axis of symmetry are
listed, using VPI numbering. This refers to interactions between subunits Al and A6
in the icosahedral virus particle in Figure 2-2. The type of interactions are indicated,
B = basic, H = hydrophobic. The phenotype of AAV mutants generated by Wu, et al.
[84] are listed, ni = non-infectious.
Amino VPI Energy
Acid numbering Type (kcal/mol) Wu, et al. mutant phenotype [84]
TRP 694 H -4.22
PRO 696 H -3.08
LYS 692 B -2.79 Mut47, ni, A20-
ARG 294 B -2.14 Mut24, ni, no capsid
ARG 298 B -1.91

participate in the strongest interaction at the icosahedral 2-fold axis of symmetry, yet the

phenotype of this mutant is the absence of capsids. While R294 (VPI numbering) is the 4th

strongest interaction at the 2-fold axis, the inability of this mutant to make capsids suggests that

this electrostatic interaction, while weaker than the primary hydrophobic interaction, is required

for capsid formation at the 2-fold axis of symmetry. With Mutl9, the interaction that is changed









dynamics of the virion (Chapter 4). Chapter 5 describes AAV Capsid Serotype Identification

(AAV-CSI). Denatured AAV capsids are subj ected to proteolysis and the AAV capsid serotype

is determined based on the fragmentation pattern. This provides a high throughput method for

capsid identity testing. Overall, the data presented provides a biophysical, biochemical, and

genetic analysis of the AAV capsid.










suggest that these intermediates, if they can be isolated may be able to be subj ected to conditions

that will allow for an in vitro assembly assay. This would be useful in shedding light on the role

that the nonstructural Rep proteins play in AAV capsid assembly. Based on available data on

assembly and packaging, with DNA packaging occurring at the 5-fold axis of symmetry, the

nonstructural Rep proteins could play a stabilizing role at the weak 2-fold axis of symmetry and

it is also possible that these weak interactions allow for structural changes to occur in the capsid

during the virus life cycle while the capsid integrity is maintained by the stronger interactions at

the 3-fold and 5-fold axis of symmetry.



Transfection

32 oC 37 oC 39.5 oC




MIut33









plM145





Figure 3-23. Transfection of Mut 33. Cells were transfected with either plM45 and UF5, or
Mut33 and UF5, and infected with adenovirus at an MOI = 2 at either 32 OC, 37 OC,
or 39.5 oC. Transfection efficiencies were similar under all conditions. Panel A.
Mut33 transfection at 32 oC. B. Mut 33 transfection at 37 oC. C. Mut33 rransfection
at 39.5 oC. D. plM45 transfection at 32 oC. E. plM45 transfection at 37 oC. F. plM45
transfection at 39.5 oC.
























vV V Pentamers
Mlonomeric Subunits Trimeric Intermediate of Trimers

Figure 2-4. Model of Parvovirus Assembly through a Trimeric Intermediate. In this model,
monomeric subunits come together to form trimers, and then five trimers come
together to form pentamers, and then pentamers add on to form the capsid. This is the
favored model for Minute Virus of Mice (MVM) assembly.

mechanism for assembly would ensure that the particles that are produced contain VPI and

would result in the formation of infectious particles. Alternatively, for every particle produced,

approximately 1 in 100 is infectious; therefore, the incorporation of VPI and VP2 in assembled

particles might be a stochastic process, as has been suggested. For AAV, since pentameric

interactions require less energy to form, these would be favored over trimers. If the virus were to

assemble its most expensive and most stable subunit first, it would risk using its resources to

build subunits and potentially never attain an intact capsid. Additionally, trimers, as the most

stable subunit, should be easy to isolate if they were the intermediate of assembly, even though

they are short-lived species. If pentamers are the intermediate of assembly for AAV, this

suggests that the site on the surface of the capsid where the nonstructural Rep protein interacts

for packaging must either not be present in pentameric intermediates or perhaps is present but

some cellular protein that is part of the packaging complex is not available at this step.










in the table except AAV-2. Residue 697 is an aliphatic residue, isoleucine for CPV, or valine for


MVM, while this is a glutamic acid residue in AAV-2.


Figure 3-9. The predicted subunit as a result of the mutations made in Mut24 is a trimer. The
residues that are mutated in Mut24 are colored differently than the main chain,
usually white, except where they would not be visible, in which case they are aqua.
Inside view. This figure was generated using the molecular modeling software Deep
View.





Green Cell Assay Infectivity Data
Transfections at 32 oC

320C Inf 370C Inf




Mut33
32 oC
Tnf


plM45
32 oC
Tnf


Figure 3-24. Green cell assay of transfections at 320 C. Lysates were prepared from the cells
transfected in Figure 3-23 and a green cell assay for infectivity was performed at 320
C, or 370 C. A. Green cell assay at 320 C of Mut33 produced at 320 C. B. Green cell
assay at 370 C of Mut33 produced at 320 C. C. Green cell assay at 320 C of plM45
produced at 320 C. D. Green cell assay at 370 C of plM45 produced at 370 C.









Table 3-7. plM45 based plasmids containing alanine substitutions for the production of mutant
AAV-2 capsids produced by Wu, et al. [89]. Ala sub = alanine substitution; ni = non-
infectious; hs = heat sensitive; pd = partially defective; hep+ = heparin positive
Mutant Type Mutation Class Phenotype
Mutl19 Ala Sub 228-232 WHCDS WACAS 4b ni; no capsid
Mut24 Ala Sub 291-295 FSPRD FSPAA 4b ni; no capsid
Mut26 Ala Sub 320-324 VKEVT VAAVT 3a hs
Mut3 3 Ala Sub 469-472 DIRD AIAA 3a hs
Mut46 Ala Sub 681-683 EIE AAA 4b Ni; no capsid

Amino Acid Alignment

A structural alignment in the region of Mutl9 with other parvovirus sequences is shown in

Table 3-9. This stretch of amino acids is not conserved among other parvoviruses. A structural

alignment in the region of Mut 24 is shown in Table 3-10. The structural alignment for Mut26

with other parvoviruses is shown in Table 3-11. The structural alignment for the amino acids

involved in Mut33 is shown in Table 3-12. The parvovirus structural capsid alignment for Mut46

are shown in Table 3-13. Additionally, an amino acid alignment was performed for the known

AAV serotypes, as well as the newly reported AAV genotypic variants. This alignment has been

included as an appendix.

For the five AAV-2 mutants listed in Table 3-7, theoretical molecular replacement was

used to generate models of the AAV mutants. Models were evaluated to determine the potential

Table 3-8. Amino acid alignment data for the specific amino acid sequence in the region of
Mut33. Amino acid sequence alignment for the capsid protein of AAV-1 through
AAV-8 that align with amino acid 469 472 of AAV-2 (VP I numbering).
Serotype Sequence
AAV 1 GMSV
AAV 2 DIRD
AAV 3 SMSL
AAV 3B SMSL
AAV 4 RPTN
AAV 5 RYAN
AAV 6 GMSV
AAV 7 TMAE
AAV 8 TMAN












VP1 isolate hu56
VP1 isolate hu57
VP1 isolate hu28
VP1 isolate hu29
VP1 isolate huT70
VP1 isolate hul3
VP1 isolate hu63
VP1 isolate hu64
VP1 isolate huT40
VP1 isolate huLG15
VP1 isolate huT17
VP1 isolate huT41
VP1 isolate huT71
VP1 isolate huT88
VP1 isolate huT32
VP1 isolate hu27
VP1 isolate hul9
VP1 isolate hu20
VP1 isolate hu21
VP1 isolate hu24
VP1 isolate hu22
VP1 isolate hu23


AAS9 93 0 3
AAS9 93 0 4
AAS 992 7 8
AAS 992 7 9
AAU 05364
AAS 992 63
AAS9 93 0 9
AAS 9 9310
AAU 053 62
AAU 05371
AAU 05358
AAU 05372
AAU 05366
AAU 05368
AAU 05360
AAS 992 7 7
AAS 992 6 9
AAS 992 71
AAS 992 7 2
AAS 992 7 5
AAS 992 7 3
AAS 992 7 4


SDIRDQSRNW
SDIRDQSRNW
SDIQDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDVRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW
SDIRDQSRNW

551
HESIINPGTA
RESIINPGTA
RESIINPGTA
RESIINPGTA
RESIINPGTA
RESIINPGTA
RESIINPGTA
RESIINPGTA
RESIINPGTA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA


LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPSYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR



MASHKDDEDK
MASHKDDEDK
MASHKDDEDK
MASHKDDEDK
VASHKDDEDK
MASHKDDKDK
MASHKDDEDK
MASHKDDEDK
MASHKDDEDK
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEER


VSKTAADNNN
VSKTAADNNN
VSKTSADNNN
VSKTSADNNN
VSKTPADNNN
VSKTSADNNN
VSKTSADNNN
VSKTSADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN
VSKTAADNNN


...EYSW
...EYSW
...EYSW
...EYSW
...EYSW
...EYSW
...EYSW
...EYSW
...DYSW
...DYSW
...DYSW
...DYSW
...DYSW
...DYSW
...DYSW
...DYSW
...DYSW
...DYSW
...DYSW
...DYSW


TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG


VSKTAADNNN S.....DYSW TGATKYHLNG
VSKTAADNNN S.....DYSW TGATKYHLNG


600
SNTALDNVMI
SNTALDNVMI
SNTALDNVMI
SNTALDNVMI
SSTALDNVMI
SNTALDNVMI
SNTALDNVMI
SNTALDNVMI
SNTALDNVMI
DNVDYSSVML
DNVDYSSVML
DNVDYSSVML
DNVDYSSVML
DNVDYSSVML
DNVDYSSVML
DNVDYSSVML
DNVDYSSVML


AAV VR195 ABA71699
AAV VR355 ABA71701
AAV1 NP 049542
AAV1 VP1 AAD27757
isolate hu48 AAS99296
AAV6 VP1 AAB95450
isolate hu43 AAS99291
isolate hu44 AAS99292
isolate hu46 AAS99294
AAV10 AAT46337
isolate rh40 AAS99244
isolate hu37 AAS99285
isolate hu42 AAS99290
isolate hu40 AAS99288
isolate hu67 AAS99312
isolate rh38 AAS99243
isolate hu41 AAS99289


FFPMSGVMIF
FFPMSGVMIF
FFPMSGVMIF
FFPMSGVMIF
FFPMSGVMIF
FFPMSGVMIF
FFPMSGVMIF
FFPMSGVMIF
FFPMSGVMIF
FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLMF


GKESA. .
GKESA. .
GKESA. .
GKESA. .
GKESA. .
GKESA. .
GKESA. .
GKESA. .
GKESA. .
GKQGA..
GKQGA..
GKQGA..
GKQGA..
GKQGA..
GKQGA..
GKQGA..
GKQGA..


VP1


VP1
VP1
VP1

VP1
VP1
VP1
VP1
VP1
VP1
VP1












Goose AAV VP1 AAA83230
GOOSE AAV VP1 NP 043515
AAV3 NP 043941


DREPERKDNN
DREPERKDNN
....DNR
....DNR
....DNR
.DDS
.DDS
.DDS
.DDS
.DDS


RGFVLPGYKY
RGFVLPGYKY
RGLVLPGYKY
RGLVLPGYKY
RGLVLPGYKY
RGLVLPGYKY
RGLVLPGYKY
RGLVLPGYKY
RGLVLPGYKY
RGLVLPGYKY


LGPGNGLDKG
LGPGNGLDKG
LGPGNGLDKG
LGPGNGLDKG
LGPGNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLYKG
LGPFNGLYKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPSNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG
LGPFNGLDKG


PPVNKADSVA
PPVNKADSVA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVDEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA
EPVNEADAAA


LEHDKAYDQQ
LEHDKAYDQQ
LEHDKAYDQQ
LEHDKAYDQQ
LEHDKAYDQQ
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEYDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!
LEHDKAYDRQ!


AAV3 Strain H
AAV3B VP1
VP1 isolate hul
VP1 isolate hu4
VP1 isolate hu2
VP1 isolate hu3
VP1 isolate hu60
VP1 isolate hu61
VP1 isolate hu25
VP1 isolate hul5
VP1 isolate hul6
VP1 isolate hul8
VP1 isolate hu7
VP1 isolate hul0
VP1 isolate hull
VP1 isolate hu9
VP1 isolate hu53
VP1 isolate hu55
VP1 isolate hu54
VP1 isolate huS17
AAV2 VP1


AAC55049
AAB 9 54 52
AAS 992 60
AAS 992 8 7
AAS 992 70
AAS 992 80
AAS9 93 0 7
AAS9 93 0 8
AAS 992 7 6
AAS 992 65
AAS 992 6 6
AAS 992 6 8
AAS 9 9313
AAS 992 61
AAS 992 62
AAS 9 9314
AAS 9 9 30
AAS9 9 302
AAS9 9 301
AAU 05370
AAC O37 80
AAS 992 8 3
AAS 992 8 4
AAS 992 9 8
AAS 992 9 9
AAS 992 9 5
AAS 992 9 3
AAS9 93 0 5
AAS 992 9 7
AAS9 93 0 3
AAS9 93 0 4
AAS 992 7 8
AAS 992 7 9
AAU 05364
AAS 992 63
AAS9 93 0 9
AAS 9 9310
AAU 053 62


.......DDS RGLVLPGYKY
.......DGS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DNS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS GGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY
.......DDS RGLVLPGYKY


VP1 isolate
VP1 isolate
VP1 islolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate


hu34
hu35
hu51
hu52
hu47
hu45
hu58
hu49
hu56
hu57
hu28
hu29


VP1 isolate huT70
VP1 isolate hul3
VP1 isolate hu63
VP1 isolate hu64
VP1 isolate huT40










Cryo-EM studies have shown that the unique N-terminus of VP I is internal to the capsid based

on additional density at the 2-fold axis of symmetry [97]. In vitro, upon heat treatment of AAV

capsids, it has also been shown that this region can be externalized. Mutagenesis experiments

have shown that this externalization occurs through the pore at the 5-fold axis of symmetry [70].

The high resolution x-ray crystallography data is unable to address these dynamic changes which

must occur as part of the virus life cycle.

For several viruses, peptide mapping has been used to address structural changes that occur

in solution. Peptide mapping was first used in 1979 to study the major capsid protein of

bacteriophage T4 [98]. Since this study, peptide mapping has been utilized to study the capsid

structure of DNA viruses, such as herpes virus, and canine parvovirus, as well as for many RNA

viruses, such as reovirus, rotavirus, vescicular stomatitis virus, influenza virus and tetraviruses.

Peptide mapping has been utilized to demonstrate that certain amino acids are surface associated.

In addition, peptide mapping has been utilized to evaluate dynamic structural changes that occur

in the capsid. In this study, peptide mapping of the AAV virion is developed as a tool for

identifying regions of the AAV capsid that are flexible, accessible, and surface associated.

Mapping of these regions of the capsid may provide a valuable tool for predicting locations on

the capsid surface that can tolerate insertions, which may be useful for improved targeting of

AAVserotypes. With 11 known AAV serotypes and up to 100 genotypic variants, this method

may provide a starting point for identifying locations on the capsid to mutate, especially if the

crystal structure is not available.

Trypsin digestion of AAV-2. Historically, AAV has been shown to be remarkably stable

and generally resistant to proteases [99]. However, occasionally, AAV-2 vector preps had

additional protein bands when analyzed for purity when assayed on polyacrylamide gels and


















VP3+ VP3










Figure 5-3. Coomassie stained samples for mass spec identification. Samples of AAV-4 and
AAV-5 that were less pure were run in parallel with purified AAV-4 and AAV-5, as
well as additional samples of AAV-8 to determine if purity is an important parameter
for this assay.

are listed across the top of the table. The column on the left hand side are the theoretical proteins,

the accession number, and the protein molecular mass. Numbers highlighted in green indicate the

number of unique fragments with a probability of over 95% upon which the AAV serotype

identification was based.

The samples that were less pure, LLL (AAV-4), MMM (AAV-4), QQ (AAV-5 VP2),

QQQ (AAV-5 VP3), RR (AAV-5 VP2), RRR (AAV-5 VP3) had detectable levels of

contaminating proteins, such as cellular and helper virus proteins used in production. In the

initial experiment to determine feasibility of this assay, LC/MS/MS was performed on VP 1, VP2

and VP3 of three AAV serotypes, AAV-1, AAV-2 and AAV-5 (impure). This method can be

used to identify contaminants; for example, the AAV Rep78 was identified in one impure prep

since it migrated with similar molecular weight as VP2 (72 kDa). This assay may be useful for

verifying that new purification methods have eliminated proteins that are similar in size to the


































Figure 3-10. Intermolecular and intramolecular residues that are disrupted as a result of changing
Arg294 and Asp295 to alanine residues. Arg294 is shown in yellow, Asp295 is
shown in white, the chain that they are attached to is shown in light blue, residues
from other chains are shown in pink or dark blue respectively. Intramolecular
interactions that are disrupted include the interaction with Ser292 and Trp234. This is
a close up of the specific interactions in Figure 3-9. Note: Tyr397 is pink, Glu689 and
Gu697 are dark blue. This figure was generated using the molecular modeling
software Deep View.





























Figure 3-13. This is a cut away view of three of the chains that make up the 5-fold axis of
symmetry. The red and aqua chains of Figure 3-12 have been removed to show the
location of the mutations. Lys321 is in aqua or white, and Glu322 is in pink. This
figure was generated using the molecular modeling software Deep View.

packaging, but this is assumed to involve residues in the region of the 5-fold pore since the

proposed model for AAV packaging suggests that the DNA is inserting into the capsid through

the 5-fold pore. Alternatively, mutating the residues at the 5-fold pore may have disrupted an as

yet undetermined site on the capsid that is required for the Rep proteins to dock. This mutation is

interesting in that infectious particles are not produced at 39.5 oC. Physical particle titer using

A20 ELISA was not obtained for this mutant so it is not known whether the lack of infectious

particles at 39.5 oC is due to a lack of capsid assembly at 39.5 OC, or an inability to package at

39.5 OC; however, based on the location of the mutation, the mutation is predicted to effect DNA

packaging. Based on the model, this mutation does not affect the 2-fold or the 3-fold axis. EM on

capsids produced at 39.5 oC would help to determine if this is a packaging mutant versus an

assembly mutant.

In this region of the capsid, there is an intermolecular interaction between Asn33 5 (yellow)

and Asn334 (blue), as shown in Figure 3-14. Although the intermolecular interaction between

















































Mutl9 ni, no capsid D231:K692 Mut47 ni, A20-
Q297:1P696
Q297:4Q699
F712:W694
F712:P696
F365:W694
]kht40 -ni, hep-, full E563:Y704
N301: Q699
Y700:1P696
Mut47 -ni, A20T R693:R294 Mut24 -ni, no capsid
Y720:W694
T713:N695


Table 3-6 Continued
B-A
B-B
B -FI
B-A
B-A
H P
H P
H-P
P A
P H
A-B
A-B
P P
P-P
P P
P-P
P-H
P-P
P H
P-P
P-P
P--H
P H
P--FI
11-H1
H-H
H P
P-P
H H
A B
A B
P--H
P-P
H H
H H
H H
A-P
P P
P--H
B-1B
P--H
P-P


Mut24 -ni, no capsid











]kht47 -ni, A20f















Mut24 -unchanged
Idid24 -unchanged
Mut24 -unchanged


R294:E689
R294:R693
IL294:1P696
R294:E697
R298:E689
I698:T701
I698:S702
I698:Y704
Y704:E563
Y704:I698
E689:R294
E689:R298
Q699: Q297
Q699:N301
Q699:Q699
Q699:T701
19695:V710
N695:T713
T701:I698
T701:Q699
T701:T701
S702:1P696
S702:I698
S292:W694
P293:W694
P293:P696
V710:N695
N3 02:N3 02
P366:W694
E697:R294


Mut47 -ni, A20Y
]kht47 -ni, AOR


Mut47 -ni, AOR




Mut40 ni, hep-, full

Mut24 -ni, no capsid


Mut24 ni, no capsid











VP1 isolate huT70
VP1 isolate hul3
VP1 isolate hu63
VP1 isolate hu64
VP1 isolate huT40
VP1 isolate huLG15
VP1 isolate huT17
VP1 isolate huT41
VP1 isolate huT71
VP1 isolate huT88
VP1 isolate huT32


AAU 05364
AAS 992 63
AAS9 93 0 9
AAS 9 9310
AAU 053 62
AAU 05371
AAU 05358
AAU 05372
AAU 05366
AAU 05368
AAU 05360
AAS 992 7 7
AAS 992 6 9
AAS 992 71
AAS 992 7 2
AAS 992 7 5
AAS 992 7 3
AAS 992 7 4


YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGH
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY
YFGYSTPWGY

351
EVTTSDGVTT
EVTTNDGVTT
EVTTNDGVTT
EVTTNDGVTT
EVTTNDGVTT
EVTTNDGVTT
EVTTNDGVTT
EVTTNDGVTT
EVTTNDGVTT
EVTQNEGTKT
EVTQDEGTKT
EVTQNEGTKT
EVTQNEGTKT
EVTQNEGTKT
EVTQNEGTKT
EVTQNEGTKT
EVTQNEGTKT
EVTQNEGTET
EVTQNEGTKT
EVTQNEGTKT
EVTQNEGTKT


FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF
FDFNRFHCHF



IANNLTSTVQ
IANNLTSTVQ
IANNLTSTVQ
IANNLTSTVQ
IANNLTSTVQ
IANNLTSTVQ
IANNLTSTVQ
IANNLTSTVQ
IANNLTSTVQ
IANNLTSTIQ
IANNLTSTIQ
IANNLTSTIQ
IANNLTSTIQ
IANNLTSTIQ
IANNLTSTIQ
IANNLTSTIQ
VANNLTSTIQ
IANNLTSTIQ
IANNLTSTIQ
IANNLTSTIQ
IANNLTSTIQ


SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN
SPRDWQRLIN


VFSDSEYQLP
VFSDSEYQLP
VFSDSEYQLP
VFSDSEYQLP
VFSDSEYQLP
VFSDSEYQLP
VFSDSEYQLP
VFSDSEYQLP
VFSDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP
VFTDSEYQLP


NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGSRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL
NNWGFRPKRL



YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGR
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC
YVLGSAHQGC


NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
NFKLFNIQVK
SFKLFNIQVK
GFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK
SFKLFNIQVK

400
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
LPPFPADVFM
PPPFPADVFM
PPPFPADVFM
PPPFPADVFM


VP1
VP1
VP1
VP1
VP1
VP1
VP1


isolate
isolate
isolate
isolate


hu27
hul9
hu20
hu21


isolate hu24
isolate hu22
isolate hu23


AAV VR195 ABA71699
AAV VR355 ABA71701
AAV1 NP 049542


AAV1 VP1 AAD27757
ate hu48 AAS99296
AAV6 VP1 AAB95450
ate hu43 AAS99291
ate hu44 AAS99292
ate hu46 AAS99294
AAV10 AAT46337
ate rh40 AAS99244
ate hu37 AAS99285
ate hu42 AAS99290
ate hu40 AAS99288
ate hu67 AAS99312
ate rh38 AAS99243
ate hu41 AAS99289
ate hu66 AAS99311
ate hul7 AAS99267
,late hu6 AAS99306
ate rh25 AAS99242


VP1 isol

VP1 isol
VP1 isol
VP1 isol

VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 isol
VP1 iso
VP1 isol










98. Steven AC, Carrascosa JL: Proteolytic cleavage and structural transformation: their
relationship in bacteriophage T4 capsid maturation. JSupramnolStruct 1979, 10:1-11.


99. Berns KI, Rose JA: Evidence for a single-stranded adenovirus-associated virus
genome: isolation and separation of complementary single strands. J Virol 1970,
5:693-699.


100. Wobus CE, Hugle-Dorr B, Girod A, Petersen G, Hallek M, Kleinschmidt JA: Monoclonal
antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping
and identification of capsid domains involved in AAV-2-cell interaction and
neutralization of AAV-2 infection. J Virol 2000, 74:9281-9293.


101. Weichert WS, Parker JS, Wahid AT, Chang SF, Meier E, Parrish CR: Assaying for
structural variation in the parvovirus capsid and its role in infection. Virology 1998,
250:106-117.


102. Sommer JM, Smith PH, Parthasarathy S, Isaacs J, Vijay S, Kieran J, Powell SK,
McClelland A, Wright JF: Quantification of adeno-associated virus particles and empty
capsids by optical density measurement. Mol1 Ther 2003, 7: 122-128.


103. Mohiuddin I, Loiler S, Zolotukhin I, Byrne BJ, Flotte TR, Snyder RO: Herpesvirus-based
infectious titering of recombinant adeno-associated viral vectors. Mol1 Ther 2005,
11:320-326.


104. Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM: Novel adeno-associated
viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U
SA 2002, 99: 11854-11859.


105. Reddy VS, Nataraj an P, Okerberg B, Li K, Damodaran KV, Morton RT, Brooks CL, 3rd,
Johnson JE: Virus Particle Explorer (VIPER), a website for virus capsid structures
and their computational analyses. J Virol 2001, 75: 11943-11947.


106. Francis JD, and Snyder, R.O. : Production of Research and Clinical Grade
Recombinant Adeno-associated Viral Vectors. In Laboratory Techniques in
Biochemistry & M~olecular Biology. Volume 31. Edited by Berns KI, and Flotte, T.R. .
Amsterdam: Elsevier, ; 2005: 19-56


107. Muzyczka N, Warrington KH, Jr.: Custom adeno-associated virus capsids: the next
generation of recombinant vectors with novel tropism. Hum Gene 7Jher 2005, 16:408-
416.









Mut26 is able to package viral DNA, although recently other investigators have produced data

using 5-fold pore mutants that suggest that mutations in this region of the capsid effect DNA

packaging. It is unknown where the nonstructural Rep proteins interact with the capsid for


Figure 3-12. Residues at the 5-fold symmetry axis that are changed in Mut26. The aqua residue
is Lys321. The pink residue is Glu322. Yellow arrows point in the direction of the
residues that have been mutated, but the amino acids that have been mutated are at the
5-fold pore. Note: The orientation on this figure is different from Mutl9 and Mut24.
This figure is from outside of the capsid looking at the 5-fold pore. This figure was
generated using the molecular modeling software Deep View.









SDS and MeOH at 45 oC. Undigested control virions treated with SDS and MeOH at 450C, and

trypsinized virions treated with SDS and MeOH at 37 OC were not detectable by the B1

antibody.

This data indicates that the digested AAV-2 capsids appear intact by EM, buoyant density

on cesium chloride gradients, as well as A20 native dot blot analysis. The internal disposition of

the maj ority of the C-terminal proteolytic fragment (~70% of residues 589 73 5, Figure 4-7)

ensures that the fragment remains associated with the capsid in 3.3 M CsCl during purification,

as well as in 0.1% SDS and 20% MeOH used in the immuno-dot blot (Figure 4-3C), and during

heparin chromatography. However, heating the trypsinized virions in the presence of 0.6M DTT

for mass spectroscopy and N-terminal sequencing, or heating in the presence of SDS and

reducing agent for denaturing gel electrophoresis was able to dissociate the VP1,2,3T fragment

from the core capsid. Since no cysteine residues are found in the VP1,2,3T C-terminal fragment,

the interaction of this fragment with the capsid is due to non-covalent interactions that are

disrupted when the capsid structure is denatured by heat and reducing agent.

The crystal structure of AAV-2 did not show a break in the polypeptide chain at position

R588. This suggests that the maj ority of the 60 R588 sites remain uncleaved by the mild trypsin

treatment used during the purifications reported here, and for samples used for crystallization of

AAV-2, which generated the additional bands observed in Figures 4-1 and 4-2. The capsids thus

maintain the properties and structure of the native virus while in the crystalline state but appear

to have increased flexibility as shown by differential staining in EM, and exposure of the B 1

epitope in the presence of SDS and MeOH at 450C (Fig. 4-3C). However, for future studies, it

may be prudent to avoid incorporating proteases into AAV purification schemes for structural

studies or gene therapy applications where proteolysis may not only alter the capsid structure,












VP1 isolate hu66
VP1 isolate hul7
VP1 isolate hu6
VP1 isolate rh25
VP1 isolate hu39
VP1 isolate rh49
VP1 isolate rh50
VP1 isolate rh51
VP1 isolate rh52
VP1 isolate rh64


AAS 9 9311
AAS 992 67
AAS9 93 0 6
AAS 992 4 2
AAS 992 8 6
AAS 992 4 7
AAS 992 4 8
AAS 992 4 9
AAS 992 50
AAS 992 59


RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNSGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLVNPGVA
RNSLANPGIA
RNSLANPGIA
RNSLANPGIA
RDSLVSPGVA
RDSLVNPGVA
RDSLVNPGVA
RDSLMNPGVA
RNSLVNPGVA
RNSLVNPGVA
RNSLVNPGVA
RNSLVNPGVA
RNSLVNPGVA
RNSLVNPGVA
RDSLVNPGVA
RNSLMNPGPA
RNSLMNPGPA
RNSLMNPGPA
RWSNIAPGPP
RWSALTPGPP
RWSALTPGPP
RW SNAP GTA
RW SNAP GTA
ASYQVPPQPN
ASYQVPPQPN
ASYQVNPQPN
ASYQVNPQPN
VQFAIAPDVP


MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATNKDDEDR
MATNKDDEDR
MATNKDDEDR
MATNKDDEDR
MATNKDDEDR
MATNKDDEDR
MATNKDDEDR
MATNKDDEDR
MATNKDDEDR
MATNKDDEDR
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEER
MASHKDDEDR
MATHKDDEDR
MATHKDDEDR
MATHKDDEER
MATHKDDEER
MATHKDDEER
MATHKDDEDR
MATHKDDEER
MASHKEGEDR
MASHKEGEDR
MASHKEGEDR
MATAGPSDGD
MATAG PADSK
MATAG PADSK
MATAANDATD
MATAANDATD
GMTNNLQGSN
GMTNNLQGSN
GMTNTLQGSN
GMTNTLQGSN
SMCNHLEGTN


FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLMF
FFPSSGILMF
FFPSSGILMF
FFPSSGILMF
FFPSSGILMF
FFPSSGILMF
FFPSSGILMF
FFPSSGILMF
FFPSSGILMF
FFPSSGILMF
FFPSSGILMF
FFPSNGILIF
FFPSNGILIF
FFPVTGSCFW
FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLMF
FFPSSGVLIF
FFPSSGVLIF
FFPSSGVLIF
FFPSSGVLIF
FFPSSGVLIF
FFPSSGVLIF
FFPSSGVLIF
FFPSSGVLIF
FFPLSGSLIF
FFPLSGSLIF
FFPLSGSLIF
FS.NAQLIFP
FS.NSQLIFA
FS.NSQLIFA
FS.QAQLIFA
FS.QAQLIFA
TYALENTMIF
TYALENTMIF
RYALENTMIF
RYALENTMIF
MIALDNSLIF


GKQGA...GR
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQNA...AR
GKQNA...AR
.QQNA...AR
GKQGA...GK
GKQGA...GK
GKQGA...GK
GKQGA...GN
GKTGA...TN
GKTGA...TN
GKTGA...1UJ
GKTGA...1UJ
GKTGA...1UJ
GKTGA...TN
GKTGA...1UJ
GKQGT...GR
GKQGT...GR
GKQGT...GR
GPSVT...GN
GPKQN...GN
GPKQN...GN
GPNIT...GN
GPNIT...GN
NSQPANPGTT
NSQPANPGTT
NAQNATPGTT
NAQNATPGTT
RDVSTAPGDT


DNVDYSSVML
DNVDYSSVML
DNVDYSSVML
DNVDYSSVML
DNVDYSNVML
DNMGYSNVML
DNVDYSNVML
DNVDYSNVML
DNVDYSNVML
DNVDYSNVML
DNVDYSNVML
DNVDYSNVML
DNVDYSNVML
DNVDYSNVML
DNADYSDVML
DNADYSDVML
DNADYSDVML
DNVEYTNVML
DNVEYTNVML
DNVEYTNVML
DGVDYSQVLI
KTT.LENVLM
KTT.LENVLM
KTT.LENVLM
KTT.LENVLM
KTT.LENVLM
KTT.LENVLM
KTT.LENVLM
DNVDADKVMI
DNVDADKVMI
DNVDADKVMI
TTTSANNLLF
TATVPGTLIF
TATVPGTLIF
TTTDANNLMF
TTTDANNLMF
ATYLEGNMLI
ATYLEGNMLI
SVYPEDNLLL
SVYPEDNLLL
TQYNINQVIV


VP1
VP1
VP1
VP1



VP1
VP1
VP1
VP1


isolate rh53 AAS99251
isolate rh61 AAS99257
isolate rh58 AAS99255
isolate rh57 AAS99254
AAV8 VP1 AANO3857
AAV8 YP 077180
isolate rh43 AAS99245
isolate pil AAS99238
isolate pi3 AAS99240
isolate pi2 AAS99239


VP1 isolate rh1 AAS99241
AAV7 VP1 AANO3855
AAV7 YP 077178


VP1
VP1
VP1
VP1


isolate rh48
isolate rh62
isolate rh55
isolate rh54


AAS 992 4 6
AAS 992 5 8
AAS 992 5 3
AAS 992 52
AAS 992 5 6
AAS 992 64
AAS 992 81
AAS 992 8 2
AAT4 63 3 9


VP1 isolate rh60
AAV9 VP1
VP1 isolate hu31
VP1 isolate hu32
AAV11


AAV4 NP 044927
AAV4 VP1 AAC58045
BOVINE AAV AAR26465
BOVINE AAV YP 024971
AAV5 VP1 AAD13756
AAV5 YP 068409
CAPRINE AAV1 AAU84890
GOAT AAV VP1 ABC69726
RAT AAV1 AAZ79676



















































AAV4 NP 044927
AAV4 VP1 AAC58045
BOVINE AAV AAR26465
BOVINE AAV YP 024971
AAV5 VP1 AAD13756
AAV5 YP 068409
CAPRINE AAV1 AAU84890
GOAT AAV VP1 ABC69726
RAT AAV1 AAZ79676
MOUSE AAV1 AAZ79672


isolate hul7 AAS99267
isolate hu6 AAS99306
isolate rh25 AAS99242
isolate hu39 AAS99286
isolate rh49 AAS99247
isolate rh50 AAS99248
isolate rh51 AAS99249
isolate rh52 AAS99250
isolate rh64 AAS99259
isolate rh53 AAS99251
isolate rh61 AAS99257
isolate rh58 AAS99255
isolate rh57 AAS99254
AAV8 VP1 AANO3857
AAV8 YP 077180
isolate rh43 AAS99245
isolate pil AAS99238
isolate pi3 AAS99240
isolate pi2 AAS99239
isolate rh1 AAS99241
AAV7 VP1 AANO3855
AAV7 YP 077178


NNMSAQAKNW
NNMSAQAKNW
NNMSAQAKNW
SNMSAQARNW
SNMSAQARNW
SNMSAQARNW
SNMSAQARNW
SNMSAQARNW
SNMSAQARNW
SNMSAQARNW
SNMSAQARNW
SNMSAQARNW
SNMSAQARNW
NTMANQAKNW
NTMANQAKNW
NTMANQAKNW
QNMSAQAKNW
QNMSAQAKNW
QNMSAQAKNW
SSMANQARNW
STMAEQAKNW
STMAEQAKNW
TTMAEQAKNW
TTMAEQAKNW
TTMAEQAKNW
STMAEQAKNW
SNMSAQARNW
SNMAVQGRNY
SNMAVQGRNY
SNMAVQGRNY
GDFAFYRKNW
TNFSNFKKNW
TNFSNFKKNW
TNFSGYRKNW
TNFSGYRKNW
GRYANTYKNW
GRYANTYKNW
GRYANTYKNW
GRYANTYKNW
HNYAQQYKNW
TNLPEQERNW


LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
LPGPCYRQQR
VPGPCYRQQR
LPGPCFRQQR
LPGPCFRQQR
LPGPCFRQQR
LPGPCFRQQR
LPGPCFRQRR
LPGPCFRQQR
LPGPCYRQQR
IPGPSYRQQR
IPGPSYRQQR
IPGPSYRQQR
LPGPCVKQQR
LPGPSIKQQG
LPGPSIKQQG
LPGPMMKQQR
LPGPMMKQQR
FPGPMGRTQG
FPGPMGRTQG
FPGPMGRTQG
FPGPMGRTQG
LTGAFQRNQD
LPGPGIRNQA


VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTLSQNNN
VSTTTGQNNN
VSTTTGQNNN
VSTTTGQNNN
VSTTVSQNNN
VSTAVSQNNN
VSTTVSQNNN
VSTTTNQNNN
VSKTLDQNNN
VSKTLDQNNN
VSKTLDQNNN
VSKTLDQNNN
VSKTLDQNNN
VSKTLDQNNN
VSTTLSQNNN
VSTTVTQNNN
VSTTVTQNNN
VSTTVTQNNN
FSKTASQNYK
FSKTANQNYK
FSKTANQNYK
FSKTASQNYK
FSKTASQNYK
WNLGSG..VN
WNLGSG..VN
WNTSSGSSTN
WNTSSGSSTN
YNYVSG.TSN
WFNSATGNNP


S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFTW
S.....NFTW
S.....NFTW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....NFAW
S.....EAW
S.....EFAW
S.....EFAW
IPAS GGNALL
IPATGSDSLI
IPATGSDSLI
IPQGRNNSLL
IPQGRNNSLL
RASVSAF ...
RASVSAF ...
RVSVNNF. ..
RVSVNNF. ..
YKGVVGS. ..
LTG.....TW


TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TAGTKYHLNG
TAGTKYHLNG
TAGTKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGAAKFKLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
TGATKYHLNG
PGASSWALNG
PGASSWALNG
PGASSWALNG
KYDTHYTLNN
KYETHSTLDG
KYETHSTLDG
HYETRTTLDG
HYETRTTLDG
ATTNRMELEG
ATTNRMELEG
SVSNRMNLEG
SVSNRMNLEG
NQNNLQRIEN
QYSNKYVLEN


isolate rh48
isolate rh62
isolate rh55
isolate rh54


VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1
VP1



VP1
VP1
VP1
VP1
VP1



VP1
VP1
VP1
VP1
VP1


AAS 992 4 6
AAS 992 5 8
AAS 992 5 3
AAS 992 52
AAS 992 5 6
AAS 992 64
AAS 992 81
AAS 992 8 2
AAT4 63 3 9


AAV9 VP1
VP1 isolate hu31
VP1 isolate hu32
AAV11


isolate rh60









[60]. Pulse-chase experiments showed that empty particles rapidly accumulate (10 20 minutes),

but that mature "full" virions accumulated more slowly (4 to 8 hours). They also showed that the

number of empty viral particles decreases at the same rate as the number of DNA-containing

mature virions increases over the course of infection. Additionally, de la Maza and Carter

showed that DI particles, AAV genomes with deletions, are packaged into apparently normal

capsids, indicating that full-length viral genomic DNA is not required for the assembly or

structural integrity of the AAV capsid [61]. In the absence of capsid assembly, ssDNA does not

accumulate, further suggesting that empty capsids form first. Interactions between the preformed

AAV capsid and Rep52 provides a mechanism where the nonstructural protein's helicase activity

inserts the viral DNA [62]. Data based on MVM and AAV suggest that the DNA is inserted into

the capsid at the 5-fold pore [63, 64]. Within the cell, capsid assembly occurs at centers within

the nucleus where Rep proteins, capsid proteins and DNA are co-localized [65]. Empty AAV

capsids can be produced by expressing the AAV cap gene in insect cells using a baculovirus

expression system [66] or in mammalian cells utilizing a recombinant adenovirus expressing the

AAV capsid proteins. These systems have advanced structural studies of the AAV capsid as a

result of the large amount of empty capsids or virus-like particles (VLPs) that can be produced.

Studies of AAV assembly have demonstrated that VP3 alone is sufficient to form VLPs [67], but

VP I is required for infectivity [68]. Subsequently, it was shown that the unique N-terminus of

VPI has phospholipase A2 activity and contains a nuclear localization signal (NLS) [69]. Studies

have demonstrated that the unique N-terminus of VP I is inside the capsid near the 2-fold axis of

symmetry and becomes externalized through the 5-fold pore during viral trafficking in the cell

[70]. The N-terminus of VP2 also has a NLS and may play a role in transporting VP3 into the

nucleus; however, it has been shown that the N-terminus of VP2 is nonessential and that









Mut24 291 -295 FSPRD FSPAA

Mut24 is an alanine substitution mutation, where the charged residues Arg294 and Asp295,

have been changed to alanine [89]. Utilizing the same methods described for Mutl9, the residues

that are mutated in Mut24 were evaluated. Like Mutl9, the phenotype for Mut24 is an inability

to make capsids. The molecular modeling software was used as a tool to predict the structural

subunit that is formed. The location of this mutant affects both the 2-fold axis and the 5-fold axis

of symmetry. Based on the location of the mutations at the 5-fold axis, pentamers may not be

able to be formed. This mutation is further from the 5-fold pore than Mutl9, so due to the

location of the mutation, there may be enough significant interactions at the 5-fold axis to

perhaps hold pentamers together. This is demonstrated in Figure 3-7. The 2-fold axis of

symmetry is shown in Figure 3-8. The residues that have been mutated are likely to produce

significant changes at the 2-fold axis. The mutation at the 2-fold axis occurs at a place in the

capsid where three different chains come together, the two that make up the 2-fold, (light blue

and dark blue residues) one of which is also a part of the 3-fold axis (dark blue), and a second

chain of the 3-fold axis (orange) that sits above the residue that makes up the 2-fold axis (light

blue). If the second chain of the 3-fold axis is disrupted then trimers would not be stable and the

predicted subunit formed would be either monomers or pentamers, if the other interactions at the

5-fold axis are significant enough to hold a pentamer together. Alternatively, the mutation may

only affect the residues at the 2-fold axis and the second chain of the 3-fold axis (orange) may be

unaffected. If this is the case, then trimers will be the predicted subunit. This is shown in Figure

3-9. Based on the information in Figure 3-10, there are quite a few residues that interact with

Arg294 and Asp295. These are also shown in Table 3-14. Based on the information in this table,

there are a couple of things to note. First, Arg294 is a positively charged basic residue that

interacts with two negatively charged glutamic acid residues. Changing Arg294 to an Ala294










28. Peden CS, Burger C, Muzyczka N, Mandel RJ: Circulating anti-wild-type adeno-
associated virus type 2 (AAV2) antibodies inhibit recombinant AAV2 (rAAV2)-
mediated, but not rAAV5-mediated, gene transfer in the brain. J Virol 2004, 78:6344-
6359.


29. Jiang H, Couto LB, Patarroyo-White S, Liu T, Nagy D, Vargas JA, Zhou S, Scallan CD,
Sommer J, Vijay S, Mingozzi F, High KA, Pierce GF: Effects of transient
immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer
in rhesus macaques and implications for human gene therapy. Blood 2006, 108:3321-
3328.


30. Wang Z, Kuhr CS, Allen JM, Blankinship M, Gregorevic P, Chamberlain JS, Tapscott SJ,
Storb R: Sustained AAV-mediated dystrophin expression in a canine model of
Duchenne muscular dystrophy with a brief course of immunosuppression. Mol1 Ther
2007, 15: 1160-1166.


31. Snyder RO, Francis J: Adeno-associated viral vectors for clinical gene transfer studies.
Curr Gene 7ler 2005, 5:311-321.


32. Snyder RO: Adeno-associated virus-mediated gene delivery. J Gene M~ed 1999, 1:166-
175.


33. Timpe J, Bevington J, Casper J, Dignam JD, Trempe JP: Mechanisms of adeno-
associated virus genome encapsidation. Curr Gene 7ler 2005, 5:273-284.


34. Becerra SP, Koczot F, Fabisch P, Rose JA: Synthesis of adeno-associated virus
structural proteins requires both alternative mRNA splicing and alternative
initiations from a single transcript. J Virol 1988, 62:2745-2754.


35. Grieger JC, Samulski RJ: Packaging capacity of adeno-associated virus serotypes:
impact of larger genomes on infectivity and postentry steps. J Virol 2005, 79:9933-
9944.


36. Halbert CL, Allen JM, Miller AD: Efficient mouse airway transduction following
recombination between AAV vectors carrying parts of a larger gene. Nat Biotechnol
2002, 20:697-701.









differences in conformation when the AAV capsid structures are compared [50]. These loops

intertwined to form the characteristic AAV surface protrusions that decorate the core capsid and

provide the immunogenic, tropic, and proteolytic determinants. The cleaved AAV-2 C-terminal

protein fragment, residues 589 to 735, is able to remain associated with the capsid because

thecleavage site is on the outer surface of a 3-fold protrusion, not part of the core. The eight

stranded beta barrel forms the core stable capsid that remain intact following trypsin cleavage.

Of note are the surface accessible R and K residues in the G-H loop that are common in AAV-1,

AAV-2, and AAV-5 (Figure 4-8), but are not cleaved by trypsin. Thus it is possible that

proteolytic digestion may be limited by steric constraints to those residues found on loops that

protrude from the surface. Results in solution confirm that the region of the G-H loop containing

residue R588 is surface accessible as seen in the crystal structure, and the presence of heparin is

not required for exposure. The B1 epitope (residues 726-733) is located on the "wall/floor" of

the dimple at the icosahedral 2-fold axis of the AAV-2 capsid, close to a "buried" stretch of G-H

loop amino acids leading up to the tryptic cleavage site (Figure 4-8 I-III). Interestingly, the B1

epitope, found on the thinnest region of the AAV-2 capsid, contains residues that are exposed on

both the interior and exterior surfaces (Figure 4-8 III). The isolation of numerous AAV serotypes

and genotypic variants [23-26, 104], and the observation that each virus serotype has unique

cellular recognition and transduction phenotypes dictated by the capsid protein sequence has

generated a need to fully characterize the capsids of these viruses, including their structural

features. Here we use proteolytic digestion combined with available information on antigenic

epitopes and mass spectroscopy to analyze the AAV capsid in solution. We show that

proteolysis is able to generate a characteristic cleavage pattern that can be used to distinguish full






































IVP3


P
AB


AB


A69
AB


AB


-VP1
- VP2, VP1T


-VP3T


25 kDa
2 0 kDka

15 kDa


-a
1 2 3 4 SGC


-VP1,2,3 T


Af A69
I I


IVP1


Figure 4-2. Tryptic mapping of full AAV-2 capsids. Purified full AAV-2 virions (F-
T/iodixanol/Heparin) were treated with trypsin, and the capsid proteins were probed
with the indicated anti-AAV antibodies (AAV-2 polyclonal, Al, B l, A69). The
epitope locations of the monoclonal antibodies and the likely trypsin digestion site are
indicated on the schematic diagram. A, AAV-2 full capsids; B, AAV-2 full capsid
digested with trypsin for 24hours. The assignment of the tryptic fragments are given
on the right. VP IT is the digestion product of VP 1, VP2T is the digestion product of
VP2, VP3T is the digestion product of VP3, and VP 1,2,3T is the common C-terminal
trypsin digestion product of VP 1, VP2, and VP3.

VP I indicated that trypsin digestion at amino acid R566, R585, and R588 would produce

fragments of 18.8, 16.8, and 16.4 kDa, respectively, which are within the range expected for the

VP 1,2,3T C-terminal fragment that is recognized by B1 antibody (Figure 4-2, lane 4). The mass

of thi s fragment was confirmed to b e 1 6,46 1 Da by matrix-as si sted laser desorpti on/i onizati on


150 kDa
100 kDa
75 kDa -

50 kDa

37 kDa-


-
rl- 4 As
































Figure 3-16. Mut33 pentamer demonstrating that this mutation is not involved in the interactions
that make up the icosahedral 5-fold symmetry axis; therefore, as a result of this
mutation, pentamers will still be formed. This view is from the outside looking in.
This figure was generated using the molecular modeling software Deep View.

mutation in Asp472, and this is probably the reason for the phenotype. The interactions that are

disrupted are summarized in Table 3-16.

This capsid mutant was determined to be heat sensitive using the GFP fluorescent cell

assay to determine infectious titer. Mut33 has a titer that is about 1 log lower than wild-type

when the crude lysate was used to infect 293 cells at 32 oC. There was no titer at 39.5 OC [89].

Based on this, it can be assumed that Mut33 is able to package viral DNA. Since at the

permissive temperature packaging can occur, this suggests that an interaction between Rep and

VP3 that is needed for packaging is not disrupted as a result of these mutations. For this mutant,

the prediction would be that pentamers would be stable, as would dimers, but that trimers would

not be stable. This supports the model of assembly for AAV that was presented in Chapter Two











LIST OF TABLES


Table page

2-1 Parvovirus subunit association energies .............. ...............29....

3-1 The Hyve strongest interactions at the icosahedral 5-fold axis of symmetry ......................36

3-2 Specific amino acid interactions at the icosahedral 5-fold axis of symmetry ........._.........36

3-3 The Hyve strongest interactions at the icosahedral 3-fold axis of symmetry ......................39

3-4 Specific amino acid interactions at the icosahedral 3-fold axis of symmetry ........._._........39

3-5 The Hyve strongest interactions at the icosahedral 2-fold axis of symmetry ......................45

3-6 Specific amino acid interactions at the icosahedral 2-fold axis of symmetry ........._._........45

3-7 plM45 based plasmids for the production of mutant AAV-2 capsids .............. .............49

3-8 Amino acid alignment data for the amino acid sequence in the region of Mut33 .............49

3-9 Parvovirus structural capsid protein alignment for Mutl9 residues .............. .................50

3-10 Parvovirus structural capsid protein alignment for Mut24 residues .............. .................50

3-11 Parvovirus structural capsid protein alignment for Mut26 residues .............. .................50

3-12 Parvovirus structural capsid protein alignment for Mut33 residues .............. .................50

3-13 Parvovirus structural capsid protein alignment for Mut46 residues .............. ..............51

3-14 Evaluation of the effect of the mutations in Mut24 on amino acid interactions ................64

3-15 Evaluation of the effect of the mutations in Mut26 on amino acid interactions ................69

3-16 Evaluation of the effect of the mutations in Mut3 3 on amino acid interactions ................73

4-1 Predicted AAV VP I tryptic fragment mass from cleavage in the G-H loop ................... ..91

5-1 AAV Serotype Amino Acid Identity Table for AAV Serotypes 1 11 ................... .......114












MUSCOVY DUCK VP1 YP 068411
MUSCOVY DUCK PARVOVIRUS YP 068413
Goose AAV VP1 AAA83230
GOOSE AAV VP1 NP 043515
AAV3 NP 043941


VEEPVNMAPA KKSS...... .GKLTDHDPI VKKP.KLSEE NSPSP.....


..........
VEDPVNTAPA
VEDPVNTAPA
VEEAAKTAPG
VEEAAKTAPG
VEEAAKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
LRKPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VGEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEGPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VGEPVKTAPG
VGEPVKTAPG
VGEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG
VEEPVKTAPG


.N.........
KKNT. .....
KKN...... P
KK.GAVDQSP
KK.GAVDQSP
KK.RPVDQSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSPP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP
KK.RPVEHSP


..........
.GKLTDHYPV
Q.GKLDHYPGV
QE.PDSSSGV
QE.PDSSSGV
QE.PDSSSGV
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
AE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
AE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
AE.PDSSSGT
AE.PDSSSGT
AE.PDSSSGT
AE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
VE.PDSSSGT
AE.PDSSSGT
AE.PDSSSGT
AE.PDSSSGT
AE.PDSSSGT


.KK........
VKKP.KLTEE
VKKP.KLTEE
GKSG.KQPAR
GKSG.KQPAR
GKSG.KQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.QRPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.NQPAR
GKAG.NQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.HQPAR
GKAG.HQPAR
GKAG.HQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.QQPAR
GKSG.QQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.QQPAR
GKAG.NQPAR
GKAG.QQPAR
GKAG.NQPAR
GKAG.NQPAR
GKSG.NQPAR
GKSG.NQPAR
GKSG.NQPAR
GKAG.QQPAR


...........
VSAGG.....
VSANGG....
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..
KRLNFGQT..


AAV3 Strain H
AAV3B VP1
VP1 isolate hul
VP1 isolate hu4
VP1 isolate hu2
VP1 isolate hu3
VP1 isolate hu60
VP1 isolate hu61
VP1 isolate hu25
VP1 isolate hul5
VP1 isolate hul6
VP1 isolate hul8
VP1 isolate hu7
VP1 isolate hul0
VP1 isolate hull
VP1 isolate hu9
VP1 isolate hu53
VP1 isolate hu55
VP1 isolate hu54
VP1 isolate huS17
AAV2 VP1


AAC55049
AAB 9 54 52
AAS 992 60
AAS 992 8 7
AAS 992 70
AAS 992 80
AAS9 93 0 7
AAS9 93 0 8
AAS 992 7 6
AAS 992 65
AAS 992 6 6
AAS 992 6 8
AAS 9 9313
AAS 992 61
AAS 992 62
AAS 9 9314
AAS 9 9 30
AAS9 9 302
AAS9 9 301
AAU 05370
AAC O37 80
AAS 992 8 3
AAS 992 8 4
AAS 992 9 8
AAS 992 9 9
AAS 992 9 5
AAS 992 9 3
AAS9 93 0 5
AAS 992 9 7
AAS9 93 0 3
AAS9 93 0 4
AAS 992 7 8
AAS 992 7 9
AAU 05364
AAS 992 63
AAS9 93 0 9


VP1 isolate
VP1 isolate
VP1 islolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate
VP1 isolate


hu34
hu35
hu51
hu52
hu47
hu45
hu58
hu49
hu56
hu57
hu28
hu29


VP1 isolate huT70
VP1 isolate hul3
VP1 isolate hu63











AVIAN AAV ATCC VR865 AAT48613
AVIAN AAV ATCC VR865 NP 852781
AVIAN AAV Strain DA1 AAT48615
AVIAN AAV Strain DA1 YP 077183
DUCK AAV Strain FM AAA83225
MUSCOVY DUCK PARVOVIRUS YP 068412
MUSCOVY DUCK VP1 YP 068411
MUSCOVY DUCK PARVOVIRUS YP 068413
Goose AAV VP1 AAA83230
GOOSE AAV VP1 NP 043515
AAV3 NP 043941
AAV3 Strain H AAC55049
AAV3B VP1 AAB95452
VP1 isolate hul AAS99260
VP1 isolate hu4 AAS99287
VP1 isolate hu2 AAS99270
VP1 isolate hu3 AAS99280
VP1 isolate hu60 AAS99307
VP1 isolate hu61 AAS99308
VP1 isolate hu25 AAS99276
VP1 isolate hul5 AAS99265
VP1 isolate hul6 AAS99266
VP1 isolate hul8 AAS99268
VP1 isolate hu7 AAS99313
VP1 isolate hul0 AAS99261
VP1 isolate hull AAS99262
VP1 isolate hu9 AAS99314
VP1 isolate hu53 AAS99300
VP1 isolate hu55 AAS99302
VP1 isolate hu54 AAS99301
VP1 isolate huS17 AAU05370
AAV2 VP1 AACO3780
VP1 isolate hu34 AAS99283
VP1 isolate hu35 AAS99284
VP1 islolate hu51 AAS99298
VP1 isolate hu52 AAS99299
VP1 isolate hu47 AAS99295
VP1 isolate hu45 AAS99293
VP1 isolate hu58 AAS99305
VP1 isolate hu49 AAS99297
VP1 isolate hu56 AAS99303


PFHSMFAHNQ
PFHSMFAHNQ
PFHSMFAHNQ
PFHSMFAHNQ
PFHSMFAHSQ
PFHSMFAHSQ
PFHSMFAHSQ
PFHSMFAHSQ
PFHSMFAHSQ
PFHSMFAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHCQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
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PFHSSYAHSQ
PFHSSYAHSQ
PFHSGYAHSQ
PFHSSYAHSQ
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PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ
PFHSSYAHSQ


TLDRLMNPLV
TLDRLMNPLV
TLDRLMNPLV
TLDRLMNPLV
DLDRLMNPLL
DLDRLMNPLL
DLDRLMNPLL
DLDRLMNPLL
DLDRLMNPLV
DLDRLMNPLV
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLV
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLLNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
GLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLGRLMNPLI
SLGRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI
SLDRLMNPLI


DQYLWAFSSV
DQYLWAFSSV
DQYLWAFSSV
DQYLWAFSSV
DQYLWNFSEV
DQYLWNFSEV
DQYLWNFSEV
DQYLWNFSEV
DQYLWNFNEV
DQYLWNFNEV
DQYLYYLNRT
DQYLYYLNRT
DQYLYYLNRT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNKT
DQYLYYLNRT
DQYLYYLNRT
DQYLYYLNRT
DQYLYYLNRT
DQYLYYLNRT
DQYLYYLNRT
DQYLYYLNKT
DQYLYYLSRT
DQYLYYLSRT
DQYLYYLSRT
DQYLYYLSTT
DQYLYYLSTT
DQYLYYLSTT
DQYLYYLSTT
DQYLYYLSRT
DQYLYYLSRT
DQYLYYLSRT


SQAGS...SG
SQAGS...SG
SQTGS...SG
SQTGS...SG
NGG.......
NGG.......
NGG.......
NGG.......
DSS. ......
DSS. ......
QGTTSGTTNQ
QGTTSGTTNQ
QGTTSGTTNQ
Q.TNSGTLQQ
Q.TNSGTLQQ
Q.TNSGTLQQ
Q.TNSGTLQQ
Q.TNSGTLQQ
Q.TNSGTLQQ
Q.TNSGTLQQ
Q.SNSGTLQQ
Q.SNSGTLQQ
Q.SNSGTLQQ
Q.SNSGTLQQ
Q.SNSGTLQQ
Q.SNSGTLQQ
Q.SNSGTLQQ
Q.TASGTQQ.
Q.TASGTQQ.
Q.TASGTQQ.
Q.SASGTVQQ
N.TPSGTTTQ
N.TPSGTTTQ
N.TPSGTTTQ
N.TPSGTTTQ
N.TPSGTTTQ
N.TPSGTTTQ
N.TPSGTTTQ
N.TPSGTTTQ
N.TPSGTTTQ
N.TPSGTTTQ


RALHYS RAT K
RALHYS RAT K
RALNYS RAT K
RALNYS RAT K
RNAQFKKAVK
RNAQFKKAVK
RNAQFKKAVK
RNAQFKKAVK
RNAQFKKAVK
RNAQFKKAVK
SRLLFSQAGP
SRLLFSQAGP
SRLLFSQAGP
SRLLFSQAGP
SRLLFSQAGP
SRLLFSQAGP
SRLLFSQAGP
SRLLFSQAGP
SRLLFSQAGP
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SRLLFSQAGP
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SRLLFSQAGP
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SRLLFSQAGP
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SRLLFSQAGP
SRLLFSQAGP
SRLLFSQAGP
SRLQFSQAGA
SRLQFSQAGA
SRLQFSQAGA
SRLQFSQAGA
SRLQFSQAGA
SRLQFSQAGA
SRLQFSQAGA
SRLQFSQAGA
SRLQFSQAGA
SRLQFSQAGA




Full Text

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1 STUDIES OF THE ADENO-ASSOCIATED VIRUS CAPSID By KIM MARIE VAN VLIET A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Kim Marie Van Vliet

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3 To those individuals who have provided inspira tion for me in leading by example, striving to perform to the highest standard of excellence in whatever work they agree to do while still adhering to the core values of honesty, integrity and ethics

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4 ACKNOWLEDGMENTS This section of my dissertation should probabl y be its own chapter because there are so many individuals who have been instrumental in assisting me with finding the needed elements to complete this work. First, I thank the Vector Core Lab at the Univer sity of Florida. Mark Potter for providing some of the initial virus prep s used for the experiments described in Chapter 4 to map the AAV capsid in solution. When thes e samples were needed, I had already learned how to prepare and purify AAV, but having so me virus samples to start the preliminary experiments allowed me to begin purifying additional virus while the preliminary data was being collected. I thank Tina and Glenn Philipsburg, D eana Sanders, Irina Korytov, Stefanie Shoja and Jenna Potter for their technical advice and training when I firs t arrived. During the course of experiments, numerous technical issues often arose that need to be sorted out at that particular moment, and the availability of a group of AAV production a nd purification specialists who were willing to share their knowledge, as well as equipment, was very much appreciated. I also thank Sergei Zolotukhin and George Aslinidi for their a ssistance, advice and training. Being in the same lab space provi ded the opportunity to watch and learn new techniques, as well as provided the use of th eir equipment for tran sforming electrically competent bacteria, the use of the DNA sequenc ing gel apparatus, the Bio-Rad econo-FPLC system, as well as other equipment that comes st andard in most labs, lik e a pH meter. I also learned that even though they both speak the Ru ssian language, Ukrainians and Russians are not the same. This is especially true during World Cup Soccer season. GOOOOOOOOOOOAAAA AAAAL!!!!!!! I thank Dr. Greg Erdos and the Interdiscip linary Center for Biotechnology Research (ICBR) Electron Microscopy Core Lab for the us e of the electron microscope. I thank Karen

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5 Kelley and Lynda Schneider for electron microsc opy training, as well as for shooting publication quality images of my grids. I thank the ICBR Proteomics Core Lab for mass spectrometry and protein sequencing. I thank Stanley Stevens and Scott McClung for the mass spectrometry work described in Chapter 4. I thank Sofie Alvarez, Scott McClung and Cindy Croft for the great scientific environment in the ICBR Proteomi cs Core Lab and assistance with the mass spectrometry work described in Chapter 5. I th ank Li Liu for bioinformatics assistance with setting up a protein database that will be useful in the future. I thank Dr. Barry Byrnes Lab for sharing the liquid nitrogen dewar fo r cryo-storage of our cell lines, their plate reader for determining the c oncentration of purified proteins, as well as for allowing me to use the incubators and tissue cult ure hoods in their lab. I th ank Denise Coutier for allowing me to use their rotor for the ultracentrifuge to purif y AAV on cesium chloride. I thank Dr. Terry Flottes Lab for allowing me to use their spectrophotometer, especially after the Muzyczka Lab and their equipment moved to the Genetics Institute. I thank Kevin Nash for allowing me to use the do t/slot blot apparatus as well as for digging through the -80 C freezer to find the constructs used in this wo rk. I thank both Kevin Nash and Wei Jun for allowing me to temporarily borrow equipment that was needed for this work. The Muzyczka lab functioned as a supply center fo r this work as most protocols that were performed are done routinely in th eir lab and they were usually the first stop in the game of scientific Go Fish, which starts with the words Do you have a ______. I thank Bert Flanagan, and Brian ODonnell for providing me access to the polio virus room, as well as allowing me to incubate my ssDNA virus in the same room as their RNA virus work. The use of several incubators at various te mperatures was very useful for the limited data that was generated for the temperature sensi tive mutants of AAV. This also corrected the

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6 problem of CO2 lines blowing off incubators, th e temperature mysterious ly being increased, and other issue that occurred in prior shared spac e. The polio virus room is a limited access room, and interestingly my streak of bad luck ended when I moved my sensitive experiments to their tissue culture room. Special thanks to Greg Tyle r for providing access to the microscope with the camera in the Genetics Institute to image cells transduced with AAV vectors harboring the gene for green fluorescent protein. A pictur e really is worth a thousand words. I thank the members of the Snyder Lab past and present, Shalini Ahnand, Traci Mayfield, Veronique Blouin, Imran Mohiuddin, and Yasmin Mohiuddin. Special th anks to Veronique Blouin for her scientific curiosity and the gift of gab, particular ly for informing me that the AAV4 virus that she sent to me had a mutation, wh ich everyone else was aware of except for me. I was asked to expand the number of serotypes fo r AAV capsid serotype identification to include AAV4 and AAV8, in addition to AAV1, AAV2, and AAV5. The mutation in the AAV4 virus was in a basic residue and these amino acids we re critical for the experiment that I was performing. After I gave my AAV8 prep to Nico le Brument for her use when she arrived, and spent my time and efforts training her on how to produce AAV8, I thank her for proving an aliquot of AAV8 for this work. I thank Mavis Agbandje-McKenna for providing scientific discussions as well as serving as a co-mentor. I thank her also for providing a highly pure, highly concentrated sample of wildtype AAV4 when I learned that the AAV4 that I received from Veronique had a mutation. I was expecting to search through crys tal trays for AAV4 samples that did not crystallize or samples that partially crystallized that could be rehydrated and instead was given a pure sample of wild type AAV4 which is a very valuable reagent. I also thank Lakshmanan Govindasamy for his training and advice, as well as Hyun Joo Nam. I thank the members of the McKenna Lab both

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7 past and present for the collegial atmosphere and scientific envi ronment that their lab afforded. Mavis for tolerating my lit reviews arriving the mi nute before they were due, and Rob McKenna for providing a critical review of my work as well as insightful discussions regarding capsid assembly and sharing his knowledge of bacteriophage X174 assembly. I thank Nick Muzyczka for providing techni cal advice, for providing precise concise information regarding this work, as well as prov iding insightful discussions that guided this work. I thank Jorg Bungert for his time and advi ce. I thank Richard Snyder for the resources to perform this work, as well as for th e opportunity to work in his lab. I acknowledge the great support that I have r eceived from the Department of Molecular Genetics and Microbiology, particul arly Joyce Conners, the best gr aduate student support person ever, and for always having my back. Special thanks to the fiscal office for helping me to get the reagents that I needed for this work, particular ly Michele Ramsey, Jeanine Spencer, and Elisha Richey. I thank Carolyn Baum for her assistance w ith ordering reagents, as well as helping me to find the appropriate people to handle various things that I needed to get done. I thank my mentor, Dr. Michael Roner of the Un iversity of Texas, for providing advice and helping to guide my decisions. For never sayi ng I told you so, as well as for standing by me through the really brill iant experiments and the other ones. Special thanks to the men of Bio-Rad, Al Gu arino (aka Alberto) and Scott Moore (aka Alejandro), for providing numerous reagents and equipment th at we purchased, as well as that we borrowed for various aspects of this work. I also thank Scott Jameson from Invitrogen for providing reagents for this work, and for providing more when I hadnt quite decided if I liked the first set. I also thank Steve Muir for providi ng reagents and samples used in Western blotting

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8 applications from Pierce Biotec hnology early in this work, as well as samples from Millipore used later in this work. Finally, I thank my parents, family and friends for always being supportive of my decisions, even when financially they dont make very much sense. I thank them for always being there when I needed to vent, as we ll as for understanding my busy schedule.

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9 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ........11 LIST OF FIGURES................................................................................................................ .......12 ABSTRACT....................................................................................................................... ............16 CHAPTER 1 INTRODUCTION..................................................................................................................18 The Family Parvoviridae........................................................................................................18 The AAV Genome................................................................................................................. .20 Vectors........................................................................................................................ ............21 2 MODELS OF AAV CAPSID ASSEMBLY...........................................................................24 Capsid Structure............................................................................................................... .......24 Capsid Assembly and Packaging............................................................................................25 Virus Assembly for ssDNA Viruses.......................................................................................27 Assembly via a Pentam eric Intermediate...............................................................................28 Assembly via a Trimeric Intermediate...................................................................................29 3 AAV-2 ASSEMBLY AND SUBUNIT INTERACTIONS....................................................35 Introduction................................................................................................................... ..........35 AAV-2 Mutant Studies...........................................................................................................48 Amino Acid Alignment..........................................................................................................49 Modeling....................................................................................................................... ..........51 The Mutants.................................................................................................................... ........52 Mut 19 228 232 WHCDS WACAS...........................................................................52 Mut24 291 -295 FSPRD FSPAA.................................................................................58 Mut26 320 324 VKEVT VAAVT.............................................................................64 Mut33 469 472 DIRD AIAA.....................................................................................69 Mut46 681 683 EIE AAA..........................................................................................73 Methods for Mutant Studies...................................................................................................78 4 STUDIES OF THE AAV CAPSID IN SOLUTION .............................................................86 Introduction................................................................................................................... ..........86 Proteolytic structural mapping........................................................................................86 Trypsin digestion of AAV-2............................................................................................87

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10 Fine mapping of the trypsin cleavage site.......................................................................89 Trypsin-Treated Virons Remain Intact............................................................................92 5 MASS SPECTROMETRY FOR AAV CAPSID SEROTYPE IDENTIFICATION...........110 AAV-CSI Introduction.........................................................................................................110 Results and Discussion.........................................................................................................115 Materials and Methods.........................................................................................................123 Viruses and cell lines.....................................................................................................123 Protein gel electrophoresis............................................................................................124 Mass Spec In-gel digestion protocol.............................................................................124 Mass spec protocol........................................................................................................125 Database Searching.......................................................................................................126 Criteria For Protei n Identification.................................................................................126 APPENDIX: AAV CAPSID ALIGNMENT...............................................................................127 LIST OF REFERENCES.............................................................................................................179 BIOGRAPHICAL SKETCH.......................................................................................................193

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11 LIST OF TABLES Table page 2-1 Parvovirus subunit association energies............................................................................29 3-1 The five strongest interactions at th e icosahedral 5-fold axis of symmetry......................36 3-2 Specific amino acid interactions at th e icosahedral 5-fold axis of symmetry....................36 3-3 The five strongest interactions at th e icosahedral 3-fold axis of symmetry......................39 3-4 Specific amino acid interactions at th e icosahedral 3-fold axis of symmetry....................39 3-5 The five strongest interactions at th e icosahedral 2-fold axis of symmetry......................45 3-6 Specific amino acid interactions at th e icosahedral 2-fold axis of symmetry....................45 3-7 pIM45 based plasmids for the production of mutant AAV-2 capsids...............................49 3-8 Amino acid alignment data for the ami no acid sequence in the region of Mut33.............49 3-9 Parvovirus structural capsid protein alignment for Mut19 residues..................................50 3-10 Parvovirus structural capsid protein alignment for Mut24 residues..................................50 3-11 Parvovirus structural capsid protein alignment for Mut26 residues..................................50 3-12 Parvovirus structural capsid protein alignment for Mut33 residues..................................50 3-13 Parvovirus structural capsid protein alignment for Mut46 residues..................................51 3-14 Evaluation of the effect of the muta tions in Mut24 on amino acid interactions................64 3-15 Evaluation of the effect of the muta tions in Mut26 on amino acid interactions................69 3-16 Evaluation of the effect of the muta tions in Mut33 on amino acid interactions................73 4-1 Predicted AAV VP1 tryptic fragment mass from cleavage in the G-H loop.....................91 5-1 AAV Serotype Amino Acid Identity Table for AAV Serotypes 1 11..........................114

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12 LIST OF FIGURES Figure page 1-1 The single-stranded DNA genome of AAV-2...................................................................21 2-1 The structure of a monomeric subunit of AAV-2, as determined by Xie, et al.................25 2-2 Icosahedral virus partic le diagram from VIPERdb............................................................28 2-3 Model of Parvovirus Assembly th rough a Pentameric Intermediate.................................30 2-4 Model of Parvovirus Assembly through a Trimeric Intermediate.....................................32 3-1 Residues at the five fold symmetr y axis that are changed in Mut19.................................53 3-2 Residues at the two fold symmetry axis that are changed in Mut19.................................54 3-3 The predicted subunit as a result of th e mutations made in Mut19 is a trimer..................55 3-4 Residues that may be responsible for the phenotype of Mut19.........................................55 3-5 Intermolecular residues that play a role in the phenotype of Mut19.................................57 3-6 Asp231 has been changed to Ala231, as in Mut19............................................................57 3-7 Residues at the 5-fold symmetry axis that are changed in Mut24.....................................59 3-8 Residues at the 2-fold symmetry axis that are changed in Mut24.....................................60 3-9 The predicted subunit as a result of th e mutations made in Mut24 is a trimer..................61 3-10 Intermolecular and intramolecu lar residues that are disrupted..........................................62 3-11 Mut24 interactions that are effected by the mutation........................................................63 3-12 Residues at the 5-fold symmetry axis that are changed in Mut26.....................................65 3-13 Cut-away view of the 5-fold axis of symmetry.................................................................66 3-14 Interactions with the residues involved in Mut26..............................................................67 3-15 Model of Mut26 after the mutation....................................................................................68 3-16 Mut33 pentamer............................................................................................................ .....70 3-17 Mut33 residues............................................................................................................ .......71 3-18 Interactions in th e region of Mut33...................................................................................72 3-19 Model of residues mutated in Mut33.................................................................................72

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13 3-20 The location of residues that are mutated in Mut46..........................................................74 3-21 Intramolecular interactio ns with the residues that are changed in Mut46,........................75 3-22 Intramolecular interactio ns after the residues in Mut46 have been changed.....................76 3-23 Transfection of Mut 33.................................................................................................... ..82 3-24 Green cell assay of transfections at 32 C.........................................................................83 3-25 Transfections at 37 C.................................................................................................... ....84 3-26 Green cell assay after transfection at 39.5 C....................................................................85 4-1 Western blot of several AAV-2 preps................................................................................89 4-2 Tryptic mapping of full AAV-2 capsids............................................................................90 4-3 Trypsinized AAV-2 virions remain intact.........................................................................93 4-4 Proteolysis distinguishes full and empty AAV-2 particles................................................97 4-5 AAV-2, AAV-1 and AAV-5 capsids can be distinguished proteolytically.......................99 4-6 AAV-2, AAV-1, and AAV-5 have different susceptibility to Chymotrypsin.................101 4-7 Capsid structure........................................................................................................... ....102 4-8 AAV-1, AAV-2 and AAV-5 Homology models.............................................................104 5-1 Phylogenetic Tree of AAV Serotypes 1 11...................................................................115 5-2 Coomassie stained samp les for mass spectrometry.........................................................116 5-3 Coomassie stained samples for mass spectrometry identification...................................118 5-4 Mass spec data analyzed using the software program Scaffold.......................................119 5-5 Mass spectrometry data for K544E AAV-4 mutant and wt AAV-4................................122

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14 LIST OF ABBREVIATIONS A Acidic amino acid interaction Angstrom AAV Adeno-Associated Virus AAV-CSI Adeno-Associated Virus-Ca psid Serotype Identification B Basic amino acid interaction CPV Canine Parvovirus Cryo-EM Cryo-Electron Microscopy cs cold-sensitive DI particle Defective Interfering Particle DNA Deoxyribonucleic acid FPV Feline Parvovirus H Hydrophobic amino acid interation hepheparin negative, viru s is unable to bind heparin hep+ heparin positive, virus binds heparin hs heat-sensitive lip mutant Low infectious particle phenotype MS Mass Spectrometry Mut Mutant MVM Minute Virus of Mice ni non-infectious P Polar, uncharged amino acid interaction pd partially defective POTs Pentamers of Trimers PPV Porcine Parvovirus

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15 TOPs Trimers of Pentamers VIPERdb Virus Particle Explorer Database wt wild-type

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16 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STUDIES OF THE ADENO-ASSOCIATED VIRUS CAPSID By Kim Marie Van Vliet August 2007 Chair: Richard O. Snyder Major: Medical Sciences Mol ecular Genetics an d Microbiology Adeno-associated virus (AAV) is a promising ge ne transfer vector that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The AAV capsid plays an essential role in cell binding, internalization, and trafficking, which are critical processes for gene delivery to specific cells. The structural proteins of the capsid spontaneously self-assemble into preformed shells, and then DNA packaging occurs. Although the viral capsid pr oteins self-assemble, little is known about this process. Potential subunits of assembly have been isolated by other investigators and this data was utilized to develop the two general m odels for Parvovirus assembly discussed here. Specific interactions that co mprise the AAV-2 capsid subunit in terfaces are analyzed with respect to several capsid muta nts and their impact on the formation of a macromolecular structure. A better understanding of specific inte ractions required for AAV capsid assembly will allow for the development of customized targeting AAV vectors in the future. The AAV capsid is remarkably stable, which is a desirable characteristic for gene therapy vectors; however, the work presented here dem onstrates that AAV serotypes exhibit differential susceptibility to proteases. The capsid fragmentat ion pattern upon limited proteolysis, as well as the susceptibility of the serotype s to a series of proteases, provid es a unique fingerprint for each

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17 serotype that can be used for capsid identity va lidation. In addition, prot ease susceptibility is utilized to study dynamic structural changes of intact capsids in solution. A high-throughput method for AAV capsid serotype identification ( AAV-CSI) utilizing denatured capsid proteins was also developed. This will be useful to verify customized gene therapy vectors that will be produced in the future, which may contain components of more than one AAV serotype.

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18 CHAPTER 1 INTRODUCTION The Family Parvoviridae Adeno-associated virus (AAV) is a member of the family Parvoviridae The family Parvoviridae is divided into two subfamilies, Densovirinae which are parvoviruses that infect arthropods, and Parvovirinae which are parvoviruses that infect vertebrates. The subfamily Parvovirinae is further subdivided into five genera: 1) Parvovirus which includes MVM and CPV 2) Erythrovirus which includes B19 3) Amdovirus which includes Aleutian Mink Disease Virus (AMD) 4) Bocavirus which includes Bovine Parvovirus (BPV), Minute Virus of Canines (MVC) and human Bocavirus (HBoV) [1], and 5) Dependovirus which includes AAV. A newly discovered parvovirus, parv4, has been shown to be unrelated to the known parvoviruses, and may become a new genus [2]. The recently discovered parvoviruses, parv4 and HBoV, are considered emerging infectious pathogens and like B19 are implicated in disease in humans. Unlike parv4, HBoV and B19, AAV is not associat ed with disease in humans. Dependoviruses, like AAV are unique in that the virus is rep lication deficient and except under special circumstances, it requires a helper virus, such as adenovirus or herpes virus for replication [3]. AAV is currently being utilized for gene ther apy applications and ha s been well tolerated in human clinical trials. Add itional features of AAV vectors that make them promising gene therapy vectors include: 1) Stable long-term expre ssion of transgenes in several tissues including the lung, liver, brain, muscle, vacular endothelium and hematopoietic cells [4-11]. 2) Ability to transduce both dividing and non-dividing cells [ 12]. 3) Episomal maintenance: wild-type AAV exhibits site-specific integration of its genome into chromosome 19; however, the majority of vector genomes appear to be maintained episomally [13-15]. Therefore, the risk of integration is minimal as compared to retroviral vectors that require integration for gene expression, exhibit

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19 random integration into the host genome, and ha ve the potential to activ ate proto-oncogenes [1618]. 4) Low immunogenicity in animal models recently, it was reported that AAV2 vectors can activate functional cytotoxic CD8+ T cells specific to the AAV2 capsid in mice after intramuscular injection [19, 20]. Rece nt clinical trial data suggests that T cell mediated immunity to AAV capsid antigens caused de struction of AAV-2 transduced hepatocytes in humans [6, 21]. Studies in mice have shown that AAV-2 capsids are able to activat e cytotoxic T cells but did not render the transduced hepatocytes as effective cyto lytic targets [22]. This suggests that currently available animal models may not be sufficient for studying th e immune response to the AAV capsid that has been shown to occur in human patients. In addition, humoral immune responses are generated to the AAV capsid that may result in viral neutralization [21]. There are 11 known serotypes of AAV with nearly 100 recently discov ered genotypic variants [23-27]. The use of other serotypes of AAV may circum vent the humoral response and allow for repeated treatments if necessary [28]. However, im muno suppressive therapy may be needed during treatment with AAV to mitigate the host immune response in hu man patients, as well as animals[29]. Recent data in a canine model of Duchenne Muscular Dy strophy demonstrated that a brief course of an immunosuppressive regimen was well tolerate d and sufficient to permit sustained AAVmediated dystrophin expression [30]. A recent st udy evaluating the immu ne response to AAV-8 expressing F.IX in rhesus macaques, the na tural host for AAV-8, evaluated preexisting neutralizing antibodies to AAV-8, a standard T-cell immunosuppressive regimen, as well as efficacy and safety of administration. This st udy showed that low titers of preexisting antibody abrogate transduction, and the use of an immuno suppressive regimen did not induce toxicity, or impair AAV transduction or F.IX synthesis [29] AAV vectors may also be useful for providing long term gene expression in immunocompromi sed hosts, which is an advantage over other

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20 vector systems. 5) Physiochemical stability: AAV virions are highly stable over a wide range of pH and temperature, a feature that is importa nt for production and purification methods for clinical grade AAV vectors, as well as for stability of the final vector product [31]. The AAV Genome The genome of AAV-2 is composed of 4679 ba ses of linear, single-stranded DNA. There are two genes, rep and cap which are flanked by the inverted terminal repeats, ITRs. The ITRs consist of 145 nucleotides on the 3 end and 5 end of the genome, which due to base pairing, forms a Y or a T-shaped structure. Th ese are the only sequences required in cis for viral DNA replication and packaging. The rep gene encodes four non-stru ctural proteins, Rep78, Rep68, Rep52 and Rep40, which play a role in viral genome replication, transcrip tional regulation [32], as well as packaging. Rep78 and Rep68 are transl ated from mRNAs transcribed from the p5 promoter, while Rep52, and Rep40 are derived fro m mRNAs transcribed from the p19 promoter. Alternative splicing replaces a 92 amino acid C-te rminal element in Rep78 and Rep52 with a 9 amino acid element in Rep68 and Rep40 [33]. The cap gene encodes the three structural proteins of the AAV capsid, VP1 (87 kDa), VP2 (72 kDa), and VP3 (63 kDa) translated from the p19 promoter. Differential splicing yi elds major and minor spliced pr oducts. VP1 is translated from the minor spliced mRNA, yielding less VP1 protei n. VP2 and VP3 are both translated from the more abundant major spliced mRNA; however, VP 2 is translated less efficiently because it initiates at an ACG codon, while VP3 is translated very efficiently because of a favorable Kozak context [34]. As a result, the AAV capsid proteins which differ only in their N-terminal region, are present in the mature virion in a ratio of 1:1:10 (VP1:VP2:VP3). The single stranded DNA genome of AAV and its products ar e depicted in Figure 1-1.

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21 Figure 1-1. The single-stranded DNA genome of AAV-2. The inverted terminal repeats (ITRs) flank the two open reading frames rep and ca p. The rep gene encodes four nonstructural proteins, Rep78, Rep68, Rep52 and Rep40. The cap ge ne encodes three structural proteins VP1, VP2 and VP3. The location of the promoters, p5, p19, and p40 are depicted by arrows. Vectors Three features of AAV-2 which may limit its use as a gene therapy vector include: 1) its small size, with an ability to package approxi mately 4.7 kb, 2) the difficulty and expense of production of large quantities of high titer virus for clinical trials, and 3) the wide range of tissue tropism of AAV-2, as a result of using heparin su lfate proteoglycan as a receptor, which could result in expression of the transgene in non-target tissues. Studies have ad dressed the small size of the AAV capsid and an evaluation of the pack aging limits of AAV has shown that the AAV capsid is capable of packagi ng and protecting DNA up to 6.0 kb; however, post-entry, capsids with DNA larger than the AAV genome are prefer entially degraded by the proteosome. It has been shown that proteosome inhibitors can be used to overcome this barrier [35]. The p5 p19 p40 ITR ITR Rep Cap 87 kDa 72 kDa 63 kDa PolyA VP1 VP2 VP3 Rep78 Rep68 Rep52 Rep40

PAGE 22

22 development of self-complementary vectors in or der to introduce larger DNA sequences than can be packaged in a single AAV capsid has provide d a method to overcome the packaging limit of the AAV capsid [36-38]. This provides a mech anism to potentially expand the number of diseases that AAV may be utilized to treat. Improved production methods for AAV have resu lted in the ability to produce the large amounts of virus required for clinical trials. Al though virus for clinical trials is currently produced by transient transfec tion of monolayers of cells, im proved production methods have allowed for AAV to be produced in other systems such as baculovirus and herpes virus [39], which provide scalability, and will allow for virus to be produced with higher titers and greater yields [40-45]. Studies have al so evaluated the natural tropism for AAV serotypes, as well as specific targeting of AAV vectors. A greater understanding of the AAV capsid structure, and the interaction of the subunits that assemble to form the capsid, as well as th e regions of the capsid surface that play a role in the various stages of the life cycle of the virion, including attachment to the cellular receptor, intrace llular trafficking, as well as sp ecific requirements for viral packaging, will aid in the production of customized and improved viral vect ors, with higher titers and specific targeting. The chapters that follow provide an in de pth analysis of the AAV capsid, beginning with parvovirus capsid assembly models (Chapter 2) as well as data regarding several AAV capsid mutants (Chapter 3) and their phenotypes. Th e 11 capsid serotypes of AAV exhibit different tissue tropism and different trans duction efficiencies. Chapters 4 a nd 5 provide methods to verify the AAV capsid of the final vector product that will be admininstered to patients. Intact AAV capsids were evaluated in solution using limite d proteolysis to determine the AAV serotype based on the fragmentation pattern [46]. This met hod was also used to evaluate the structural

PAGE 23

23 dynamics of the virion (Chapter 4). Chapter 5 describes AAV Capsid Serotype Identification (AAV-CSI). Denatured AAV capsids are subjected to proteolysis and the AAV capsid serotype is determined based on the fragmentation pa ttern. This provides a high throughput method for capsid identity testing. Overall, the data pres ented provides a biophysical, biochemical, and genetic analysis of the AAV capsid.

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24 CHAPTER 2 MODELS OF AAV CAPSID ASSEMBLY Capsid Structure The adeno-associated virus capsid is approximate ly 25 nm in diameter and is composed of 60 subunits arranged in T=1 icosahedral symmetr y. Because AAV has a number of features that make it attractive as a gene tr ansfer vector, many studies have focused on the basic biology of the virus, including studies that address the structural characteristics. Cryo-EM or crystal structures for AAV-2, AAV-4, and AAV-5 have b een determined [47-51], and the crystal structure for AAV-8 is currently in progress [45]. Dependoviruses share the same capsid subunit fold as the other members of the family Parvoviridae including the insect densoviruses, and the autonomously replicating parvoviru ses such as canine parvovirus (CPV) and minute virus of mice (MVM), even though AAV shares low capsid primary sequence identity (7-22%) [52-54]. The monomeric subunit of AAV has a conserved -barrel core that is common in viral capsid proteins. The structure of a monomeric subunit of AAV-2 as determined by Xie, et al. [48] is shown in Figure 2-1. The motif is an eight-stranded anti-parallel -barrel motif (jelly-roll -barrel), with the beta strands labeled B-I [52]. The -strand labeled A is also presen t in all parvoviruses, including AAV. AAV has long loop insertions between the strands of the core -barrel that are labeled according to the beta strands that they flank. These long interstrand loops contain beta ribbons and elements of secondary structure that form much of the outer su rface features of the AAV capsid. The GH loop is the longest in terstrand loop and three VPs interact extensively at each icosahedral 3-fold axis of symmetry forming a pr ominent spike. Five DE loops from each viral protein form antiparallel beta ribbons at the 5-fold axis of symmetry that results in a cylindrical

PAGE 25

25 A B D C E F G H I B I DB C 1C0 EF 2G H 3G H 2GH12 G H 1 3 A B D C E F G H I B I DB C 1C0 EF 2G H 3G H 2GH12 G H 1 3 Figure 2-1. The structure of a monomeric subunit of AAV-2, as determined by Xie, et al. [55]. The beta ribbons are depicted in black a nd labeled A through I, and helices are shown in grey. This image was produced using the AAV-2 coordinates from the Protein Databank, (PDB Accession #1lp3), with th e molecular modeling software PyMOL (www.pymol.org ) provided by DeLano Scientif ic, Palo Alto, CA [56]. structure that surrounds a canyon-like depression. At the 2-fold axis of symmetry there is a small depression, often referred to as the 2-fold di mple [57, 58]. Analysis of newly discovered AAV genotypes identified a total of 12 hypervariable regions on the AAV capsid [59]. Overlaying these regions onto the X-ray crystallographic m odel of AAV-2 showed that these regions are exposed on the capsid surface. Most of the va riability is located between the G and H strands, which are implicated in the formation of the vall ey at the icosahedral 3-fold axis of symmetry and the protrusions that surround it. These surface features of the virus are responsible for the interactions of the capsid with cellula r receptors, as well as antibodies. Capsid Assembly and Packaging In 1980, Myers and Carters work provided evid ence that the structural proteins for AAV assemble into empty capsids, and then the genom e is packaged into these preformed capsids

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26 [60]. Pulse-chase experiments showed that empty particles rapidly accumu late (10 20 minutes), but that mature full virions accumulated more sl owly (4 to 8 hours). They also showed that the number of empty viral particles decreases at the same rate as the number of DNA-containing mature virions increases over the course of in fection. Additionally, de la Maza and Carter showed that DI particles, AAV genomes with de letions, are packaged in to apparently normal capsids, indicating that full-length viral genom ic DNA is not required for the assembly or structural integrity of the AAV capsid [61]. In the absence of capsid assembly, ssDNA does not accumulate, further suggesting that empty capsids fo rm first. Interactions between the preformed AAV capsid and Rep52 provides a mechanism where th e nonstructural protei ns helicase activity inserts the viral DNA [62]. Data based on MVM and AAV suggest that the DNA is inserted into the capsid at the 5-fold pore [63, 64]. Within the cell, capsid assembly occurs at centers within the nucleus where Rep proteins, capsid protei ns and DNA are co-localized [65]. Empty AAV capsids can be produced by expressing the AAV cap gene in insect cells using a baculovirus expression system [66] or in mammalian cells utilizing a recombinant adenovirus expressing the AAV capsid proteins. These systems have advanced structural studies of the AAV capsid as a result of the large amount of empty capsids or virus-like particles (VLPs) that can be produced. Studies of AAV assembly have demonstrated that VP3 alone is sufficient to form VLPs [67], but VP1 is required for infectivity [68]. Subsequently it was shown that the unique N-terminus of VP1 has phospholipase A2 activity and contains a nuclear localization signa l (NLS) [69]. Studies have demonstrated that the unique N-terminus of VP 1 is inside the capsid near the 2-fold axis of symmetry and becomes externalized through the 5-fold pore during viral trafficking in the cell [70]. The N-terminus of VP2 also has a NLS and may play a role in transporting VP3 into the nucleus; however, it has been shown that the N-terminus of VP2 is nonessential and that

PAGE 27

27 infectious virus can be produced that lack VP 2 entirely [71]. Additiona lly, the N-terminus of VP2 has been replaced with green fluorescent protein and these capsids still assemble and maintained infectivity. This demonstrates that VP2 can tolerate peptide insertions and may be useful for incorporating peptides into the cap sid for cell-specific ta rgeting of AAV. Virus Assembly for ssDNA Viruses Spontaneous assembly of macromolecular stru ctures such as a virus capsid is a process that is not clearly understood. For a T=1 icosah edral capsid, such as AAV, 60 subunits must come together in the correct orientation to provi de a stable capsid, and AAV assembles with high fidelity. There are features on th e surface of the AAV capsid that distinguish the icosahedral 5fold, 3-fold or 2-fold axis of symmetry, for exam ple, the 5-fold pore, the 3-fold mounds or the 2fold dimple. These regions on the capsid are impo rtant for various steps in the virus life cycle. The 3-fold mounds are implicated in receptor bi nding, and the 5-fold pore has been implicated in both externalization of the unique N-terminus of VP1 as we ll as DNA packaging. In vitro studies have identified a number of potential assembly intermediates [60, 72]. Studies of CPV structure estimating the free en ergy of association have revealed that the formation of 3-fold and 5-fold contacts likely takes precedence over 2-fold interactions [52, 53]. For AAV-2, the crystal structure is known, and the individual am ino acid interactions at the interface between subunits have be en analyzed and will be discussed further in Chapter 3. The strongest interactions for AAV-2 are at the 3-fold axis of symmetry, followed by the 5-fold axis of symmetry, and the weakest inte ractions are at the 2-fold axis of symmetry. The image in Figure 2-2 depicts an icosahedral virus partic le (taken from VIPERdb) [73]. The subunit association energies for AAV-2 and other parvoviruses ar e shown in Table 2-1. This data verifies that the subunit association ener gies for other parvoviruses for the 5-fold, 3-fold and 2-fold interactions are comparable (data from VIPERdb) [73].

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28 The potential intermediates of assembly for AAV could include pentameric subunits, 12 of which must come together to make an intact capsid, trimeric subunits, 20 of which comprise an intact capsid, or dimeric subunits 30 of which must come together to produce an intact capsid. The two favored hypotheses for intermediates in assembly are: 1. Assembly via a pentameric intermediate or 2. Assembly via a trimeric intermediate. Assembly via a Pentameric Intermediate If a pentameric intermediate is the asse mbly intermediate, pentamers form first (intrapentameric interactions), and then interpentameric inte ractions form and subsequent pentamers are added. This model requires a trimer of pentamers or TOPs, to produce the 5-fold, 2-fold and 3-fold interactions. In X174, a ssDNA bacteriophage, the first detectable intermediates in assembly are pentamers of the F protein [74]. If the mechanism of assembly for ssDNA viruses is conserved, the hypothesis rega rding AAV assembly is that pentamers form Figure 2-2. Icosahedral virus part icle diagram from VIPERdb [73] The 5-fold interactions are the interactions between A1 and A2, the 2-fold interactions are the interactions between A1 and A6, and the 3-fold interac tions are the interactions between A1 and A7.

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29 Table 2-1. Parvovirus Subunit Association Energies expressed in kcal/mol and Buried Interface Surface in 2, calculated in VIPERdb [73]. For the parvoviruses listed in this table, the strongest interactions occur at the icos ahedral 3-fold axis of symmetry, followed by the icosahedral 5-fold axis of symmetry, followed by the weakest interactions, which are at the icosahedral 2-fold axis of symmetry. Protein Databank accession numbers are listed in parenthese s below the virus abbreviation. 5-fold (A1:A2) Association energies kcal/mol 2-fold (A1:A6) Association energies kcal/mol 3-fold (A1:A7) Association energies kcal/mol 5-fold Buried Surface ( 2) 2-fold Buried Surface ( 2) 3-fold Buried Surface ( 2) AAV-2 (1lp3) -98.0 -64.0 -209.0 4961.0 3127.0 10437.0 CPV (2cas) -95.0 -76.0 -233.0 4654.0 3873.0 10899.0 MVM (1mvm) -97.0 -73.0 -218.0 4875.0 3749.0 10614.0 FPV (1fpv) -98.0 -78.0 -236.0 4793.0 3908.0 10971.0 PPV (1k3v) -94.0 -76.0 -235.0 4577.0 3914.0 11153.0 first, then the 3-fold interactions, followed by the 2-fold interactions. Due to the 5-fold symmetry inherent in icosahedral geomet ry, pentamer formation is like ly to be a common feature in initiation of assembly of severa l other small icosahedral viruses. This was suggested to be the case for assembly of bacteriophage MS2 [75], and Norwalk virus [76]. Additionally, for the assembly of blue tongue virus core protein vp3, Gr imes et al. [77] proposed that 5-fold decamers preassemble, and then associate to form the inner capsid. Zlotnick et al. [ 78] also describe the mechanism of capsid assembly for an icosahedra l plant virus as occurring via a pentameric intermediate. Assembly via a pentameric intermediate is shown in Figure 2-3. Assembly via a Trimeric Intermediate If a trimeric intermediate is the assembly intermediate, trimers form first, and then intertrimeric interactions form and then subs equent trimers are added. This model requires pentamers of trimers or POTs, to produce the 5-fol d, 3-fold and 2-fold interactions. There is data

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30 for other parvoviruses such as MV M that suggest that trimers are the intermediate, and that VP1 and VP2 are both capable of translocating into th e nucleolus where virus assembly occurs [79] when singly expressed in transfec ted cells; however, only VP2 is ab le to assemble into capsid by itself. Recent data for MVM suggests that trimer s are actually transported through the nuclear pore complex, not intact capsids [80]. For MVM, there are 2 proteins, VP1 and VP2, present in a ratio of 1:5 in the assembled capsid. Because trim ers are more stable than pentamers and dimers, it is thought that this energetical ly expensive subunit forms firs t and is transported through the nuclear pore. How the stoichiometry of the viral stru ctural proteins (1 VP1: 5 VP2) is maintained in the assembled MVM capsid is un clear. If pentamers are the structural intermediate, as in X174, then the stoichiometry of 1 VP1: 4 VP 2 would approximate the observed 1:5 ratio; however, recent data does not support the hypothesis that MVM assembles via a pentameric Figure 2-3. Model of Parvovirus Assembly through a Pentameric Intermedia te. In this model, monomeric subunits come together to form pentamers, and then 3 of these pentamers come together to form trimers of pentamers (TOPs), and then pentamers add on to form the capsid. This is the favored m odel for Adeno-Associated Virus (AAV) assembly. Monomeric Subunits Pentameric Intermediate Trimers of Pentamers Macromolecular Assembly

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31 intermediate. The model for assembly via a trim eric intermediate is shown in Figure 2-4. A trimeric intermediate for MVM would ensure th at a macromolecular capsid was produced before DNA could be packaged, since the 5-fold pore where DNA packaging occurs, requires pentamers of trimers for its formation. For AAV, if pentamers are the intermediate in assembly and are transported across the nuclear pore complex as pentamers or as dimers of pentamers, with 1 VP1, or 1 VP2 subunit contained in each pentamer, this would be consis tant with a stoichiometry of 1:1:8 (6 VP1: 6 VP2: 48 VP3). This model also would allow for th e N-terminus of VP1 and VP2 to be at the 2 fold axis. For example, in Figure 2-2, VP1 might be subunit A1 or A5, and VP2 might be subunit A6 or A7. Ruffing, et al. [66] showed that coexpr ession of VP1 or VP2 with VP3 in insect cells resulted in accumulation of VP3 in the nucleus. Th is suggests that capsid proteins need to form complexes for efficient nuclear accumulation and pa rticle assembly. If th e intermediate in AAV assembly is a trimer, it is unclear how the st oichiometry of VP1:VP2:VP3 is maintained. Girod, et al. [69] showed th at the unique region of VP1 contains a phospholipase A2 domain (PLA2) that is required for infectivity. While assembly a nd packaging were not affected by mutations in this region, inf ectivity was reduced in these lip mutants [68]. It has also been suggested by Zadori, et al. [ 81] that the PLA2 domain is so metimes exposed, possibly through the 5-fold channel. Recent studies with capsid mutants also support the N-terminus of VP1 or VP2 being exposed through the 5-fold pore [82]. VP 3 is transcribed more efficiently than VP1, or VP2 and it has been proposed that the lack of availability of more significant quantities of VP1 and VP2 results in the ratio of 1:1:10 in intact particles; howev er, a more ordered, less stochastic

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32 Figure 2-4. Model of Parvovirus Assembly through a Trimeric Intermediate. In this model, monomeric subunits come together to form trimers, and then five trimers come together to form pentamers, and then pentamers add on to form the capsid. This is the favored model for Minute Virus of Mice (MVM) assembly. mechanism for assembly would ensure that th e particles that are produced contain VP1 and would result in the formation of infectious part icles. Alternatively, for every particle produced, approximately 1 in 100 is infectious; therefore, the incorporation of VP1 and VP2 in assembled particles might be a stochastic process, as has been suggested. For AAV, since pentameric interactions require less energy to form, these would be favored over trimers. If the virus were to assemble its most expensive and most stable subu nit first, it would risk using its resources to build subunits and potentially neve r attain an intact capsid. Additionally, trimers, as the most stable subunit, should be easy to isolate if they were the intermediate of assembly, even though they are short-lived species. If pentamers ar e the intermediate of assembly for AAV, this suggests that the site on the surface of the capsi d where the nonstructura l Rep protein interacts for packaging must either not be present in pent americ intermediates or perhaps is present but some cellular protein that is pa rt of the packaging complex is not available at this step. Monomeric Subunits Trimeric Intermediate Pentamers of Trimers

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33 In terms of building a macromolecular structur e, both of the models, either pentamers of trimers (POTs), or trimers of pentamers (TOPs) demonstrate that the important interactions for achieving an intact capsid are the two-fold interactions. Although th ey are the weakest interactions, there are many of them In addition, when either the pe ntamer or the trimer is added, these weak two-fold interactions would enable a new subunit to attach, deta ch, reattach, in effect allowing it to be sampled so that it is added in the correct orientation (like a puzzle piece). The assembly model favored for AAV based on all of th e data is trimers of pentamers (TOPs). This model allows for two pentamers to come together, so intrapentameric interactions form first, then interpentameric interactions second, which are st abilized by the two-fold interactions at the pentameric interfaces, and as the trimer of pentam ers come together, these interactions allow for the energentically expensive three-fold intratri meric interactions. This model of AAV assembly incorporates what is currently known about a ssembly, capsid stability and disassembly. The AAV capsid is stable over a wide range of pH and temperature. It has been shown that upon treating the AAV capsid at 65 C for 30 minutes that its possible to expose the unique Nterminus of VP1, and VP2 [70]. However, upon h eat treatment of the AAV capsid, experiments to date to isolate an assembly intermediate have been unsuccessful. This s uggests that this is an all-or-none phenomenon. The energy required to break the strong inte ractions at the 3-fold axis of symmetry results in complete disruption of the AAV capsid. This is consistant with a pentameric intermediate, where the strongest inte raction at the 3-fold ax is of symmetry would form last, and for capsid disassembly to occur, woul d need to be disrupted first. If trimers are the assembly intermediate, and they form pentamers of trimers, and then the weak 2-fold interactions form last, in a disassembly model, these should be relatively easy to disrupt giving either a

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34 pentameric or trimeric subunit. Computer mode ling data for AAV also supports pentamers as the intermediate in assembly [83].

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35 CHAPTER 3 AAV-2 ASSEMBLY AND SUBUNIT INTERACTIONS Introduction Understanding the capsid subunit interactions that are critical for AAV virion assembly will allow for the production of customized vectors in the future. An analysis of the types of residues located at the 5-fold axis of symmetry, the 3-fold axis of symmetry, as well as the 2-fold axis of symmetry provides a starting point for evaluating residues that are important for virus assembly. The interactions at the 5-fold axis of symmetry are shown in Table 3-1, with the strongest interactions listed first. The specific amino acids involved in the strongest intrapentermic interactions at the 5-fold axis of symmetry are shown in Table 3-2. For the mutants produced by Wu, et al. [84], an analysis of the phenotype when that amino acid has been mutated is also included. The strongest interaction energies for the 3-fo ld axis of symmetry are listed in Table 3-3. The specific residues involved in these interac tions, as well as the phenotype of capsids with mutations in these residues are listed in Table 34. The mutations at the 3-fold axis may provide information regarding assembly and packaging sin ce mutants have been constructed in which the strongest interactions at the 3-fold axis have been disrupted. One of these mutants (Mut31) results in the formation of empty particles but is unable to package DNA. This would be consistent with a conserved mechanism for packaging in small DNA viruses, like X174 where DNA is inserted at the 3-fold, and exits the viri on thru the 5-fold pore. However, due to other mutant studies, the most likely site for DNA packaging for AAV is through the 5-fold pore [85]. The interactions at the 3-fold axis of symmetry are very strong and would represent a significant barrier to packaging. Thes e interactions are probab ly responsible for the re markable stability of

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36 Table 3-1. The five strongest interactions at the icos ahedral 5-fold axis of symmetry (VIPERdb). The amino acids that are the strongest at th e icosahedral 5-fold axis of symmetry are listed, using VP1 numbering. This refers to interactions between subunits A1 and A2 in the icosahedral virus particle in Figure 2-2. The type of inter actions are indicated, B = basic, H = hydrophobic, P= Polar, uncharged. The phenotype of AAV mutants generated by Wu, et al. [84] are listed, wt = wild type. Amino Acid VP1 numbering Type Energy (kcal/mol)Wu, et al. mutant phenotype [84] ARG 404 B -3.76 LYS 665 B -3.57 Mut45, wt PHE 661 H -3.11 TYR 397 P -3.07 MET 402 H -2.14 Table 3-2. Specific amino acid interactions at the icosahedral 5-fold axis of symmetry (VIPERdb). Specific amino acids at the icos ahedral 5-fold axis of symmetry are listed, using VP1 numbering, with the stronge st interactions listed first, and the weakest interactions listed la st. This list include s all interactions between subunits A1 and A2 in the icosahedral virus particle depicted in Figure 2-2. The type of interactions are indicate d, A = acidic, B = basic, H = hydrophobic, P = Polar, uncharged. The phenotype of AA V mutants generated by Wu, et al. are listed, either in the column to the left of the amino aci d list if the mutation was made in the 1st amino acid of the pair listed, or in the column to the right of the amino acid list if the mutation was made in the 2nd amino acid of the pair listed. cs = cold-sensitive, hep+ = heparin positive, hs = heat-sensitive, ni = non-infectious, pd = partially defective, wt = wild-type. Amino acids that were not mutated by Wu, et al. [84], but flank the mutated amino acids are designated unchanged. Type of Interaction Mutant Phenotype (Wu, et al.[84]) VP1 numbering Mutant Phenotype (Wu, et al.[84]) B H B H B P B P B P B P B P R404:V221 R404:G222 R404:S224 R404:S225 R404:N317 R404:Q319 R404:T405 B A Mut45 wt K665:D368 H P H H H H H H F661:Y252 F661:G360 F661:M371 F661:P373

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37 Table 3-2 Continued P H P A P P P P P A P H Y397 :C230 Y397 :D231 Y397 :S232 Y397 :S292 Y397 :D295 Y397:P366 Mut19 unchanged Mut19 ni, no capsid Mut19 unchanged Mut24 unchanged Mut24 ni, no capsid H P H P H P H P H P H P M402:S224 M402:N227 M402:W228 M402:F316 M402:N317 M402:Q677 Mut19 unchanged H H H P H H F392:F365 F392:T713 F392:V714 P P P P P P T337:Q319 T337:N334 T337:T405 H A P399:D231 Mut19 ni, no capsid H H H H H H H P P657:A248 P657:P250 P657:M371 P657:Y673 A H A H A H A H E396:W228 E396:F365 E396:P366 E396:A367 Mut19 unchanged H H H H H P P654:A248 P654:V369 P654:T675 P H P H P P N656:V323 N656:I332 N656:Y673 Mut26 unchanged P B P P P H Mut28 cs Q385:K706 Q385:S707 Q385:V708 Mut48 pd, hep+ P P P P Q259:N709 Q259:T716 H H H H H H F666:A248 F666:V369 F666:M371 B P Mut29 wt R389:Y704 H P A663:Q359 H B H H I670:K321 I670:I332 Mut26 hs

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38 Table 3-2 Continued P P P P T651:T246 T651:Q677 P H P H P H Mut21 unchanged Y257:F365 Y257:A367 Y257:V714 H P H H P652:T246 P652:V369 H H L336:V221 B H Mut21 pd, unstable capsid K258:G718 H H A386:V710 H P V387:Y704 H H Mut21 unchanged L256:G718 H H V221:V221 P B N382:K706 Mut48 pd, hep+ P P P P N326:T329 N326:T331 P H P P P H Y275:V710 Y275:T713 Y275:V714 P P Q401:N227 P B P P N335:K321 N335:N334 Mut26 hs P P S338:Q319 H P Mut18 unchanged A218:N223 H H H H C394:F365 C394:P366 P H S400:W228 Mut19 unchanged P H S662:M371 P P N407:N223 H H F669:V369 P H S390:V710 P P P P T339:S224 T339:N317 H H A655:I332 P H Q341:W228 Mut19 unchanged H H F273:V710

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39 Table 3-3. The five strongest interactions at the icos ahedral 3-fold axis of symmetry (VIPERdb). The amino acids that are the strongest at th e icosahedral 3-fold axis of symmetry are listed, using VP1 numbering. This refers to interactions between subunits A1 and A7 in the icosahedral virus particle in Figure 2-2. The type of inter actions are indicated, B = basic, H = hydrophobic, P = Polar, uncharged. The phenotype of AAV mutants generated by Wu, et al. [84] are listed, wt = wild type. Amino Acid VP1 numbering Type Energy (kcal/mol)Wu, et al. [84] mutant phenotype LYS 692 B -4.88 Mut47, ni, A20(Hartladge, Warrington, Muzyczka, unpublished data) ARG 432 B -3.92 Mut31, ni, empty particle ARG 471 B -3.79 Mut33, hs TYR 441 P -3.44 PRO 602 H -3.07 Table 3-4. Specific amino acid interactions at the icosahedral 3-fold axis of symmetry (VIPERdb). Specific amino acids at the icos ahedral 3-fold axis of symmetry are listed, using VP1 numbering, with the stronge st interactions listed first, and the weakest interactions listed la st. This list include s all interactions between subunits A1 and A7 in the icosahedral virus particle depicted in Figure 2-2. The type of interactions are indicate d, A = acidic, B = basic, H = hydrophobic, P = Polar, uncharged. The phenotype of AAV mutants ge nerated by Wu, et al. [84] are listed, either in the column to the left of the am ino acid list if the mutation was made in the 1st amino acid of the pair listed, or in the co lumn to the right of the amino acid list if the mutation was made in the 2nd amino acid of the pair listed. ala ins = alanine insertion, cs = cold-sensitive, hep= hepari n negative, hep+ = heparin positive, hs = heat-sensitive, ni = non-infectious, pd = pa rtially defective, wt = wild-type. Amino acids that were not mutated by Wu, et al.[ 84], but flank the mutated amino acids are designated unchanged. Type of Interaction Mutant Phenotypes (Wu, et al. [84]) VP1 numbering Mutant Phenotype (Wu, et al. [84]) B P B P B H Mut47 ni, A20K692:Q349 K692:Y397 K692:F398 B P B H B P B B B P Mut31 ni, empty particle R432:Y272 R432:L378 R432:N381 R432:R513 R432:S515 Mut22 unchanged Mut36 pd, hep+ Mut36 unchanged B P B A B P B H Mut33 hs R471:S267 R471:D269 R471:N270 R471:W502 Mut22 ni, full particle Mut22 unchanged

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40 Table 3-4 Continued P B P H P P P H P H P H Y441:R286 Y441:A357 Y441:Q359 Y441:C361 Y441:F542 Y441:P616 Mut23 ni, no capsid H H H H H H H H H B P602:P481 P602:V600 P602:W606 P602:F628 P602:H629 Mut43 unchanged H B H P H B H P H A L583:R484 L583:Q486 L583:R487 L583:T573 L583:E574 H H H H H H H H H H W477:A619 W477:I621 W477:P632 W477:P630 W477:L633 H P H P H P W694:F392 W694:C394 W694:Y397 H P H P H H L437:S276 L437:Q374 L437:G376 H H H P H H L460:V488 L460:N495 L460:I554 Mut39 unchanged P H P H P P P H P H Y443:I541 Y443:G543 Y443:S547 Y443:V552 Y443:V557 Mut38 unchanged Mut39 unchanged H H H H H H H H F462:I541 F462:I554 F462:V557 F462:I559 Mut39 unchanged Mut39 unchanged H P H B H P H P H P V579:Y483 V579:R484 V579:T506 V579:N596 V579:T597

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41 Table 3-4 Continued H H H H H H H H L442:A357 L442:L540 L442:F542 L442:M634 H P H B H P H H I438:Y281 I438:H358 I438:Q359 I438:P373 H H H P H H H H P436:I260 P436:Y272 P436:G376 P436:L378 Mut22 unchanged H H H P H H H H L601:P481 L601:Y483 L601:P521 L601:G599 H H H P H P L445:V488 L445:S501 L445:Q536 B B B P R566:R389 R566:N511 Mut29 wt P P P P P P P P Q584:Q486 Q584:N495 Q584:N496 Q584:Y500 B P B P R475:Y508 R475:S515 Mut36 unchanged A P A P Mut37 ni, full particle D528:N382 D528:N511 P B P P S580:R484 S580:Q486 P H P H P H P H N476:A619 N476:P632 N476:L633 N476:M634 P P P P S458:N495 S458:N497 P A P P P P T448:E499 T448:Y500 T448:S501 P P P H P B P P P H N435:Y281 N435:V353 N435:H358 N435:Y375 N435:G376

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42 Table 3-4 Continued P P P H P P P P S427:T379 S427:L380 S427:S391 S427:Y393 H P H H H H H H L478:Y508 L478:V517 L478:P521 L478:L633 Mut36 unchanged H H L735:P622 H B I698:R389 Mut29 wt H H H P H B V605:P622 V605:T624 V605:H627 Mut43 unchanged Mut43 wt H P H P A590:Q486 A590:Y500 H P H H P479:Y508 P479:L510 H P H B H H M434:S356 M434:H358 M434:L378 B P B P Mut47 ni, A20R693:S391 R693:Y393 P H Q428:P351 P H P P N734:P351 N734:Y393 A H A H Mut33 hs D472:W502 D472:L516 Mut36 unchanged A P A H A B A P D431:Y508 D431:L510 D431:R513 D431:S515 Mut36 pd, hep+ Mut36 unchanged H P Mut41 unchanged G586:N496 P P P A Q461:N551 Q461:D553 Mut38 unchanged Mut39 pd, unstable P P P H P H S474:N518 S474:P519 S474:M634 H H H H V600:V600 V600:F628 Mut43 unchanged B B R733:H623 A B E574:H509 Mut35 Ala ins, cs, hepsurface A P A B D439:Q359 D439:K549 Mut38 wt

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43 Table 3-4 Continued B A B P R447:E499 R447:N551 Mut38 unchanged H P H H W606:T624 W606:G626 Mut43 unchanged Mut43 unchanged A P Mut37 ni, full D529:N382 B A R459:D553 Mut39 pd, unstable P H T592:G504 P H Y444:L516 Mut36 unchanged H H Mut43 wt F628:F628 Mut43 unchanged P P P H N695:S391 N695:F392 B H Mut37 ni, full K527:G512 P H P B Q440:A357 Q440:H358 P B S578:K507 P P P P Y576:Y483 Y576:Y508 P P P P Q589:N496 Q589:S498 P A P P N449:E499 N449:N497 P P P B Q464:Q359 Q464:K549 Mut38 wt P B P P T581:R484 T581:N596 P P P P S463:T550 S463:N551 Mut38 unchanged Mut38 unchanged A B Mut40 ni, hep-, full particle, surface E564:R389 Mut29 wt H P H P Mut33 hs I470:N270 I470:Y272 Mut22 unchanged Mut22 unchanged H P A467:Y272 Mut22 unchanged P A P P Mut47 ni, A20N690:E347 N690:Q349 Mut27 hs H H G603:L633 P P Mut32 wt T456:N497 P P P P P H Q598:Y483 Q598:T597 Q598:G599 P B Q575:H509 Mut35 ala ins, cs, hep-, surface P P S446:N551 Mut38 unchanged H H G599:G599

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44 Table 3-4 Continued H B A425:R389 Mut29 wt P A S422:D625 Mut43 wt B H H629:G626 Mut43 unchanged P B Y424:H623 B P H426:T624 Mut43 unchanged H P H P V595:Y483 V595:T597 P P Mut47 unchanged S691:Y393 A A Mut42 ni, no capsid D608:D625 Mut43 wt P P T732:S391 P P P A Mut42 ni, no capsid Q607:T624 Q607:D625 Mut43 wt H P H H L430:Y508 L430:L510 H B G466:K549 Mut38 wt P B Q473:H358 P P N582:Q486 P P T568:T624 Mut43 unchanged B A R729:D625 Mut43 wt the AAV capsid, and disassembly experiments to date suggest it is not possi ble to gently disrupt the capsid and isolate an intermediate of assemb ly. The energy required to disrupt the 3-fold interactions results in the capsi ds being broken down completely into monomers when heated and treated with SDS (data not shown). For MVM, one study evaluate d critical residues involved in inter-trimeric cont acts and showed that both hyd rophobic interactions and hydrogen bonding between interfacial side chains could potentially make major contributions toward holding trimeric subassemblies together. These inter-trimeric contacts were nearly identical for both strains of MVM, MVMi which is pathoge nic and MVMp which is nonpathogenic [86]. The five strongest interactions at the icosahedral 2-fold axis of symmetry are listed in Table 3-5. The specific residues and t ypes of interactions that occur at the icosahedral 2-fold axis of symmetry are listed in Table 3-6. While the strongest interaction at the 2-fold axis is a hydrophobic interaction, electrostati c interactions contribute 13.63 kcal/mol per subunit at the 2-

PAGE 45

45 fold axis. Hydrophobic interactions contribute -12.79 kcal/mol per subunit at the 2-fold axis and there are nearly twice as many interactions as co mpared with electrostatic interactions. This is consistent with the suggestion that weak electrostatic in teractions provide ca psid stability. Polar interactions contribute -8.75 kcal/mol per subun it and there are a sim ilar number of polar interactions as hydrophobic interactions. Based on this analysis, the majority of interactions at the 2-fold axis of symmetry are hydrophobic interactions and pol ar hydrogen bonding interactions; however, although fewer in number, th e electrostatic interact ions have a greater energy contribution at the 2-fold axis of symmetry. In additio n, for both Mut19 and Mut24, when charged residues are changed to alanine, the resulting phenotype is the abse nce of capsids, and no infectious particles. Particularly with the re sidues involved in Mut24, the residues in Mut24 that are unchanged in this mutant (FSPRD FSPAA), S292 and P293 (VP1 numbering), Table 3-5. The five strongest interactions at the icos ahedral 2-fold axis of symmetry (VIPERdb). The amino acids that are the strongest at th e icosahedral 2-fold axis of symmetry are listed, using VP1 numbering. This refers to interactions between subunits A1 and A6 in the icosahedral virus particle in Figure 2-2. The type of inter actions are indicated, B = basic, H = hydrophobic. The phenotype of AAV mutants generated by Wu, et al. [84] are listed, ni = non-infectious. Amino Acid VP1 numbering Type Energy (kcal/mol) Wu, et al. mutant phenotype [84] TRP 694 H -4.22 PRO 696 H -3.08 LYS 692 B -2.79 Mut47, ni, A20ARG 294 B -2.14 Mut24, ni, no capsid ARG 298 B -1.91 participate in the strongest interaction at the icosahedral 2-fold axis of symmetry, yet the phenotype of this mutant is the absence of capsids. While R294 (VP1 numbering) is the 4th strongest interaction at the 2-fold axis, the inabilit y of this mutant to make capsids suggests that this electrostatic interaction, while weaker than the primary hydrophobic interaction, is required for capsid formation at the 2-fold axis of symmet ry. With Mut19, the inter action that is changed

PAGE 46

46 at the 2-fold axis of symmetry effects the 3rd strongest interaction and the resulting phenotype is the absence of capsids, and no inf ectious virions. The residue i nvolved in this interaction (K692 VP1 numbering), only interacts with D231 (VP1 numbering), wh ereas other amino acid residues involved in the 3 strongest interac tions at the 2-fold axis interact with several amino acids that could perhaps compensate for a change. This is c onsistent with the models of assembly presented in Chapter 2, which suggest that the interactions at the 2-fold axis of symmetry are critical for assembling into a macromolecular structure. Table 3-6. Specific amino acid interactions at the icosahedral 2-fold axis of symmetry (VIPERdb). Specific amino acids at the icos ahedral 2-fold axis of symmetry are listed, using VP1 numbering, with the stronge st interactions listed first, and the weakest interactions listed la st. This list include s all interactions between subunits A1 and A6 in the icosahedral virus particle depicted in Figure 2-2. The type of interactions are indicate d, A = acidic, B = basic, H = hydrophobic, P = Polar, uncharged. The phenotype of AAV mutants ge nerated by Wu, et al. [84] are listed, either in the column to the left of the am ino acid list if the mutation was made in the 1st amino acid of the pair listed, or in the co lumn to the right of the amino acid list if the mutation was made in the 2nd amino acid of the pair listed. ala ins = alanine insertion, cs = cold-sensitive, hep= hepari n negative, hep+ = heparin positive, hs = heat-sensitive, ni = non-infectious, pd = pa rtially defective, wt = wild-type. Amino acids that were not mutated by Wu, et al. [84], but flank the mutated amino acids are designated unchanged. Type of Interaction Mutant Phenotype (Wu, et al. [84]) VP1 numbering Mutant Phenotype (Wu, et al. [84]) H P H H H H H H H H H P W694:S292 W694:P293 W694:F365 W694:P366 W694:F712 W694:Y720 Mut24 unchanged Mut24 unchanged H H H B H P H P H P H H P696:P293 P696:R294 P696:Q297 P696:Y700 P696:S702 P696:F712 Mut24 unchanged Mut24 ni, no capsid B A Mut47 ni, A20K692:D231 Mut19 ni, no capsid

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47 Table 3-6 Continued B A B B B H B A Mut24 ni, no capsid R294:E689 R294:R693 R294:P696 R294:E697 Mut47 ni, A20Mut47 ni, A20B A R298:E689 Mut47 ni, A20H P H P H P I698:T701 I698:S702 I698:Y704 P A P H Y704:E563 Y704:I698 Mut40 ni, hep-, full A B A B Mut47 ni, A20E689:R294 E689:R298 Mut24 ni, no capsid P P P P P P P P Q699:Q297 Q699:N301 Q699:Q699 Q699:T701 P H P P N695:V710 N695:T713 P H P P P P T701:I698 T701:Q699 T701:T701 P H P H S702:P696 S702:I698 P H Mut24 unchanged S292:W694 H H H H Mut24 unchanged Mut24 unchanged P293:W694 P293:P696 H P V710:N695 P P N302:N302 H H P366:W694 A B E697:R294 Mut24 ni, no capsid A B Mut19 ni, no capsid D231:K692 Mut47 ni, A20P H P P Q297:P696 Q297:Q699 H H H H F712:W694 F712:P696 H H F365:W694 A P Mut40 ni, hep-, full E563:Y704 P P N301:Q699 P H Y700:P696 B B Mut47 ni, A20R693:R294 Mut24 ni, no capsid P H Y720:W694 P P T713:N695

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48 AAV-2 Mutant Studies Five AAV-2 mutants (Mut19, Mut24, Mut26, Mut33, and Mut46), described by Wu, et al. [84] were analyzed in these studies. These are listed in Table 3-7. Amino acid alignments were performed to determine if these residues are highly conserved for AAV serotype 1 through AAV serotype 8. The AAV capsid sequences for AAV serotype 1 through AAV serotype 8 have been aligned using several soft ware programs, including Clusta l W, and Palign (Pcgene), as described by Chiorini, et al. [87] Gao, et al. [24], and Hauck, et al. [88]. In evaluating the capsid sequence for Mut19 (WHCDS), this sequence is conserved for AAV serotype 1 through AAV serotype 8. The capsid sequence in the region of Mut24 (FSPRD) is also conserved for AAV serotype 1 through AAV serotype 8, except fo r AAV-5, which has the sequence WSPRD. The amino acid that is different in AAV-5 when comp ared to the other AAV serotypes, is still a hydrophobic amino acid with a large bulky aromatic side group. The amino acid sequence in the region of Mut26 (VKEVT) is also conserve d for AAV serotype 1 through AAV serotype 8, except for AAV-3, which has the sequence VRGVT. Instead of a lysine, AAV serotype 3 has an arginine in that position, which is still a basic, positively charged amino acid. The most significant change in the region of Mut26 is th e use of a glycine instead of the negatively charged glutamic acid that the other serotypes use. The amino acid sequ ence in the region of Mut33 (DIRD) is highly divergen t, as shown in Table 3-8, with isoleucine in AAV-2 being the most conserved residue when compared to the other AAV serotypes. While the other AAV serotypes use an amino acid other than isoleu cine, which is used in AAV-2, all of the AAV serotypes maintain a hydrophobic amino acid here The amino acid sequence in the region of Mut46 (EIE) is conserved for AAV serotype 1 through AAV serotype 8, with the exception of AAV4 which has a glutamine instead of a glutam ic acid. Unlike glutamic acid, glutamine is an uncharged amino acid; however, like glutam ic acid, it is a hydrophilic residue.

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49 Table 3-7. pIM45 based plasmids containing alanine substitutions for the production of mutant AAV-2 capsids produced by Wu, et al. [89]. Al a sub = alanine substitution; ni = noninfectious; hs = heat sensitive; pd = partially defective; hep+ = heparin positive Mutant Type Mutation Class Phenotype Mut19 Ala Sub 228-232 WHCDS WACAS 4b ni; no capsid Mut24 Ala Sub 291-295 FSPRD FSPAA 4b ni; no capsid Mut26 Ala Sub 320-324 VKEVT VAAVT 3a hs Mut33 Ala Sub 469-472 DIRD AIAA 3a hs Mut46 Ala Sub 681-683 EIE AAA 4b Ni; no capsid Amino Acid Alignment A structural alignment in the region of Mut19 with other parvovirus sequences is shown in Table 3-9. This stretch of amino acids is not conserved among other parv oviruses. A structural alignment in the region of Mut 24 is shown in Table 3-10. The structural alignment for Mut26 with other parvoviruses is show n in Table 3-11. The structural alignment for the amino acids involved in Mut33 is shown in Table 3-12. The parvovirus structur al capsid alignment for Mut46 are shown in Table 3-13. Additionally, an ami no acid alignment was performed for the known AAV serotypes, as well as the newly reported AAV genotypic variants. This alignment has been included as an appendix. For the five AAV-2 mutants listed in Tabl e 3-7, theoretical molecular replacement was used to generate models of the AAV mutants. Mode ls were evaluated to determine the potential Table 3-8. Amino acid alignment data for the specific amino acid sequence in the region of Mut33. Amino acid sequence alignment for the capsid protein of AAV-1 through AAV-8 that align with amino acid 469 472 of AAV-2 (VP1 numbering). Serotype Sequence AAV 1 GMSV AAV 2 DIRD AAV 3 SMSL AAV 3B SMSL AAV 4 RPTN AAV 5 RYAN AAV 6 GMSV AAV 7 TMAE AAV 8 TMAN

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50 Table 3-9. Parvovirus structural capsid protein alignment for Mut19 residues. A structural alignment was performed in DALI for other parvoviruses whose structures have been solved and the amino acids were evaluate d to determine if the region is highly conserved. The amino acids are color code d based on general properties, pink are aromatic amino acids, green are neutral ami no acids, grey are aliphatic amino acids, red are acidic amino acids and aqua are basic amino acids. Parvovirus Sequence AAV2 (PDB#1lp3) W H C D S Porcine Parvovirus (PDB#1k3v) F NNQT MVM (PDB#1mvm) Y D NQT CPV (PDB#4dpv) F NNQT Table 3-10. Parvovirus structural capsid protein alignment for Mut24 residues. A structural alignment was performed in DALI for other parvoviruses whose structures have been solved and the amino acids were evaluate d to determine if the region is highly conserved. The amino acids are color code d based on general properties, pink are aromatic amino acids, green are neutral ami no acids, grey are aliphatic amino acids, red are acidic amino acids and aqua are basic amino acids. Parvovirus Sequence AAV2 (PDB#1lp3) F S P R D Porcine Parvovirus (PDB#1k3v) F N P A D MVM (PDB#1mvm) L Q P S D CPV (PDB#4dpv) F N PG D Table 3-11. Parvovirus structural capsid protein alignment for Mut26 residues. A structural alignment was performed in DALI for other parvoviruses whose structures have been solved and the amino acids were evaluate d to determine if the region is highly conserved. The amino acids are color code d based on general properties, pink are aromatic amino acids, green are neutral ami no acids, grey are aliphatic amino acids, red are acidic amino acids and aqua are basic amino acids. Parvovirus Sequence AAV2 (PDB#1lp3) V K E V T Porcine Parvovirus (PDB#1k3v) L K T I T MVM (PDB#1mvm) L K T V T CPV (PDB#4dpv) L K T V S Table 3-12. Parvovirus structural capsid protein alignment for Mut33 residues. A structural alignment was performed in DALI for other parvoviruses whose structures have been solved and the amino acids were evaluate d to determine if the region is highly conserved. The amino acids are color code d based on general properties, pink are aromatic amino acids, green are neutral ami no acids, grey are aliphatic amino acids, red are acidic amino acids and aqua are basic amino acids. Parvovirus Sequence AAV2 (PDB#1lp3) D I R D Porcine Parvovirus (PDB#1k3v) T E A T MVM (PDB#1mvm) S E A I CPV (PDB#4dpv) T E A T

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51 Table 3-13. Parvovirus structural capsid protein alignment for Mut46 residues. A structural alignment was performed in DALI for other parvoviruses whose structures have been solved and the amino acids were evaluate d to determine if the region is highly conserved. The amino acids are color code d based on general properties, pink are aromatic amino acids, green are neutral ami no acids, grey are aliphatic amino acids, red are acidic amino acids and aqua are basic amino acids. Parvovirus Sequence AAV2 (PDB#1lp3) E I E Porcine Parvovirus (PDB#1k3v) T L T MVM (PDB#1mvm) K L T CPV (PDB#4dpv) K LV effect of the mutation. This allows for a predicti on of the role that th ese regions of the capsid play in assembly and/or packaging. Modeling The structure of AAV-2 capsid mutants we re modeled and evaluated based on the available coordinates for wild-type AAV-2 VP3 (1l p3) to visualize the lo cation of the mutation on the capsid. The resources provided by the European Bioinformatics Institute (EBI) European Molecular Biology Laboratory (EMBL) we re utilized to generate these models. The primary program utilized for this was Deep Vi ew, also called Swiss PDB Viewer, or SPDBV. Deep View provides a user friendly interface for viewing and analyzing protein and nucleic acid structures. It also provides some advanced features including an in terface for theoretical modeling, and visualization of electron density maps and elect rostatic surfaces. Deep View provides automatic structure retrieval by PDB-ID as well as measurement of bonds, angles, and distances between atoms. Using Deep View, it is possible to compare st ructural details, to superimpose a structure onto a nother structure for comparison, as well as model amino acid mutations. Swiss PDB Viewer is the primary t ool for accessing Swiss-Model, which is an automated homology modeling server at Glaxo Welcome Experimental Research in Geneva. Utilizing both Swiss PDB Viewer and Swiss-Model, it is possible to model mutations as well as

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52 build in and modify surface loops for evaluating the impact of potential modifications that may be used for specific targeting of AAV vectors, as well as to build in missing amino acids on the N-terminus of VP3, or VP2, or VP1. Available da ta suggests that the N-terminus of VP-2 is located inside at the 2-fold axis; however, it has also been reported that the N-terminus is on the outside of the capsid. Utilizing the model of AAV-2 (PDB accession #1lp3), the appropriate mutations (Mut19, Mut24, Mut26, Mut33, and Mut46, from Wu, et al.[ 89]) were modeled, and the various structural units, dimer, trimer, pentamer and intact cap sids, were built from the monomer. A comparison was made between the wild-type ca psid and the mutants to correlate the effect of the mutation on capsid assembly and/or packaging. This may beco me a useful tool in the area of AAV targeting, as the current data suggests that regions of the cap sid that tolerate some mutations do not tolerate others. The Mutants Mut 19 228 232 WHCDS WACAS Mut19 is an alanine substitution mutation, where the charged residues His229 and Asp231 have been changed to alanine [89]. Utilizing the molecular modeling software DeepView, as well as PyMOL, the residues that are mutated in Mut19 were evaluated. Since the phenotype of Mut19 is an inability to make capsids, this mole cular modeling software was used as a tool to predict the structural subunit that is formed. The location of this mutation affects both the 2-fold axis and the 5-fold axis of symm etry. As a result of the 5-fold interaction, it is unlikely that pentamers would be a stable subunit, as the muta tions are at the interf ace between the monomers that make up the 5-fold axis of symmetry. This is demonstrated in Figure 3-1. The interaction at the 2-fold axis of symmetry between Asp231 and Lys692 is shown in Figure 3-2. Interestingly, when Lys692 was changed to alanine, which is Mut 47 [89], capsids were unable to be detected

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53 Figure 3-1. Residues at the five fo ld symmetry axis that are changed in Mut19. The aqua residue is His229. The pink residue is Asp231. Yello w arrows point to the location of the mutation. This figure was generated using the molecular modeling software DeepView. using the conformational antibody A20; however, capsid proteins were subsequently detected using B1 antibody and the buoyant density in ces ium chloride for thes e capsid proteins was consistent with the production of intact capsids (Hartladge, Warrington and Muzyczka, unpublished data). Figure 3-3 shows that the 3-fold in teractions are still inta ct as a result of the amino acid changes in Mut19. Based on this, the predicted subunit of asse mbly that should be produced from Mut19 is a trimer. To determine what interactions were disrupted by changing His229 and Asp231 to alanine, the amino acids WH CDS were selected in the model.Amino acids were added to the view within 4 of the se lected residues. Based on this, intermolecular distances and intramolecular distances we re determined. The model shows several intramolecular interactions be tween His229 and Thr242, Thr243 a nd Thr244, as seen in Figure 3-4; however, when His229 was changed to an alan ine, these interactions are still maintained (data not shown). This suggests th at the lack of capsids is prob ably not due to disrupting the

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54 intramolecular interactio ns that are involved in proper prot ein folding. Mut19 occurs at the beginning of the first beta-sheet structure in VP3. If an intramol ecular interaction was responsible for the lack of capsi ds, the prediction would be that monomers would be formed that are unable to associate into a capsid structure. The intermolecular inte ractions that may be disrupted in Mut19 are shown in Figure 3-5. In addition to Lys692, the interaction with Pro399 may also be disrupted as a result of Asp231 being changed to alanine. Figure 3-2. Residues at the two fold symmetry axis that are changed in Mut19. The aqua residue is His229. The pink residue is Asp231. The residue shown in yellow is Lys692. The residues that are mutated in Mut19 are colored differently than the main chain, usually white, except where they would not be visible, in which case they are aqua. Yellow arrows point to the region of the mutation. This figure was generated using the molecular modeling software Deep View.

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55 Figure 3-3. The predicted subunit as a result of the mutations made in Mut19 is a trimer. Yellow arrows point to the region of the mutation. The pink, peach and dark blue subunits comprise a trimer. Figure 3-4. Residues that may be responsible fo r the phenotype of Mut19. Lys692 is shown in purple, His229 is aqua, and Asp231 is pi nk. The blue chain shows intramolecular interactions. The yellow and pur ple chains demonstrate inte rmolecular interactions. This figure was generated using the mo lecular modeling software Deep View.

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56 This interaction also involves the 2-fold axis of symmetry. If this inter action is responsible for the phenotype of Mut19, the expected subunit would al so be a trimer. Anothe r possibility is that both Lys592 and Pro399 are needed to interact wi th Asp231. Using the methods established here, it is not possible to determine if both interactions are require d for intact capsid formation. However, Pro399 is 3.60 away from Asp231, while Lys692 is only 2.78 away. The model of Mut19 after the amino acids are change d to alanine is shown in Figure 3-6. Another approach to evaluating the effect of the alanine mutation is to do a structural alignment of several parvovirus cap sid proteins and look at whethe r or not the residues that are changed in Mut19 are conserved. The capsid protei ns of several parvoviruses were aligned, and then evaluated to determine if the residues th at were mutated in Mut19 are conserved among other parvoviruses. These are s hown in Table 3-9. The table in cludes AAV, Porcine Parvovirus, MVM, and CPV. Based on the table, the residues in Mut19 appear to be unique to AAV. AAV residue in this position. Based on the structural alignment, the prediction would be that this residue might be able to be changed without affec ting the overall structure, since there is such a wide range of amino acids in this position for other parvoviruses. AAV is also the only parvovirus in the table that has a negatively ch arged acidic residue at Asp231. Based on this, the prediction would be that changing Asp231 to alan ine, should be tolerated by AAV, as the other parvoviruses listed have neutral am ino acids in this position. Howe ver, the model indicates that the negatively charged Asp231 interacts with th e positively charged Lys692. In the capsid protein alignment, amino acid 692 in porcine pa rvovirus and MVM are neut ral residues, N and T respectively, while CPV, like AAV-2 also has a positively charged basic residue (H) in this position. Based on the data, the charge-charg e interaction between Asp231 and Lys692 is probably necessary for AAV capsid stability.

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57 Figure 3-5. Intermolecular residues are show n between the yellow chain, and the Asp231. Residues that also may play a role in the phenotype of Mut19 include Tyr397 and Pro399. This figure was generated using th e molecular modeling software Deep View. Figure 3-6. Asp231 has been changed to Ala231, as in Mut19. Note that the primary interaction that is disrupted is the intermolecula r interaction with Lys692; however, an intermolecular interaction with Pro399 is al so disrupted. One or both interactions may result in the inability to form intact ca psids. This figure was generated using the molecular modeling software Deep View.

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58 Mut24 291 -295 FSPRD FSPAA Mut24 is an alanine substitution mutation, where the charged residues Arg294 and Asp295, have been changed to alanine [89]. Utilizing the same methods described for Mut19, the residues that are mutated in Mut24 were evaluated. Like Mut19, the phenotype for Mut24 is an inability to make capsids. The molecular modeling software was used as a tool to predict the structural subunit that is formed. The location of this mutant affects both the 2-fold axis and the 5-fold axis of symmetry. Based on the location of the mutati ons at the 5-fold axis, pentamers may not be able to be formed. This mutation is further from the 5-fold pore than Mut19, so due to the location of the mutation, there may be enough signif icant interactions at the 5-fold axis to perhaps hold pentamers together. This is demons trated in Figure 3-7. The 2-fold axis of symmetry is shown in Figure 3-8. The residues that have been mutate d are likely to produce significant changes at the 2-fold axis. The mutation at the 2-fold axis occurs at a place in the capsid where three different chains come together the two that make up the 2-fold, (light blue and dark blue residues) one of which is also a pa rt of the 3-fold axis (dark blue), and a second chain of the 3-fold axis (orange) that sits above the residue that makes up the 2-fold axis (light blue). If the second chain of the 3-fold axis is disrupted then tr imers would not be stable and the predicted subunit formed would be either monomers or pentamers, if the ot her interactions at the 5-fold axis are significant enough to hold a pent amer together. Alternatively, the mutation may only affect the residues at the 2fold axis and the second chain of the 3-fold axis (orange) may be unaffected. If this is the case, then trimers will be the predicted subunit. This is shown in Figure 3-9. Based on the information in Figure 3-10, ther e are quite a few residue s that interact with Arg294 and Asp295. These are also shown in Table 314. Based on the information in this table, there are a couple of th ings to note. First, Arg294 is a pos itively charged ba sic residue that interacts with two negatively charged glutam ic acid residues. Changing Arg294 to an Ala294

PAGE 59

59 disrupts these interactions, as the side chain in alanine is much smaller and is now not in close enough proximity to interact. In addition, this ch arge:charge interaction is also disrupted and may be required to hold th e monomers together. Figure 3-7. Residues at the 5-fold symmetry axis that are change d in Mut24. The view is from the inside of the capsid. The aqua resi due is Arg294. The pink residue is Asp295. This figure was generated using the mo lecular modeling software Deep View. When Glu689 was changed to an alanine in Mut47 [89], the phenotype is a non-infectious capsid that is A20 negative. The lack of infectivity provides furt her evidence for the importance of the interaction between Ar g294 and Glu689. When Asp295 is changed to an alanine, the intermolecular intera ction between it and Tyr397 is disrupted. This residue is from the light pink chain that is part of the 3-fold axis of symmetry shown in Figure 3-11. Based on this, the predicted subunits formed by Mut24 are monomers, assuming that the mutation at the 5-fold axis of symmetry prevents pentameric interactions. To further evaluate the mu tations that were made

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60 in Mut24, the parvovirus capsid alignment data was used to evaluate if these residues are conserved. This is shown in Table 3-10. Figure 3-8. Residues at the 2-fold symmetry axis that are changed in Mut24. The aqua residue is Arg294. The pink residue is Asp295. This fi gure was generated using the molecular modeling software Deep View. Generally, there is an aromatic phenylalanine residue at position 291, with the exception of MVM which has a lysine in that position. Next ther e is a neutral residue at position 292, then a proline at 293, and then Asp at 295, for all of the am ino acids in the table. While the aspartic acid residue is conserved when compared to other parvoviruses in the table, the residue that it interacts with, Tyr397, is unique to AAV-2. Based on the alignment data at residue 397, the other parvoviruses in the table have an N at that pos ition. In addition, AAV-2 is the only parvovirus in the table that has a charged basic residue at position 294. Porcine parvovirus has an alanine in this position, and because of this it might be ex pected that an alanine at this position in AAV-2 would be tolerated. Residue 689 in the alignment is an arginine for all of the parvoviruses listed

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61 in the table except AAV-2. Residue 697 is an aliphatic residue, isoleu cine for CPV, or valine for MVM, while this is a glutam ic acid residue in AAV-2. Figure 3-9. The predicted subunit as a result of the mutations made in Mut24 is a trimer. The residues that are mutated in Mut24 are colored differently than the main chain, usually white, except where they would not be visible, in which case they are aqua. Inside view. This figure was generated us ing the molecular modeling software Deep View.

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62 Figure 3-10. Intermolecular and in tramolecular residues that are di srupted as a result of changing Arg294 and Asp295 to alanine residues. Arg294 is shown in yellow, Asp295 is shown in white, the chain that they are att ached to is shown in light blue, residues from other chains are shown in pink or dark blue respectively. Intramolecular interactions that are disrupt ed include the interaction w ith Ser292 and Trp234. This is a close up of the specific interactions in Figure 3-9. Note: Tyr397 is pink, Glu689 and Gu697 are dark blue. This figure was generated using the molecular modeling software Deep View.

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63 Figure 3-11. Mut24 interactions that are effected by the muta tion. Theoretical model of the interactions that are change d in Mut24 when Arg294 is changed to Ala294 (yellow) and Asp295 is changed to Ala295 (white). This figure was generated using the molecular modeling software Deep View.

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64 Table 3-14. Evaluation of the effect of the mutations introduced in Mut24 on amino acid interactions. The amino aci ds that interact with Arg294 and Asp295 were modeled using SPDB Viewer and the distances befo re the mutation were introduced were determined. The type of interaction, eith er intermolecular or intramolecular, was evaluated. The amino acids were mutated using the molecular modeling software Deep View for visualization. The interactions were evalua ted after the mutations were introduced to determine which interactions we re disrupted as a result of the mutation. Figure 3-10 and Figure 3-11 show the intera ctions before and after the mutation respectively, and the colors in parenthesis above refer to the residues in these figures. Amino Acid that is changed Distance () Interacting residue Was interaction disrupted? Type of interaction Arg294 2.75 Glu689 (blue) Yes Intermolecular Arg294 2.91 Glu697 (blue) Yes Intermolecular Arg294 3.36 Glu697 (blue) Yes Intermolecular Arg294 2.79 Trp694 (blue) No Intermolecular Arg294 2.66 Ser292 (lt blue) No Intramolecular Arg294 2.54 Pro293 (lt blue) No Intramolecular Arg294 2.85 Gln297 (lt blue)No Intramolecular Arg294 2.98 Arg298(lt blue) No Intramolecular Asp295 2.52 Tyr397 (pink) Yes Intermolecular Asp295 2.76 Trp234 (lt blue) Yes Intramolecular Asp295 2.83 Ser292 (lt blue) Yes Intramolecular Asp295 2.44 Ser292 (lt blue) No Intramolecular Asp295 3.30 Ser292 (lt blue) No Intramolecular Asp295 2.66 Arg298(lt blue) No Intramolecular Mut26 320 324 VKEVT VAAVT Mut26 is an alanine substitution mutation, where the charged residues Lys321 and Glu322, have been changed to alanine [89]. Utilizing th e same methods described for Mut19 and Mut24, the residues that are mutated in Mut26 were evalua ted. Mut26 is located near the pore at the 5fold axis of symmetry as shown in Figure 3-12 and Figure 3-13, and is NOT involved in the loop region of the 5-fold monomers that interact w ith the neighboring residue. There is a surface loop that projects from the surface of the capsid, and Mut26 is at the base of this loop. This capsid mutant was determined to be heat sensitive using the GFP fluorescent cell assay to determine infectious titer. Mut26 has a titer that is about 1 log lower than wt when the crude lysate is used to infect 293 cells at 32 C. There is no titer at 39.5 C [89]. Based on this, it can be assumed that

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65 Mut26 is able to package viral DNA, although rece ntly other investigator s have produced data using 5-fold pore mutants that suggest that muta tions in this region of the capsid effect DNA packaging. It is unknown where the nonstructural Re p proteins interact with the capsid for Figure 3-12. Residues at the 5-fo ld symmetry axis that are cha nged in Mut26. The aqua residue is Lys321. The pink residue is Glu322. Yellow arrows point in the direction of the residues that have been mutated, but the ami no acids that have been mutated are at the 5-fold pore. Note: The orientation on this figure is different from Mut19 and Mut24. This figure is from outside of the capsid looking at the 5-fold pore. This figure was generated using the molecular modeling software Deep View.

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66 Figure 3-13. This is a cut away view of three of the chains that make up the 5-fold axis of symmetry. The red and aqua chains of Figur e 3-12 have been removed to show the location of the mutations. Lys321 is in aqua or white, and Glu322 is in pink. This figure was generated using the molecular modeling software Deep View. packaging, but this is assumed to involve resi dues in the region of th e 5-fold pore since the proposed model for AAV packaging suggests that the DNA is inserting into the capsid through the 5-fold pore. Alternatively, mutating the residues at the 5-fold pore may have disrupted an as yet undetermined site on the capsid that is required for the Rep proteins to dock. This mutation is interesting in that infectious pa rticles are not produced at 39.5 C Physical particle titer using A20 ELISA was not obtained for this mutant so it is not known wh ether the lack of infectious particles at 39.5 C is due to a lack of capsid assembly at 39.5 C or an inability to package at 39.5 C; however, based on the location of the muta tion, the mutation is predicted to effect DNA packaging. Based on the model, this mutation does not affect the 2-fold or the 3-fold axis. EM on capsids produced at 39.5 C would help to determ ine if this is a packaging mutant versus an assembly mutant. In this region of the capsid, there is an in termolecular interaction between Asn335 (yellow) and Asn334 (blue), as shown in Figure 3-14. Al though the intermolecular interaction between

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67 Lys321 and Asn335 (yellow) is disrupted, there is an intermolecular interaction between Asn335 (yellow) and Asn334 (blue 2.77 A, and 3.83 A). This interaction may maintain the position of Asn335 and compensate for the intermolecular inte raction that is disrup ted between Lys321 and Asn335, allowing for capsids to be produced at th e permissive temperature. The intramolecular interactions between Glu322 and Ala333 (2.56 A, 3.18 A, 3.56 A, and 3.60 A) are maintained when Glu322 is changed to Ala322, as shown in Figure 3-15. The inte raction between Ala333 that is maintained is connected to Asn334, a nd therefore probably he lps to preserve the interaction between Asn335 (yellow) and Asn334 (blue), even though the interaction between Lys321 and Asn355 is disrupted. The intraand inte rmolecular interactions for Mut26 are listed in Table 3-15. Figure 3-14. Interactions with the residues involved in Mut26. Lys321 is white, Glu322 is pink, Asn335 is yellow, Ile332 is green. This figure was generated using the molecular modeling software Deep View.

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68 Figure 3-15. Model of Mut26 afte r the mutation. This figure was generated using the molecular modeling software Deep View. The protein capsid alignment data suggests that Lys321 is conserved among the parvoviruses listed in Table 3-11. In addition th ere is an aliphatic residue be fore Lys321, usually leucine but for AAV-2 its a valine residue, and a neutral residue (Threonine) follows Lys321 for all parvoviruses listed except AAV-2 which has a ch arged Glutamic acid in this position. Residue 323 in the alignment is an aliphati c residue, usually valine, except for MVM which has an isoleucine. Residue 324 is a neut ral residue either serine or threonine. The alignment data further confirms that Lys321 is conserved and therefore important for AAV capsid stability. However, due to the compensati ng interaction with Asn334, capsids are able to be produced at the permissive temperature. In terestingly, Asn334 is c onserved for all of the parvoviruses listed in Table 3-11.

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69 Table 3-15. Evaluation of the effect of the mutations introduced in Mut26 on amino acid interactions. The amino acids that interact with Ly s321 and Glu322 were modeled using SPDB Viewer and the distances befo re the mutations were introduced were determined. The type of interaction, eith er intermolecular or intramolecular, was evaluated. The amino acids were mutated using the molecular modeling software Deep View for visualization. The interactions were evalua ted after the mutations were introduced to determine which interactions we re disrupted as a result of the mutation. Figure 3-14 and Figure 3-15 show the intera ctions before and after the mutation respectively, and the colors in parenthesis above refer to the residues in these figures. Amino Acid that is changed Distance () Interacting residue Was interaction disrupted? Type of interaction Lys321 3.16 Asn335(yellow)Yes Intermolecular Lys321 4.30 Asn335(yellow)Yes Intermolecular Lys321 3.41 Ala333 No Intramolecular Lys321 2.38 Val320 No Intramolecular Lys321 3.39 Val320 No Intramolecular Lys321 2.96 Tyr734 No Intramolecular Lys321 3.16 Tyr734 No Intramolecular Glu322 3.90 Ile332 (green) Yes Intermolecular Glu322 2.78 Asn335 Yes Intramolecular Glu322 2.98 Asn335 Yes Intramolecular Glu322 3.65 Asn335 Yes Intramolecular Glu322 3.82 Asn335 Yes Intramolecular Glu322 3.38 Thr732 No Intramolecular Glu322 2.59 Ala333 No Intramolecular Glu322 3.56 Ala333 No Intramolecular Glu322 3.18 Ala333 No Intramolecular Glu322 3.60 Ala333 No Intramolecular Mut33 469 472 DIRD AIAA Mut33 is an alanine substitution mutation, where the charged residue Asp469, Arg471, and Asp472 have been changed to alanine. Utilizin g the same methods described for Mut19, Mut24 and Mut26, the residues that are mutated in Mut33 were evaluated. Mut33 is not involved in the 5-fold axis of symmetry or the 2-fold axis of symm etry. The lack of an effect at the 5-fold axis of symmetry is shown in Figure 3-16. The mutations ar e located at the 3-fold axis of symmetry in Mut33 as shown in Figure 3-17. There are two intr amolecular interactions that are disrupted in Asp469 as a result of the mutation, as shown in Figure 3-18. However, as shown in Figure 3-19, intermolecular interactions, as well as intramolecula r interactions are disrup ted as a result of the

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70 Figure 3-16. Mut33 pentamer demons trating that this mutation is not involved in the interactions that make up the icosahedral 5-fold symmetry axis; therefore, as a result of this mutation, pentamers will still be formed. Th is view is from the outside looking in. This figure was generated using the mol ecular modeling software Deep View. mutation in Asp472, and this is probably the reas on for the phenotype. The interactions that are disrupted are summarized in Table 3-16. This capsid mutant was determined to be heat sensitive using the GFP fluorescent cell assay to determine infectious titer. Mut33 has a titer that is about 1 l og lower than wild-type when the crude lysate was used to infect 293 cells at 32 C. Th ere was no titer at 39.5 C [89]. Based on this, it can be assumed that Mut33 is able to package viral DNA. Since at the permissive temperature packaging can occur, this suggests that an inte raction between Rep and VP3 that is needed for packaging is not disrupted as a result of these mutations. For this mutant, the prediction would be that pentamers would be stable, as would dimers, but that trimers would not be stable. This supports the model of assembly for AAV that was presented in Chapter Two

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71 Figure 3-17. Mut33 residues. The yellow re sidue is Asp469, aqua is Arg471, pink is Asp472. Note: for the pink chain, Asp472 is white. This view is from the outside looking in three fold axis. The mutation effects where the monomers come together at the icosahedral 3-fold axis of symmetry. This figure was generated using the molecular modeling software Deep View. which suggests that capsid assembly probably occurs in the following order: 5-fold interactions first, then the 2-fold interactions occur, followed by the 3-fold interactions. The 3-fold interactions in the AAV capsid ar e the strongest interactions. Be cause the thermodynamic cost is greater, these probably form last. This mutant should provide the basis for validating this hypothesis. The alignment of several parvovirus capsid prot eins in the region of Mut33 is shown in Table 3-12. The capsid protein alignment for the resi dues involved in this mutant suggests that this region is not highly cons erved among parvoviruses. The resi due that was not changed in Mut33, Ile470 is an aliphatic residue for AAV2, Porcine Parvovirus and MVM. In CPV the residue at position 470 is an arom atic residue, while in the next position there is an Ile, Ile471. MVM and CPV have a neutral re sidue at position 469, while AAV-2 is unique in that it has an acidic negatively charged residue.

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72 Figure 3-18. Interactions in th e region of Mut33. The yellow re sidue is Asp469, the aqua residue is Arg471, and the white residue is Asp472. This figure was generated using the molecular modeling software Deep View. Figure 3-19. Model of residues mutated in Mut33. This figure was generated using the molecular modeling software Deep View.

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73 Table 3-16. Evaluation of the effect of the mutations introduced in Mut33 on amino acid interactions. The amino acids that in teract with Asp469, Asp471, and Asp472 were modeled using SPDB Viewer and the distances before the mutations were introduced were determined. The type of interaction, e ither intermolecular or intramolecular, was evaluated. The amino acids were mutated using the molecular modeling software Deep View for visualization. The interactions were evalua ted after the mutations were introduced to determine which interactions we re disrupted as a result of the mutation. Figure 3-18 and Figure 3-19 show the intera ctions before and after the mutation respectively, and the colors in parenthesis above refer to the residues in these figures. Amino Acid that is changed Distance () Interacting residue Was interaction disrupted? Type of interaction Asp469(Yellow) 3.61 Asp269 (Blue) No Intermolecular Asp469(Yellow) 2.46 Tyr444 (Pink) Yes Intramolecular Asp469(Yellow) 2.73 Asp472 (White) No Intramolecular Asp469(Yellow) 3.77 Gln464 (Pink) Yes Intramolecular Asp469(Yellow) 2.69 Arg471 (Aqua) No Intramolecular Asp469(Yellow) 2.73 Asp472 (White) No Intramolecular Arg471 (Aqua) 2.95 Leu516 (Blue) No Intermolecular Arg471 (Aqua) 2.60 Asn270 (Blue) No Intermolecular Arg471 (Aqua) 3.81 Asn270 (Blue) No Intermolecular Arg471 (Aqua) 3.45 Asp269 (Blue) No Intermolecular Asp472 (White) 3.32 Trp502 (Blue) Yes Intermolecular Asp472 (White) 3.36 Trp502 (Blue) Yes Intermolecular Asp472 (White) 3.77 Asn518 (Blue) No Intermolecular Asp472 (White) 2.98 Tyr444 (Pink) Yes Intramolecular Asp472 (White) 3.10 Arg471 (Aqua) Yes Intramolecular Asp472 (White) 3.20 Asp469(Yellow)Yes Intramolecular Asp472 (White) 3.05 Arg471 (Aqua) Yes Intramolecular Asp472 (White) 3.69 Arg471 (Aqua) Yes Intramolecular Asp472 (White) 3.17 Arg471 (Aqua) Yes Intramolecular Mut46 681 683 EIE AAA Mut46 is an alanine substitution mutation, where the charged residues Glu681, Glu683, and the aliphatic residue Ile682, have been chan ged to alanine [89]. U tilizing the same methods described for the other mutants, the residues th at are mutated in Mut46 were evaluated. Like Mut19, the phenotype for Mut46 is an inability to make capsids. The location of Mut46 lies in the middle of a -sheet, which is part of the main structur al motif of the capsid protein, and is conserved among members of the family Parvoviridae Mut46 is not located at an interface at either the 2-fold axis of symmetry, or the 3-fo ld axis of symmetry, or the 5-fold axis of

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74 symmetry, as shown in Figure 3-20. This suggests th at the mutation disrupts the interaction in the -sheet structure. Because the mutation occurs in one of the -sheets, the mutation probably effects the proper folding of the monomers. Due to improper folding and the potential instability in a critical structural compone nt of the capsid, the predicted s ubunit that Mut46 will produce, if Figure 3-20. The location of residues that are mutated in Mut46. This figure provides a view from inside the capsid, with both the 5-fold pore as well as the 3-fold pore included. The residues that are mutated in Mut46 ar e colored white, except where they would not be visible, in which case they are aqua. This figure was generated using the molecular modeling software Deep View. any at all, is a misfolded mono mer. Upon closer inspection, it s hould be noted that this mutation does not appear to involve any intermolecular inte ractions; however, intr amolecular interactions are affected as shown in Figur e 3-21 and Figure 3-22. While it may appear from Figure 3-20 that this mutation is near the loop region that intrudes from one monomer into a nother, these residues are actually further apart than they appear. The intermolecula r distances between the residues that are mutated in Mut46 and the next chain ar e approximately 20 away. This further supports

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75 the hypothesis that it is the improper folding of the monomeric subunits that result in the phenotype of Mut46, which is the inability to pr oduce intact capsids. Figure 3-21 shows the intramolecular interactio ns that occur in the wild-type cap sid monomers. It should be noted however that when Glu681, Ile682, and Glu683 were changed to alanine, that many of these intramolecular interactions are st ill maintained. In Figure 3-22, these residues have been mutated to alanine and intramolecular distances have been calculated. Based on this model, the primary interaction that is disrupted in this mutant involves interactio ns with Glu683. Upon changing this residue to Ala683, there is a 2.81 interaction with Arg238 that is disrupted. After the mutation this distance is approx imately 7 There is also an inte raction between Arg683 and Val239 that is disrupted as a result of the mutation. Intere stingly, in Mut2 0 [89] Arg238 is mutated and the Figure 3-21. Intramolecular inte ractions with the residues th at are changed in Mut46, Glu681, Ile682, and Glu683. Glu681 is pi nk, Ile682 is blue, and Gl u683 is aqua. This figure was generated using the molecular modeling software Deep View.

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76 Figure 3-22. Intramolecular interactions after the residues invo lved in Mut46 have been changed to alanine. Ala681 is pink, Ala682 is blue and Ala683 is aqua. This figure was generated using the molecular modeling software Deep View. phenotype is also an inability to make capsi ds. Mut20 involves residues 235 -239 where the sequence MGDRV was changed to M GAAV and is not a focus of this study; however it provides further evidence that the inte raction between Glu683 and Arg238 may be responsible for the phenotype of this capsid mutant. The alignment of several parvovirus capsid prot eins for this region are shown in Table 313. For the other parvoviruses in the table, residue 681 is a charged residue, with the exception of Porcine Parvovirus which has a neutral residue in that position. It should be noted that while AAV has an acidic negatively charged residue for residue 681, MVM and CPV have a basic positively charged residue. For residue 682, all of the parvoviruses listed have an aliphatic residue. Both MVM and AAV have a charged resi due for residue 683; although, as with residue 681, AAV utilizes an acidic negatively charged re sidue at that position, while MVM utilizes a basic positively charged residue at that positi on. CPV has an aliphatic residue at that position,

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77 while Porcine Parvovirus has a neutral residue at th at position. So for residue 683, there is quite a bit of variability among pa rvoviruses at that position. As discussed in Chapter 2, data for MVM i ndicates that trimers are the intermediate subunit of assembly. Conventional nuclear localization sequences have been described in the Nterminal unique region of VP1 for MVM, as we ll as for AAV2. The major capsid protein, VP2 for MVM or VP3 for AAV does not possess these Nterminal sequences. In theory, parvoviruses are small enough with a capsid diam eter of approximately 25 nm to traverse the nuclear pore complex intact. It is unclear wh ether structural protei n subunits are assembled prior to traversing the nuclear pore complex or whether the structur al protein subunits are transported across the nuclear pore complex for capsid assembly inside the nucleus. For MVM, it has been shown that when singly expressed in transfec ted cells, VP1 and VP2 were able to enter the nucleus; although only VP2 assembled into capsids, which suggest ed that both VP1 and VP2 have nuclear localization signals (NLS) whose act ivity is independent of capsid assembly [90]. Lombardo, et al. [91] showed that for MVM, singly expresse d VP2, as well as VP1/VP2 oligomers target the nucleus by a structural nonconven tional nuclear localizat ion motif (NLM), which is located in the I strand, one of the 8 -barrel structural motifs that are conserved for parvoviruses (Chapter 2, Figure 2-1). For AAV, Ruffing, et al. have shown that there is also c ooperativity in nuclear transport [66]. Lombardo, et al. [91] have shown that the MVM capsi d proteins interact cooperatively in the cytoplasm to pass thr ough the nuclear pore complex. The sequence 528KGKLTMRAKLR538 near the C-terminus of VP2 in MVM is a nonconventional nuclear localization signal. For MVM, it has been suggested that the I conformation is necessary for proper protein folding of VP2 and assembly of tr imeric intermediates. Inactivation of the NLM through mutations resulted in onl y VP1 containing trimers being able to translocate into the

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78 nucleus. Functional NLMs depend on a correct three-dimensional protein conformation. For AAV2, the sequence KGKLTMRAKLR is not presen t in the C-terminal region of VP3; however, an alignment of the MVM capsid pr otein with the AAV2 capsid protein sequence reveals that this is the location of the mutations introduced in Mut46. This further supports the suggestion that the phenotype of Mut46 is the result of imprope r protein folding which disrupts the intrastrand hydrogen bondi ng interactions of the -sheet, which also may result in a defect in protein trafficking through the nuclear pore co mplex. Other mutations introduced into Mut46 by Wu et al. [89], such as a serine substituti on at amino acid position 682 (Mut46subser15), and a FLAG substitution at amino acid position 682 (Mut46s ubflg11) also resulted in a noninfectious, no capsid phenotype. Methods for Mutant Studies The capsid mutants were evaluated biochemi cally in an effort to determine their properties with respect to capsid assembly and packaging. DNA for the mutant AAV capsids was transfected and analyzed in an attempt to isolate and identify an assembly intermediate. For AAV, isolating an intermediate of assembly is complicated due to the requirement of a helper virus such as adenovirus for production of AAV. Adenovirus assembly is known to proceed via pentameric intermediates and hexon and penton prot eins isolated from AAV preps can easily be mistaken for pentameric AAV intermediates by electron microscopy. Baculovirus produced AAV vectors do not require a helper virus; however the baculovirus protei ns that are providing the helper function for AAV produ ction have not yet been elucidated and few antibodies are available for baculovirus proteins (anti-gp64). The method developed and described in Chapter 5 for AAV Capsid Serotype Identification (AAV-CSI) will be useful for verifying and validating that the assembly intermediate isolated is an actual AAV intermediate and not a contaminating subunit from the helper virus used to produce th e AAV. Of the capsid mutants described here,

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79 the temperature sensitive mutant, Mut33, provides the best opportuni ty for isolating an assembly intermediate since it is able to assemble into capsids at the permissive temperature, and once assembled has been shown to remain intact after cesium gradients. For transfections, nearly confluent HEK 293 cells were split 1:3 the day before the transfection so that they could reach 70% confluency the next da y. Mutant capsid proteins were produced by triple-transfecting HEK 293 cel ls with pIM45, UF5, and XX6 by Calcium Phosphate precipitation. The UF5 v ector contains the gene for GFP, flanked by the AAV ITRs. XX6 contains the Adenovirus helper genes requ ired for AAV replication. pIM45 contains the AAV rep and cap genes. pIM45 was used to genera te baseline data for wild-type capsids and serves as a control for these expe riments. For the transfection, fl asks were transfected at 37 C, using the calcium phosphate mediated DNA transf ection protocol and incubated at 37 C. The capsid mutants were built into the pIM 45 backbone [89]. To generate the mutants, HEK 293 cells were transfected with the pI M45 mutant plasmid (Mut19, Mut24, Mut26, Mut33, or Mut46), and UF5 and XX6. For the temper ature sensitive mutants, Mut26 and Mut33, transfections were done at 32 C, 37 C and 39.5 C. At 60 hours the cells were harvested by cen trifugation at 1,140 x g for 10 minutes. The supernatant was removed and the pellets were resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). The virus was subjecte d to 3 cycles of freezing and thawing. The resuspended pellets were frozen in an ethanol dr y ice bath, and then thaw ed at 37 C. During the 3rd thaw, the crude lysates were treated with Benz onase at a final concentr ation of 50 U/ml at 37 C for 30 minutes to degrade DNA that is not encapsidated. Crude lysa tes were clarified by centrifugation at 3,700 X g for 20 minutes to rem ove the cellular debris. The supernatant was collected and an aliquot of the lysate was flas h frozen and stored at -80 C. The lysate was

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80 subject to cesium chloride gr adients, AAV fractions were iden tified based on refractive index on cesium chloride. These fractions were dialyzed and analyzed by green cell assay on C12 cells [92]. Additional methods of production were explor ed by co-transfecting pIM45 or the pIM45 based mutant plasmid and UF5, followed by infec ting the cells with adenovirus to provide the helper function. The temperature sensitive phenotype was still present when using adenovirus to supply the helper function. This production me thod was evaluated because other investigators [64] have used this method for their temperature sensitive mutant studies. Briefly, flasks were transfected for 5-7 hours and then infected with adenovirus (MOI=2) and incubated at 32 C, or 37 C or 39.5 C. Plat es were incubated for 48 to 60 hours and cells were harvested by aspirating them off the cell surface. Cells were pelle ted by centrifugation. Pellets were resuspended in 30 mls Lysis buffer. The cell lysate was prepared by repeated freeze thaw cycles of the cell pellet. The lysate was brought to 25% with Ammonium sulfate in an oakridge tube and a 25% ammonium sulfate precipation was performed. This was incubated on ice for 1 hour, then centrifuged 10 minutes at 5000 x g (8000 rpm). The s upernatant was transferred to new oakridge tubes and brought to 50% satu ration with ammonium sulf ate to precipitate rAAV and adenovirus. These were incubated on ice for 1 hour and then centrifuged for 20 minutes at 12,000 X g (12,000 rpm). After removing the supernat ant, pellets were centrifuged one more time for 5 minutes at 5000 X g (8,000 rpm) a nd residual liquid was removed. This pellet contained AAV and Ad and was diss olved in 1.37 g/ml CsCl. Cesi um gradients were setup and underlayed with 1.5 g/ml CsCl. Centrifuga tion was performed for 36 hours at 288,000 X g (41,000 rpm). The CsCl gradient was dripped and fractions were collected. Refractometry was

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81 performed on the fractions. Positive fractions were dialyzed into column buffer to prepare the samples for heparin chromatography. Virus was puri fied on a heparin column and fractions were collected. Green cell assays were performed on the column fractions. Mut33 that formed capsids at the permissive temperature we purified by heparin chromatography; however, Mut33 produced at the non-permissive te mperature, did not bind to th e heparin column. This could occur for a number of reasons in addition to the possibility that this mutant may form pentamers at the nonpermissive temperature. Images of the tranfected cells at 32 C, 37 C and 39.5 C for Mut33 and pIM45 contols are shown in Figure 3-23. This da ta shows that the transfection efficiency is similar under the conditions described. Green cell assays at 32 C and 37 C of the cell ly sate was performed for each of the conditions. Cells tran sfected at 32 C were lysed and the infectivity assay was performed at 32 C and 37 C. As shown in Figure 3-24, virus wa s produced under these conditions. Images of the transfections at 37 C prior to harvest are shown in Figure 3-25. The green cell assay after transfec tion at 39.5 C is shown in Figure 3-26. For pIM45 virus is produced, but for Mut33, no virus is produ ced at the nonpermissive temperature. Studies of Mut33 have been ongoing, as Mut 33 may provide the best opportunity for the isolation of a potential pentameric assembly in termediate for AAV-2 that will be useful as a substrate in an in vitro assembly assay. One issu e with developing an in vitro assembly assay is the substrate that is used as the starting material. One cannot be certain that th e intermediate isolated for use in developing an in vitro assemb ly assay is not a dead-end product of assembly. If the substrate is a dead-end product of assembly then regardless of the conditions tested, those subunits will never be able to be built into a macromolecular capsid. For Mut33, the ability to produce capsids at the permissive temperature but not at the nonpermissive temperature may

PAGE 82

82 suggest that these intermediates, if they can be isolated may be ab le to be subjected to conditions that will allow for an in vitro assembly assay. This would be useful in sh edding light on the role that the nonstructural Rep protei ns play in AAV capsid assembly. Based on available data on assembly and packaging, with DNA packaging occu rring at the 5-fold axis of symmetry, the nonstructural Rep proteins could play a stabilizing role at the weak 2-fold axis of symmetry and it is also possible that these weak interactions allow for structural changes to occur in the capsid during the virus life cycle while the capsid integrity is mainta ined by the stronger interactions at the 3-fold and 5-fold axis of symmetry. Figure 3-23. Transfection of Mut 33. Cells were transfected with either pIM45 and UF5, or Mut33 and UF5, and infected with adenoviru s at an MOI = 2 at either 32 C, 37 C, or 39.5 C. Transfection efficiencies were similar under all conditions. Panel A. Mut33 transfection at 32 C. B. Mut 33 tr ansfection at 37 C. C. Mut33 rransfection at 39.5 C. D. pIM45 transfection at 32 C. E. pIM45 transfection at 37 C. F. pIM45 transfection at 39.5 C. 32 C 37 C 39.5 C Mut33 pIM45 Transfection A B C D E F

PAGE 83

83 Figure 3-24. Green cell assay of transfections at 32 C. Lysates were prepared from the cells transfected in Figure 3-23 and a green cell assay for infectivity was performed at 32 C, or 37 C. A. Green cell assay at 32 C of Mut33 produced at 32 C. B. Green cell assay at 37 C of Mut33 produ ced at 32 C. C. Green cell assay at 32 C of pIM45 produced at 32 C. D. Green cell assay at 37 C of pIM45 produced at 37 C. Mut33 32 C Tnf Green Cell Assay Infectivity Data Transfections at 32 C pIM45 32 C Tnf 453232 333232 333237 32 C Inf 37 C Inf A B C D 453237

PAGE 84

84 Figure 3-25.Transfections at 37 C. Green cell assa y of transfections at 37 C. Lysates were prepared from cells transfected at 37 C and a green cell assay for infectivity was performed at 32 C, or 37 C. A. Green cell assay at 32 C of Mut33 produced at 37 C was not performed. B. Green cell assay at 37 C of Mut33 produced at 37 C. C. Green cell assay at 32 C of pIM45 produced at 37 C. D. Green cell assay at 37 C of pIM45 produced at 37 C. 453732 333737 Mut33 37 C Tnf Green Cell Assay Infectivity Data Transfections at 37 C pIM45 37 C Tnf 32 C Inf 37 C Inf B C D 453737

PAGE 85

85 Green Cell Assay for Infectivity Figure 3-26. Green cell assay afte r transfection at 39.5 C. Green cel l assay of transfections at 39.5 C. Lysates were prepared from cells transfected at 39.5 C and a green cell assay for infectivity was performed at 32 C, or 37 C. A. Green cell assay at 32 C of Mut33 produced at 39.5 C. B. Green cell a ssay at 37 C of Mut33 produced at 39.5 C. C. Green cell assay at 32 C of pIM45 produced at 39.5 C. D. Green cell assay at 37 C of pIM45 produced at 39.5 C. E. Image of cells after infec tion with Mut33 that was produced at 39.5 C and incubated at 32 C. 4539537 4539532 3339537 Mut33 39.5 C Tnf pIM45 39.5 C Tnf 32 C Inf 37 C Inf A B C D Transfections at 39.5 C 3339532 3339532 E

PAGE 86

86 CHAPTER 4 STUDIES OF THE AAV CAPSID IN SOLUTION Introduction Proteolytic structural mapping The three-dimensional struct ure of several autonomous parvoviruses [53, 93, 94], plus t hose of AAV-2 [48], AAV-4 [50] and AAV-5 [51], have been determined by X-ray crystallography or cryoelect ron microscopy (cryo-EM). Several features of the virus cannot be determined based on the cr ystal structure. For example, the unique Nterminus of VP1, as well as the unique N-term inus of VP2, are not present in the crystal structures of AAV. This is likely due to the f act that these monomers are present in the AAV capsid in low abundance, (~5 copies each), and icos ahedral averaging is used to solve the crystal structure. In addition to bei ng present in low abundance, as discussed in Chapter 2, the Nterminus of VP1 and VP2 may be disordered in assembled capsids which would preclude crystallographic structure dete rmination of these regions. In the near future, customized AAV gene ther apy vectors may consist of modified capsids that allow for specific targeting to treat patients with various diseases. The 3D structures of the AAV capsids will provide a basis fo r rational vector design, however, the 3D structures available for autonomous parvoviruses and dependoviruse s only provide a snapshot of the capsid topology in a low energy conformation. Our knowle dge about the AAV viral capsid structure in solution is limited; however, this structure must be dynamic to carry out the various functions required for viral attachment and entry, as well as trafficking within the cell. AAV-2 has been shown to utilize heparin sulfate proteoglycan as a cell surface receptor, and the specific amino acid residues involved in this inte raction have been mapped to basi c amino acids at the three-fold axis of symmetry on the capsid surface, includi ng R585 and R588 [95, 96]. Studies of the AAV capsid proteins have shown that the unique N-terminus of VP1 is required for infectivity [69].

PAGE 87

87 Cryo-EM studies have shown that the unique N-term inus of VP1 is internal to the capsid based on additional density at the 2-fo ld axis of symmetry [97]. In vitro upon heat treatment of AAV capsids, it has also been shown that this regi on can be externalized. Mutagenesis experiments have shown that this externalizat ion occurs through the pore at the 5-fold axis of symmetry [70]. The high resolution x-ray crystallography data is unable to address these dynamic changes which must occur as part of the virus life cycle. For several viruses, peptide mapping has been us ed to address structural changes that occur in solution. Peptide mapping was first used in 1979 to study the majo r capsid protein of bacteriophage T4 [98]. Since this study, peptide mapping has b een utilized to study the capsid structure of DNA viruses, such as herpes virus, and canine parvovirus, as well as for many RNA viruses, such as reovirus, rotavirus, vescicular stomatitis virus, influenza virus and tetraviruses. Peptide mapping has been utilized to demonstrate that certain amino acids are surface associated. In addition, peptide mapping has been utilized to evaluate dynamic structur al changes that occur in the capsid. In this study, peptide mapping of the AAV virion is developed as a tool for identifying regions of the AAV capsid that are flexible, accessible, a nd surface associated. Mapping of these regions of the capsid may provide a valuable tool for predicting locations on the capsid surface that can tolerate insertions, which may be useful for improved targeting of AAVserotypes. With 11 known AAV serotypes and up to 100 genotypic va riants, this method may provide a starting point for identifying locations on the capsid to mutate, especially if the crystal structure is not available. Trypsin digestion of AAV-2. Historically, AAV has been shown to be remarkably stable and generally resistant to pr oteases [99]. However, occasio nally, AAV-2 vector preps had additional protein bands when analyzed for pur ity when assayed on polyacrylamide gels and

PAGE 88

88 stained. To determine if these additional bands were degradation products or smaller proteins that co-purified with the AAV stru ctural proteins, a western blot was performed and probed with polyclonal antibody to AAV-2. This is shown in Figure 4-1. This assay verifies that the fragments are degradation products of AAV-2 that co-purified with the intact virus. The classical method for purifying AAV-2 vectors in cluded using trypsin, deoxycholate and the performing cesium chloride gradients followed by heparin chromatography. To determine if trypsin was responsible for the additional band s, AAV2-GFP vector virion s were produced as described in the materials and methods and digest ed with trypsin. For proteolyic mapping of the AAV-2 capsid, several antibodies are available that detect various regions of the AAV capsid proteins [65, 100]. The B1 antibody epitope is on the C-terminal end of the capsid protein, and is primarily internal at the 2-fold ax is of symmetry in assembled capsids. This antibody is useful in detecting denatured AAV proteins and the epitope is highly conserved among the AAV serotypes. A1 antibody recognizes the unique N terminal region of VP1, while the A69 epitope is in the N-terminal region of VP2 for AAV-2. Polyclonal antibodies have been produced to AAV2 capsids, as well as other serotypes. Figure 42 is a western blot of AAV2-GFP digested with trypsin as described in the mate rials and methods. Odd lanes (lan e 1, 3, 5 and 7) are undigested AAV2-GFP. Even lanes (lane 2, 4, 6 and 8) have been digested w ith trypsin. Lanes 1 and 2 are probed with polyclonal antisera to AAV-2 resulting in the detection of two digestions products at ~40 kDa, and a smaller 15 to 20 kDa fragment. La nes 3 and 4 are probed with B1. This shows that the smaller 15 to 20 kDa frag ment is derived from the C-terminal region that is common to all three of the structural proteins, VP1, VP 2, and VP3. This fragment was named VP1,2,3T. The ~40 kDa N-terminal fragment is not detectable since the N-terminal fragment no longer harbors the B1 epitope. This fragment was designate d VP3T. Lanes 5 and 6 are probed with A69

PAGE 89

89 Figure 4-1. Western blot of se veral AAV-2 preps. Lane 1 AAV-2 standard. Lane 2 AAV-2 sample. Lane 3 AAV-2 sample. Lane 4 AAV-2 sample. Using polyclonal antibody, AAV fragments are detectab le in the sample lanes. antibody. This detects the unique N-terminal re gion that is common in VP1 and VP2. Lane 6 shows that upon cleavage with trypsin there is a unique fragment th at runs just smaller than VP3. A69 does not recognize VP3, so this fragment is the result of a C-terminal trypsin cleavage event that results in the N-terminal fragment shifting in size after proteolysis to approximately 50 kDa. This fragment was designated VP2T. Lanes 7 an d 8 are probed with A1 antibody. Lane 8 shows a shift after proteolysis in the size of VP1. Because it is detectab le by A1 and the epitope for the antibody is on the N-terminus of VP1, this fr agment was designated VP1T. Figure 4-2 also shows a diagram of the approximate locations of the epitopes for the various antibodies to the AAV structural proteins, as we ll as and arrow which indicates the trypsin cleavage site. Fine mapping of the trypsin cleavage site. Trypsin cleavage sites of AAV-2 VP1 were determined using the program Peptide Cutter on the ExPASy server (www.expasy.org ). Based on the potential basic residues wher e trypsin can cleave, these sequences were entered into the program Peptide Mass on the ExPASy server and the mass of each trypsin fragment was determined. This is shown in Table 4-1. Theoreti cal prediction of trypsin sites in the capsid G-H loop [48], comprising amino acid residues 416 to 645, and fragment masses for AAV-2 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 250 kDa 150 kDa 20 kDa 1 2 3 4

PAGE 90

90 Figure 4-2. Tryptic mapping of full AAV-2 cap sids. Purified full AAV-2 virions (FT/iodixanol/Heparin) were tr eated with trypsin, and the cap sid proteins were probed with the indicated antiAAV antibodies (AAV-2 polyclonal, A1, B1, A69). The epitope locations of the monoc lonal antibodies and the likely trypsin digestion site are indicated on the schematic diagram. A, AAV-2 full capsids; B, AAV-2 full capsid digested with trypsin for 24hours. The assi gnment of the tryptic fragments are given on the right. VP1T is the digestion product of VP1, VP2T is the digestion product of VP2, VP3T is the digestion product of VP 3, and VP1,2,3T is the common C-terminal trypsin digestion product of VP1, VP2, and VP3. VP1 indicated that trypsin digestion at amino acid R566, R585, and R588 would produce fragments of 18.8, 16.8, and 16.4 kDa, respectively, wh ich are within the ra nge expected for the VP1,2,3T C-terminal fragment th at is recognized by B1 antibody (Figure 4-2, lane 4). The mass of this fragment was confirmed to be 16,461 Da by matrix-assisted laser desorption/ionization

PAGE 91

91 time-of-flight mass spectroscopy (M ALDI-TOF) analysis, which is close to the theoretical mass of the C-terminal peptide that results from cleavage at R588 (Tab le 4-1). The R588 tryptic site was confirmed by N-terminal se quencing, which mapped residues 589QAATADVNTQGV600 as the terminal peptide for the VP1,2,3T fragment. Table 4-1. Predicted AAV VP1 tryptic fragment mass from cleavage in the G-H loop. Trypsin cleavage sites of AAV-2 VP1 were determined using the program Peptide Cutter found on the ExPASy server (www.expasy.org ). Based on the sites where trypsin cleaves, these sequences were entered into the program Peptide Mass on the ExPASy server and the mass of each trypsin fragment was determined. Fragment Average Mass (Da) Peptide VP1 81944.65 All Cys in reduced form 1 549 61112.40 VP1 start to 549 137 549 45757.24 VP2 start to 549 203 549 39231.12 VP3 start to 549 550 735 20850.26 C terminal end 1 556 61912.28 VP1 start to 556 137 556 46557.12 VP2 start to 556 203 556 40031.00 VP3 start to 556 557 735 20050.38 C terminal end 1 566 63128.65 VP1 start to 566 137 566 47773.49 VP2 start to 566 203 566 41247.37 VP3 start to 566 567 735 18834.01 C terminal end 1 585 65176.85 VP1 start to 585 137 585 49821.69 VP2 start to 585 203 585 43295.57 VP3 start to 585 586 735 16785.81 C terminal end 1 588 65504.19 VP1 start to 588 (VP1T) 137 588 50149.03 VP2 start to 588 (VP2T) 203 588 43622.91 VP3 start to 588 (VP3T) 589 735 16458.47 C terminal end (VP1,2,3T) 1 609 67743.68 VP1 start to 609 137 609 52388.52 VP2 start to 609 203 609 45862.40 VP3 start to 609 610 735 14218.98 C terminal end

PAGE 92

92 Trypsin-Treated Virons Remain Intact. The presence of tryptic fragments in the AAV2GFP vector preps indicated that the cleaved pro ducts remain tightly associated with the capsid, as these fragments are still present after virions are subjected to cesium chloride gradients. The buoyant density of these capsids are ~1.40 g/cm3, indicating a protein and DNA composition that is similar to other vector preps. The value is slightly less than the de nsity of wt AAV-2 (1.41 1.45 g/cm3) because the rAAV2-GFP vector is sma ller (4331 nucleotides) than the wt AAV genome (Genbank AF043303). Figure 4-3A shows Nega tive-stain EM analysis of the trypsintreated and untreated purified rAAV2-GFP samples. This confirmed the intact nature of the treated capsids (Figure 4-3A). However, the ne gative staining pattern suggested a difference between the samples in their permeability to ur anyl acetate, indicating a possible structural rearrangement or flexibility due to the cleavage event. Antibodies, such as A20, are available that recognize conformational epitopes that are present only on assembled capsids for AAV-2. Na tive immuno-dot blot analysis with the A20 anti-capsid antibody showed that virions remain intact following 24 hours of trypsin digestion. Figure 4-3B is a native immuno-dot blot of AAV2-GFP virions that have been digested with trypsin in a time course experiment for the vari ous times indicated. Control samples were heated at 65C or 75C for 30 minutes. Previously, it had been shown that treating AAV-2 65C, capsids were still intact and rec ognized by A20, but that at 75 C, capsids are no longer intact as shown by a loss of signal when probed with A20. An immunodot bot was also performed after exposing AAV2-GFP to SDS and Methanol (MeOH) This is shown in Figure 4-3C. AAV-2 virions were digested with trypsin for 5 hours or 12 hours (T5 and T12) and treated with 0.1% SDS and 20% MeOH for 2 hour s at 37 or 45 C, transferred to n itrocellulose and probed with B1 antibody. The B1 epitope was not recognized until th e virions were trpsini zed and treated with

PAGE 93

93 Figure 4-3. Trypsinized AAV-2 viri ons remain intact. A. Elect ron microscopy. Negative stain electron micrographs of untreated rAAV2 -GFP capsids (F-T/iodixanol/Heparin) or trypsin-treated rAAV2-GFP virions. The sa mples were viewed on a Hitachi H-7000 transmission electron microscope at 30,000X magnification. Each high-powered insert is 70,000X. B. Immuno dot-blot. rAAV2-GFP virions were digested with trypsin and samples were taken at the i ndicated time points (hours). An immuno dotblot was performed on undigested sample (T 0), as well as sample digested for 1, 5, 12, or 24 hours with trypsin (T1, T5, T12, T24, respectively). Duplicate samples were probed with either A20 or B1 antibodi es. Also included are native virions heated at 65oC or 75oC for 30 minutes, treatments that are known to expose the B1 epitope [70]. C. Immuno dot-blot with SDS and MeOH. To mimic the Western transfer conditions, AAV-2 virions (T0) were digested with trypsin for 5 and 12 hours (T5 and T12, respectively), and treated with 0.1% SDS and 20% MeOH for 2 hours at 37oC or 45oC, transferred to nitrocellulo se, and probed with B1 antibody. Not treated Trypsin treated

PAGE 94

94 SDS and MeOH at 45 C. Undigested control viri ons treated with SDS and MeOH at 45C, and trypsinized virions treated w ith SDS and MeOH at 37 C we re not detectable by the B1 antibody. This data indicates that th e digested AAV-2 capsids appear intact by EM, buoyant density on cesium chloride gradients, as well as A20 nativ e dot blot analysis. The internal disposition of the majority of the C-terminal proteolytic fr agment (~70% of residues 589 735, Figure 4-7) ensures that the fragment remains associated with the capsid in 3.3 M CsCl during purification, as well as in 0.1% SDS and 20% MeOH used in the immuno-dot blot (Fig ure 4-3C), and during heparin chromatography. However, heating the tryps inized virions in the presence of 0.6M DTT for mass spectroscopy and N-terminal sequencing, or heating in the presence of SDS and reducing agent for denaturing ge l electrophoresis was able to dissociate the VP1,2,3T fragment from the core capsid. Since no cysteine residue s are found in the VP1,2,3T C-terminal fragment, the interaction of this fragment with the capsi d is due to non-covalent interactions that are disrupted when the capsid structure is de natured by heat and reducing agent. The crystal structure of AAV-2 did not show a break in the polypeptide chain at position R588. This suggests that the majority of the 60 R588 sites remain uncleaved by the mild trypsin treatment used during the purifica tions reported here, and for sample s used for crystallization of AAV-2, which generated the additional bands observe d in Figures 4-1 and 4-2. The capsids thus maintain the properties and structure of the nativ e virus while in the crystalline state but appear to have increased flexibility as shown by differe ntial staining in EM, and exposure of the B1 epitope in the presence of SDS and MeOH at 45C (Fig. 4-3C). However, for future studies, it may be prudent to avoid incor porating proteases into AAV purifi cation schemes for structural studies or gene therapy applica tions where proteolysis may not onl y alter the caps id structure,

PAGE 95

95 but also chromatographic propertie s and vector potency. In retros pect, the use of trypsin during purification of rAAV-2 vectors and the heat inactivation of the helper adenovirus at 56oC, which can expose the N-terminus of AAV VP-1 [70], ma y have been responsible for the low vector titers and low transduction effici encies obtained during the initial evaluations of this vector system. Proteolytic digestion can distingu ish genome-full and empty capsids To determine if genome-full (ie. virions) and empty capsids can be distinguished by proteolysis, purified full rAAV2-GFP virions and empty AAV-2 capsids were digested with trypsin. Similar to the differential proteolytic sensitiv ity reported for members of th e autonomous parvoviruses [101], the full and empty AAV-2 capsids differed in their sensitivity to trypsin cleavage (Figure 4-4). The unique proteolytic fragment (VP2T) deri ved from VP2 of the full capsids was seen migrating slightly ahead of VP3 when probed with the polyclonal sera (Fig. 4-4A, lane 2) or the A69 monoclonal antibody (Fig. 4-4A, lane 10), bu t was not observed in the digested empty capsids (Fig. 4-4A, lanes 3 and 11) In addition to the absence of the VP2T fragment in empty capsids, full length VP2 was not de tected, indicating complete diges tion of this protein (Figure 44A, lanes 3 and 11). A trypsin digestion timec ourse showed that VP2 and the VP2T fragment are more sensitive to digestion in empty capsids compared to full capsids (Figure 4-4B). During the time course, less VP2T is seen in empty capsids than full capsids, and by 12 hours VP2T is not detected in empty capsids, whereas the VP2T fr agment is still detected in full capsids even after 24 hours of digestion, as seen in Figure 4-4A. Chymotrypsin, with a different cleavage site sp ecificity (W, F, Y, M, L) than trypsin, was also able to differentiate between the full rAAV2-GFP capsids and the AAV2 empty capsids (Figure 4-4C). The empty capsids were digested more rapidly than full capsids, as observed with

PAGE 96

96 trypsin treatment, but with this pr otease all of the VPs were dige sted within 2 hours to fragments that were either too small to be resolved by 10% SDS-PAGE or recogn ized by polyclonal sera (Figure 4-4C). Comparison of the proteolytic susceptibility of full and empty AAV-2 capsids indicated that there are recognizab le structural differences between these capsids in solution that involves VP2 (Figure 4-4). This may be due to stabilization of the capsid by packaged DNA, in addition to different conformations of the capsi d surface loops. In addition, the AAV-2 VP1 and VP2 N-termini become accessible in full particles at 65oC, as A69 and A1 antibody reactivity is seen in the 110S (full) virus sp ecies but not in empty capsids [65, 70]. It is also interesting to note that Bleker et al. [85] were able to cleave the N-termini of AAV-2 VP1 with trypsin following heating of full capsids resulting in a fragment that maintained the A69 and B1 antibody epitopes. Since we do not detect the VP 2 or VP2T bands following digestion of empty capsids, trypsin digestion for 24 hours may degrade the VP2 protein to the point where it is not recognized by either the polyclona l, B1, or A69 antibodies. A lternatively, the cleavage of both the VP2 Cand N-termini of empty capsids may re sult in a fragment that is indistinguishable from VP3T. Since differences in the parvoviru s capsid surface, consisting of the overlapping capsid protein region, is i ndistinguishable in full and empty cap sid structures determined using X-ray crystallographic or cryo-EM techniques (except for differen ces in internal regions that contact ordered DNA) [57, 58], the use of proteoly sis and specific antigenic mapping of cleavage products provides a means to distinguish thes e species in solution. In addition, proteolytic distinction between full and empty capsids can be used in conjuction with quantitative methods used to determine full and empty capsid ratios [102]. Proteolytic digestion can distinguish AAV serotypes. To further confirm that R588 is the only specific site on the AAV-2 capsid su rface susceptible to tr yptic cleavage during

PAGE 97

97 Figure 4-4. Proteolysis distinguis hes full and empty AAV-2 particles. A. Trypsin. Purified full (F-T/iodixanol/heparin) and empty AAV-2 cap sids were treated with 0.02% trypsin, and the capsid proteins were probed with th e indicated antibodies (AAV-2 polyclonal, A1, B1, A69). Lanes A, AAV-2 full capsids; lanes B, AAV-2 full capsids digested with trypsin for 24 hours; lanes C, AAV-2 em pty capsids digested with trypsin for 24 hours; lanes D, AAV-2 empty capsids. B. Trypsin time course. Purified full (FT/iodixanol/heparin) and empty AAV-2 virions were treated with trypsin for the time (hours) indicated above each lane and probe d with anti-AAV2 polyclonal antisera. C. Chymotrypsin time course. Purified full (F-T/iodixanol/heparin) and empty AAV-2 capsids were treated with chymotrypsin for the time (hours) indicated above each lane and the capsid proteins were probed with anti-AAV2 polyclonal antisera.

PAGE 98

98 proteolysis, we compared the suceptibility of rAAV1-GFP, rAAV2-GFP, and rAAV5-GFP virions to proteolytic cleava ge (Fig. 4-5A). AAV-1 provi ded an example of a highly homologous serotype to AAV-2 (83%) that doe s not contain R588 a nd AAV-5 provides an example of a less homogolous (59%) serotype to AAV-2 (Figure 4-5B). As seen in Figure 4-5A, AAV-1 and AAV-5 are resistant to trypsin digest ion after 24 hours of incubation, even though AAV-1 VP1 has 65 potential cleavage sites and AAV-5 VP1 has 60 potential cleavage sites; further supporting the iden tification of R588 as the specific tr ypsin cleavage site in AAV-2. The lack of trypsin digestion of AAV1 at other possible tryptic sites w ithin the C-terminal stretch of VP amino acids, for example, K567, R610, and K621 was not unexpected because of the lack of AAV-2 digestion at the equivalent locatio ns, R566, R609, and K620 (Figure 4-5B). A difference in the susceptibility and se nsitivity of the AAV1-GFP, AAV2-GFP, and AAV5-GFP virions to proteolytic cl eavage was also evident from digestion with chymotrypsin. Chymotrypsin digestion of AAV-2 (Figure 4-5C) ge nerated similar fragments to those seen with trypsin treatment (Figure 4-5A) and a novel 27 kDa fragment was de tected by polyclonal antisera at 5 hours that was not B1 an tibody reactive. AAV-1 was more resistant to chymotryptic cleavage compared to AAV2, with all fragments of AAV-2 being undetectable, except for the Cterminal 18kDa fragment, at 24 hours. Five Cterminal AAV-1 fragment s were generated at 12 hours, ranging in size from 30kDa to 50kDa, that are recognized by polycl onal sera; two of these fragments harbor the B1 antibody epitope, with the 30kDa fragment as the most C-terminal fragment (Figure 4-5C). Base d on primary sequence, chymotryps in is expected to cleave denatured AAV-1 VP1 148 times and AAV-2 VP1 145 times, but as shown, only a limited number of cleavage sites are ac cessible. Additionally, AAV-5 with 139 potential cleavage sites was not cleaved by chymotrypsin (Figure 4-5C). The proteolytic data was confirmed by

PAGE 99

99 AAV Serotype Position2 1 3 4 5 6 7 8 910 11A 556 K N N T N N N D K S N Q 566 R K R A Q K R K K K A R 585 R S S S S S A Q S Q N S 588 R T T N T T T T A T T T 609 R R R R R R R R R R R R 620 K K K K K K K K K K K K Figure 4-5. AAV-2, AAV-1 and AAV5 capsids can be distinguish ed proteolytically. A. Trypsin time course. Purified AAV-1 (F-T/iodixanol/Q), AAV-2 (FT/iodixanol/heparin), and AAV-5 (F-T/iodi xanol/Q) virions were incubated at 37oC without trypsin (0), or treated with 0.02% trypsin for the time ( hours) indicated above each lane. The capsid proteins of AAV-1 and AAV-2 were probed with anti-AAV2 polyclonal and AAV-5 was probed with anti-A AV5 polyclonal sera. B. Alignment. Amino acid alignment of the G-H loop [50] region 556-620 of AAV serotypes 1 to 11 and Avian AAV. Arginine or Lysine (R or K) residues, the target residues for trypsin, are shown in blue. Amino acid position numbering is based on VP1 of AAV2. correlating the effect of digestion on infectivity (Figure 4-6) where the infectious titers of the vectors following 24 hours of digestion with tr ypsin or chymotrypsin resulted in AAV-2 losing 3-4 logs of infectivity, but AAV-1 and AAV-5 lost less than 1 log, indicating that chymotrypsin cleavage of AAV-1 is at a site(s) less important for infectivity. The predictive power of the

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100 primary sequence and 3D structuredata was limited with respect to identify ing protease cleavage sites on AAV intact capsids. Numerous potential trypsin and chymotrypsin cleavage sites exist in the AAV-1, AAV-2, and AAV-5 VP1 primary sequen ces, but access to these sites could be dictated by their stable or transient presence on the virion surface. In some cases, surface loops in solution may not be exactly positioned as in the 3D structures. Analysis of the primary sequence of AAV-1, AAV-2, and AAV-5 predicted numer ous tryptic and chymotryptic sites, but the actual proteolysis generated a pattern of produc ts resulting from cleavage at specific sites. In addition, a search for the position of K and R re sidues on the AAV-2 crystal structure and a 3D model generated for AAV-1 showed residues, other than R588 in AAV-2, believed to be accessible on the capsid surface that were not clea ved (Figure 4-7A and B), which may be due to inaccessibility or steric hindrance in solution. Proteolytic analysis compleme nts 3D-structure analysis. The available crystal structure of AAV-2 [48] and homologous VP3 models, generated for AAV-1 and AAV-5 based on a structural alignment with AAV-2 [50], provi ded a means to visua lize the solution data through mapping of the trypsin cleavage site onto the capsid surface (Figure 4-7). The R588 mapped by N-terminal sequencing as the trypsin cl eavage site is located on the capsid surface in the finger-like projections surr ounding the icosahedral 3-fold axis, and forms part of the basic patch that is responsible for AAV2s heparin binding interaction [ 95, 96]. The visualization of AAV-1 and AAV-5 with AAV-2 capsids in this region clearly show ed that the cleavage event was specific, since adjacent basic residues we re not susceptible, and AAV-1 and AAV-5, which do not contain R588, are resistant. The cleaved residue R588 of AAV-2 is located in the G-H loop that protrudes from the capsid surface where it is accessible to hepa rin binding and trypsin cleavage (Figure 4-7). Capsid protein loops, including the G-H loop, are able to tolerate

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101 Figure 4-6. AAV-2, AAV-1, and AAV5 have different susceptib ility to Chymotrypsin. A Chymotrypsin time course. Purified AAV-1, AAV-2, and AAV-5 were incubated at 37oC without chymotrypsin (0) or di gested with chymotrypsin at 37oC for the time (hours) indicated above each lane. The capsid proteins of AAV-1 and AAV-2 were probed with anti-AAV2 polyclonal or B1 antisera, and AAV-5 was probed with antiAAV5 polyclonal sera. The asterisk indi cates an additional AAV-2 band observed with chymotrypsin that was not seen fo llowing trypsin digestion in Fig. 6A. B. Infectivity. Infectious titering of rAAV1-GFP, rAAV2-GFP, and rAAV5-GFP virions was performed following digestion wi th trypsin or chymotrypsin by infecting C12 cells in the presence of Adenovirus (reported as the average SDEV from three separate infections). AAV-1 and AAV-5 inf ect C12 cells less e fficiently than AAV-2 [103]. 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10 1.00E+11 0510152025HoursTiter (IU/ml) AAV1 Trypsin AAV2 Trypsin AAV5 Trypsin AAV1 Chymotrypsin AAV2 Chymotrypsin AAV5 Chymotrypsin

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102 Figure 4-7. Capsid structure. A. Capsid mono mer. Ribbon representation of an AAV-2 VP3 monomer (amino acids 217-735) rotated 90o from standard icosahedral orientation. -strands and -helices are represented as arrows and coils, respectively. Small arrows indicate the location of R588 (blue s pheres) and the B1 epitope at the extreme C-terminus (green). The 2-fold (oval), 3fold (triangle) and 5-fold (pentamer) axes are indicated. B. Side view of an AAV-2 VP3 trimer. Amino acids R585 and R588 in the three G-H loops from three VP3 monomers (gray, salmon, wheat) are represented as blue spheres and indicated by arrows.

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103 differences in conformation when the AAV capsid structures are compared [50]. These loops intertwined to form the characte ristic AAV surface protrusions that decorate the core capsid and provide the immunogenic, tropic, and proteolytic determinants. The cleaved AAV-2 C-terminal protein fragment, residues 589 to 735, is able to remain associated with the capsid because thecleavage site is on the outer su rface of a 3-fold protrusion, not part of the core. The eight stranded beta barrel forms the core stable capsi d that remain intact following trypsin cleavage. Of note are the surface accessible R and K residu es in the G-H loop that are common in AAV-1, AAV-2, and AAV-5 (Figure 4-8), but are not cleaved by trypsin. Thus it is possible that proteolytic digestion may be lim ited by steric constraints to th ose residues found on loops that protrude from the surface. Results in solution c onfirm that the region of the G-H loop containing residue R588 is surface accessible as seen in the cr ystal structure, and the presence of heparin is not required for exposure. The B1 epitope (residues 726-733) is located on the wall/floor of the dimple at the icosahedral 2-fold axis of th e AAV-2 capsid, close to a buried stretch of G-H loop amino acids leading up to the tryptic cleavage site (Figure 4-8 I-III). Interestingly, the B1 epitope, found on the thinnest regi on of the AAV-2 capsid, contains residues that are exposed on both the interior and exterior surfaces (Figure 4-8 III ). The isolation of numerous AAV serotypes and genotypic variants [23-26, 104], and the obs ervation that each virus serotype has unique cellular recognition and transduction phenotypes dictated by the capsid protein sequence has generated a need to fully charac terize the capsids of these viru ses, including their structural features. Here we use proteoly tic digestion combined with available information on antigenic epitopes and mass spectroscopy to analyze the AAV capsid in solution. We show that proteolysis is able to generate a characteristic cleavage pattern that can be used to distinguish full

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104 Figure 4-8. AAV-1, AAV-2 and AAV-5 Homology m odels. A. Capsid surface basic amino acids. AAV-1, AAV-2, and AAV-5 capsid surface structures at the 3-fold axis of symmetry w ith the indicated basic amino acids of the salmon reference monomer highlighted in blue. B. AAV-2 VP3 dimer viewed down the 2-fold axis. (I) Polyglycine trace, (II) Surface, and (III) Close-up view of the surface exterior (top) and interior (bottom) of an AAV-2 VP3 dimer (cyan and gray) showing the locations of R588 and the B1 epitope (color scheme as in A, B, and C). The re sidues in the G-H l oop are colored limon and orange in the reference monomer and 2-fold related monomer, respectively. Th e coordinate files used in A-D were based on the X-ray crystallographic st ructure of AAV-2 [48] (PDB accession N o. 1LP3) and homologous models were generated for AAV-1 and AAV-5 using a st ructure-based alignment with AAV-2. Co ordinate files for the homologous models were generated using VIPER [105]. Figures were generated using PyMOL.

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105 from empty AAV-2 capsids and diffe rentiate between two highly homologous serotypes, such as AAV-1 and AAV-2, as well as between less homo logous serotypes (AAV-5). The size and sequence of the proteolytic products in addition to the origin of the products (i.e. VP1, VP2, or VP3) were determined. Furthermore, availa ble structural information enabled the 3D visualization of the location of the AAV-2 cleavag e and B1 antigenic sites on the surface of the virion. Proteolytic analysis has practical applications. AAV-2 is currently being evaluated in a variety of human clinical trials [106] and clinical trials are be ing planned that utilize other serotypes. Prior to use in th e clinic, an important product rele ase test is the confirmation of product identity to comply with current Good Manufacturing Pract ices (cGMP) regulations. Identity release testing involves not only confirmation of the vector genome that is packaged, but also the serotype of the capsid. Since immunologi c reagents generated to one serotype have the potential to cross react with other serotypes, screening diffe rent proteases on different AAV serotypes may generate a capsid fi ngerprint database that could di stinguish the 11 serotypes, as well as the more than 100 genotypi c variants that have been recently isolated [23]. We have shown that the highly homologous AAV1 and A AV2 capsids can be distinguished from each other and from AAV5 by comparing their trypsin and chymotrypsin digestion patterns (Figure 45). An assay capable of distinguishing differe nt serotypes that utilizes the commercially available reagents applied in these studies wi ll be useful to the gene therapy community. In addition, vector targeting has gained a great deal of interest, and is intended to reduce non-specific uptake by non-target organs and increase the efficiency of uptake into target tissue. Cell-specific epitopes and recepto rs have been engineered in to the AAV capsids and have resulted in efficient targeti ng [107-109]. The sites in the AAV capsid sequence that can

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106 accommodate insertions have been identified main ly through mutagenesis studies. In the absence of 3D structure, proteolytic mapping in combin ation with mass spectroscopy and N-terminal sequencing, as is demonstrated in these studies, can identify exposed, flexible loops of the AAV capsid. These regions can be tested for their ab ility to accommodate epit ope/peptide insertions, as has been shown for AAV-2 at R587, see Girod, et al. [109]. The first mutant generated for AAV-8 is an AAV-8 capsid with His residues inse rted after amino acid 590 in the AAV8 capsid protein, which is the region in AAV-8 that is homologous to R587 in AAV2[110]. Having an additional approach to identifying insertion sites in the capsid of any AAV serotype can aid in the generation of new targeting vectors. Viruses and cell lines Full (DNA-containing) rAAV2-GFP vector virions were produced by transient transfection of HE K293 cells, lysed with 0.5% deoxyc holate (DOC), and purified on sequential CsCl density gradient s (designated as DOC/CsCl). Pu rification methods incorporated the use of Benzonase at 50U/ml for 30 minutes at 37oC and additionally utilized a final heparin chromatography step for concentration [111] (d esignated as DOC/CsCl/Heparin). rAAV2-GFP, rAAV1-GFP, rAAV5-GFP vector vi rions were also produced by transient transfection of HEK293 cells, lysed by freeze/thaw, and purified on iodixanol gradients followed by heparin affinity or Q-sepharose chromatography (d esignated as F-T/Iodixanol/Heparin or FT/Iodixanol/Q [111]. Alternatively, rAAV2-G FP and rAAV1-GFP vector virions were purified by freeze/thaw and CsCl gradients followed by hepa rin affinity or Q-sepharose chromatography (designated as F-T/CsCl/heparin or F-T/CsCl/Q). Infectivity of the AAV vectors were assayed on C12 cells in the presence of adenovirus as pr eviously described [92]. Empty AAV-2 capsids were made by infecting HEK293 cells with an Adenoviral vector expressing the AAV2 cap ORF (MOI=2). The cells were lysed by 3 freezing/th awing cycles, the cell debris was removed by

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107 centrifugation (3000g for 10 minutes), and DOC wa s added to the supernatant to a final concentration of 0.5%. The supernatant was dige sted with 50U/ml benzonase for 30 minutes at 37oC, filtered (Acrodisc 25mm PF, Pall), and purified by heparin affinity chromatography. Virions and empty capsid preparations were di alyzed into 50mM Tr is-Cl pH 8 containing 100mM NaCl using a 10,000 MWCO membrane (Pie rce), aliquoted, and stored frozen at -20oC or -80oC. Proteolytic digestion. 0.8g of virions (equivalent to ~1.2X1011 capsids) were digested with 5g (0.02% final concentratio n) of trypsin (Gibco) or 80ug of -chymotrypsin (Sigma) in a 25l reaction at 37oC for up to 24 hours. The products of proteolysis were denatured at 100oC using Laemmli sample buffer at final concentrations of 1% SDS and 655mM -mercaptoethanol, and separated by SDS-PAGE. After transferri ng the proteins to nitrocellulose (25mM Tris/192mM Glycine/0.1% (w/v) SDS/20%MeOH for 2 hours at 0.5 Amp in a Criterion-transblot apparatus (Bio Rad) that reached 45oC), they were probed with an tisera to AAV-2 (polyclonal, A1, B1, A69 [65, 100]) and AAV-5 from Progen at dilutions of 1:250 ( polyclonal) to 1:2000 (monoclonal). The AAV-2 polyclonal antiserum cross-reacts with ot her AAV serotypes, but cross-reacts weakly with AAV-5 under our conditions. Based on th e peptides used to generate the monoclonal antisera, B1 (antibody epit ope IGTRYLTR) recognizes AAV1-3, and AAV5-10; A69 (antibody epitope LNFGQTGDADSV) is sp ecific for AAV-2; and A1 (antibody epitope KRVLEPLGL) recognizes AAV-1, AAV-2, AAV-4, AAV-7, AAV-8, AAV-10, and AAV-11. Bands were visualized by chemiluminescence using HRP-conjugated secondary antibodies (Amersham) and captured on X-ray film. Mass spectroscopy and N-terminal sequencing For mass spectroscopy and N-terminal sequencing, DOC/CsCl/heparin purified rAAV2-GFP virions were digested with trypsin (0.02%

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108 trypsin for 24 hours at 37 C) and heated to 100oC for 5 minutes in the presence of 0.6M dithiothreitol (DTT). The comm on C-terminal trypsin digestio n product of VP1, VP2, and VP3 (designated as the VP1,2,3T fragment) wa s isolated by HPLC on a Vydac C4 5 m 150mm 2.1mm ID column eluted using a 15% A to 80% B buffer gradient (Buffers : A is 0.1% Tri Fluoro Acetic Acid (TFA) in H2O and B is 0.1% TFA in Acetonitrite (CH3CN)) over 30min at a flow rate of 200 l/min. Detection was at 215 nm and column peak #3 of 5 peaks (~0.5ml fractions) contained VP1,2,3T as verified by Western blotting with polyclonal and B1 antisera. For matrix assisted laser desorption ionization time of flight ma ss spectroscopy (MALDI-TOF), the VP1,2,3T fragment was reconstitu ted in 50% acetonitrile/0.1% acetic acid and evaluated using an Applied Biosystems QSTAR with electros pray ionization and the Bayesian Protein Reconstruction algorithm was used to deconvolut e the mass. N-terminal sequence of the VP1,2,3T fraction was obtained using an Applied Biosystems 494/HT PROCISE Protein Sequencing System with sta ndard liquid blot cycles. Electron microscopy (EM) Purified rAAV2-GFP virions were digested with 0.02% trypsin for 24 hours at 37oC. 3-5 l of treated and untreated samples (at approximately 0.05mg/ml) were loaded onto 400 mesh carbon-coat ed formar copper grids (Ted Pella, Inc., Redding, CA), and negatively stained with 2% ur anyl acetate. The grids were viewed on a Hitachi H-7000 transmission electron microscope at 30,000X and 70,000X magnification. Immuno dot-blot. The immuno dot-blot procedure was esse ntially as described in Bleker, et al. [112]. Whatman filter paper No. 3 (Whatman International, Ltd., Maidstone, England) and supported nitrocellulose, 0.2 m pore size, (Bio-Rad, Hercules, CA) were soaked briefly in TBS (50 mM Tris, 100 mM NaCl, pH 8.0) prior to as sembling the dot-blot manifold (Schleicher and Schuell). After proteolysis, 25 l samples were directly applied to the wells of the dot-blot

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109 manifold and allowed to adsorb to the membrane In some cases, the trypsinized virions were treated with 0.1% SDS and 20% MeOH for 2h at 37oC or 45oC and then applied. Excess fluid was drawn through the membrane by vacuum filtr ation. Each well was washed with 100ul of TBS. The membrane was removed from the ma nifold and blocked with TBS/0.05% Tween 20 (TTBS) + 5% milk for 1 hour. Membranes were then probed with antisera to AAV-2 for 1 hour, in TTBS+5% milk at antibody dilutions of 1:2000, washed 3 times for 5 minutes each with TTBS + 0.05% milk, and were probed with secondary antibody, HRP-linked anti-mouse antibody diluted 1:5000 in TTBS+5% milk for 1 hour Membranes were washed 3 times for 5 minutes each with TTBS + 0.05% milk prior to de tection using Pierce S uperSignal West Pico Chemiluminescent Substrate. 3D-structure analysis Potential protease cleavage sites were analyzed using the protein sequences of AAV-1 (NP049542), AAV-2 (AAC03780), AAV-3 (NP043941), AAV-4 (NP044927), AAV-5 (YP068409), AAV-6 (NP045758), AAV-7 (YP077178), AAV-8 (YP077180), AAV-9 (AAS99264), AAV-10 (A AT46337), AAV-11 (AAT46339), and AAAV (AAT48613). Structure analysis to map potential tr yptic cleavage sites on the AAV-2 and AAV1 capsids utilized available coordinates for the AAV-2 VP3 m onomer [48] (PBD Accession No. 1LP3) and homologous models were generated for AAV-1 VP3 and AAV-5 VP3 based on a structure-guided sequence alignment with AAV-2 [50]. VIPER was used to apply icosahedral symmetry operators to the VP3 coordinates to ge nerate 3D models [73]. The coordinates were visualized using the program PyMOL (http://www.pymol.org DeLano Scientific, San Carlos, CA).

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110 CHAPTER 5 MASS SPECTROMETRY FOR AAV CAPSID SEROTYPE IDENTIFICATION AAV-CSI Introduction There are 11 serotypes of AAV, each with unique properties. AAV-2 was the first to be discovered, is the most studied and the majority of clinical trial data has come from studies with AAV-2. Currently, there are two Phase 1 clinical tr ials with AAV-2, one to deliver RPE65 to eye in patients, which was shown to restore vision in animal models, and one to provide aromatic Lamino acid decarobxylase to patients with Parkins ons disease. However, serotypes other than AAV-2 have been shown to have unique ce llular receptors and different transduction efficiencies. AAV-4 has also been utilized to deliver transgenes to the retina of the eye. In addition, AAV-8 has been shown to have highe r transduction efficiency than AAV-2. AAV-1 has been shown to be superior for transducing skel etal muscle and is currently in Phase 1 clinical trials to investigate its use in the treatmen t of limb girdle muscular dystrophy type 2D (LGMD2D), as well as for the treatment of alpha 1-antitrypsin (AAT) deficiency. Recently, over 100 AAV genotypic variants have been discovered [23, 26, 104], which provide the potential for an expanding number of AAV serotypes that could be developed into gene therapy vectors in the future. A PCR -based assay using unique primers to the cap genes of different serotypes has been developed to distinguish th e serotype of wt virus fo r AAV-2, AAV-3B or AAV-6 [113]; however, in gene therapy vectors, the rep and cap genes have been removed and replaced with a therapeutic gene, so this assa y cannot be used to determine the capsid serotype. For patient safety, the therapeutic transgene is sequenced from viral DNA to demonstrate that the gene therapy vector harbors th e correct transgene. However, there are very few methods available to verify the capsid serotype of the therapeuti c AAV vector. One recently developed method for

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111 verifying the capsid serotype involves partia l proteolysis of the cap sid and analyzing the fragments [46] as described in Chapter 4, or al ternatively, other investig ators have developed a serotype specific ELISA utilizing antibodies that react to intact capsid proteins [114]. Several monoclonal antibodies for AAV-2 capsids have b een described, such as A20 which recognizes intact AAV-2 or AAV-3 capsids [72], mAb C37 and C24 which selectively react with AAV-2 [100], and mAb D3 which shows br oad reactivity with different serotypes [100]. Antibody B4 is a monoclonal antibody that recognizes intact AAV-5 capsids; however, it has not been tested for serotype specificity [115]. Ot her monoclonal antibodies have recently been generated that recognize AAV1/6 (ADK1a and b), AAV-4 (ADK4) and AAV-5 (ADK5a and b) [114] for the purpose of serotype identity testing. ADK1a and b, ADK4 or ADK5 a and b were each shown to recognize a serotype specific conformational epitope and do not cross-react with serotypes AAV1-6, 8 and 9, with the exception of ADK1 a and b which also reacts with AAV-6, whose primary sequence differs from AAV-1 by six am ino acids. In 1999, Grimm, et al. developed a capture ELISA for AAV-2 using the monoclona l antibody A20 [116]. Recently, newly generated monoclonal antibodies have been utilized to de velop a capture ELISA assay for serotypes 1/6, 4 and 5. However, with the potential of over a hundred AAV serotypes, it will be difficult to generate unique conformational an tibodies that do not cross-re act with other serotypes. In addition to using different serot ypes, capsids may be specifically altered for improved targeting to transduce specific cells and capsids have been modified to express peptide fragments on the AAV surface. Methods for determining AAV serotype identity utilizing intact capsids were described in Chapter 4. The focus of this chapter includes techniques to determine the AAV capsid serotype identity using denatured viral capsid proteins. Th e western blot assay fo r evaluating proteolytic

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112 cleavage fragments of intact cap sids described in Chapter 4 is dependent upon an available antibody, as well as the antibody ep itope(s) not being the site of cleavage. This assay, while highly reproducible, is depende nt on the AAV capsid concentrati on (substrate concentration), enzyme concentration and the length of the digest. The specific ac tivity of the protease can also vary between lots or between suppliers. Addition ally, the purity of the preparation can effect digestion conditions. This assay relies on differe ntial proteolytic cleavag e of the capsid of different AAV serotypes and the resulting unique fragmentation patter ns provide capsid identification; however, capsids of serotypes that are resistant to se veral proteases or capsids that are cleaved to such an extent th at the antibody epitopes are not dis tinguishable, will not be able to be differentiated. The methods described in Chapter 4, as well as the AAV ELISA that has been developed by other investigators are valuable assays and are the only methods developed to date that allow for an eval uation of intact capsids. Recently, mass spectrometry (MS) techniques have been shown to provide useful, robust and rapid proteomics approaches to identify vira l proteins. It has been used to identify viral proteins produced during the c ourse of infection for Mareks Disease Virus (MDV), an alpha herpes virus [117]. Recent advanc es in MS techniques have allowed for its use as a method to detect viruses that are human pa thogens, like Norovirus [118]. MS applications have been used to identify the viral proteins a ssociated with viral particles fo r mimivirus [119], the vaccinia virus mature virion [120], murine cytomegalovi rus virions [121], bacter iophage MS2 [122] and Sulfolobus turreted icosahedral virus (STIV) [123]. It was also demonstrated that mass spectrometry could be used to monitor hepatitis C virus (HCV) genotype 1a, which exists within as a heterogeneous population of quasispecies, in a clinical patient [ 124]. Mass spectrometry methods have also been used to investigate dynamic interactions that occur for viruses in

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113 solution, such as Cowpea chlorotic mottle viru s (CCMV), which has a dynamic structural transition in response to changes in pH, ionic strength, and divale nt cation concentrations, which results in swelling of the CCMV capsid. Closed and swollen CCMV capsid pa rticles subjected to trypsin digestion provides info rmation regarding protein dynamics, and allowed for a comparison of the solution phase properties of the particle s [125]. For AAV, we have utilized protease digestion to evaluate the solution phase propert ies of intact empty a nd full (DNA-containing) capsids, as well as shown that different serotypes display differential susceptibility to proteases [46]. For AAV-2, high resolution mass spectrometry has been utilized to characterize capsid protein glycosylation [126]; however, for AAV, spect rometry has not been previously utilized to evaluate the capsid serotype. Currently, we are developing methods for AAV se rotype identity testi ng that incorporate the need for a quick and reliable assay to confirm serotype identit y. In this work, we show that denatured capsid proteins digested with tryps in and subjected to mass spectrometry can be utilized for serotype identity testing. Differen ces in the primary amino acid sequence of AAV serotypes provide fragments of different masses when digested with tr ypsin, and these unique fragments are detectable by mass spectrometr y. A serotype identity table for AAV-1, AAV-2, AAV-4, AAV-5 and AAV-8 is shown in Tabl e 5-1. A phylogenetic tree of AAV-1 through AAV-11, which depicts relatedness of AAV serotypes, is shown in Figure 5-1. We show that the highly homologous serotypes, AAV-1 and AAV-2 can be distinguished from each other, as well as from less homologous

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114 AAV1 AAV2 AAV3 AAV3B AAV4 AAV5 AAV6 AAV7 AAV8 AAV9 AAV10 AAV11 AAV1 100% 83% 87% 87% 64% 58% 99% 85% 84% 82% 85% 68% AAV2 83% 100% 87% 88% 61% 58% 84% 83% 83% 82% 84% 64% AAV3 87% 87% 100% 99% 63% 59% 87% 84% 86% 84% 86% 65% AAV3B 87% 88% 99% 100% 64% 59% 87% 85% 86% 84% 86% 66% AAV4 64% 61% 63% 64% 100% 54% 64% 65% 64% 64% 65% 82% AAV5 58% 58% 59% 59% 54% 100% 58% 59% 58% 57% 58% 55% AAV6 99% 84% 87% 87% 64% 58% 100% 85% 84% 82% 85% 68% AAV7 85% 83% 84% 85% 65% 59% 85% 100% 88% 82% 88% 68% AAV8 84% 83% 86% 86% 64% 58% 84% 88% 100% 85% 93% 67% AAV9 82% 82% 84% 84% 64% 57% 82% 82% 85% 100% 86% 65% AAV10 85% 84% 86% 86 % 65% 58% 85% 88% 93% 86% 100% 68% AAV11 68% 64% 65% 66 % 82% 55% 68% 68% 67% 65% 68% 100% Table 5-1. AAV Serotype Amino Acid Identity Table for AAV Serot ypes 1 11. AAV Serotypes were aligned and an identity table was constructed using Vector NTI (Invitr ogen). Numeric values represent perc ent identity. AAV-1 is 84% identical to AAV-8, and 83% identical to AAV-2, repres enting highly homologous serotypes. AAV-4 is 65% identical to AAV-1, 64% identical to AAV-8, 61% identical to AAV-2, 54% identical to AAV-5 representi ng a less homologous serotype. AAV-5 is 54 59% identical to the othe r serotypes, also representing a less homologous serotype. Accession numbers for proteins used in the alignment are: AAV-1 VP1 sequences NP_049542 and AAD27757, which are identical, AAV-2 VP1 AAC03780, AAV-3 VP1 sequences NP_043941 and AAV-3 Strain H AAC55049 whic h are identical, AAV-3B VP1 AAB95452, AAV-4 VP1 sequences NP_044927 and AAC58045 whic h are identical, AAV-5 VP1 sequences YP_068408 and AAD13756 which are identical, AAV-6 VP1 AAB 95450, AAV-7 VP1 sequences AAN03855 and YP_077178 which are identical, AAV-8 VP1 sequences YP_077180 and AAN0 3857 which are identical, AAV-9 VP1 AAS99264, AAV-10 AAT46337, AAV-11 AAT46339.

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115 Figure 5-1. Phylogenetic Tree of AAV Serotypes 1 11. serotypes such as AAV-4, AAV-5 and AAV-8. Uni que fragments were detected for all AAV serotypes tested which allowed for serotype identification. Results and Discussion Prior to generating an AAV vector, one factor th at should be considered is the desired target since different AAV serotypes have unique tissu e tropisms. After the AAV vector has been produced, it harbors the therapeutic gene a nd the ITRs, and is lacking other viral DNA sequences. As a result, there are few methods ava ilable for capsid serotype identification (CSI) of the final AAV vector. Upon evaluation of seve ral different AAV serot ypes in an SDS-PAGE gel, the electrophoretic mobility of the VPs in the gel can provide some limited information about the AAV serotype. The electrophoretic mo bility of viral proteins for AAV-1, and AAV-2 run faster than the viral prot eins for AAV-5 in an SDS-PAGE gel [111]. In a 10% Criterion minigel format (Bio-Rad, Hercules, CA), VP 3 of AAV-4 and AAV-8 migrate similarly to AAV5, this is shown in Figure 5-2. To obtain info rmation about the serotype, this requires a comparison of several AAV samples run in pa rallel, with known serotype samples for comparison. From an unknown sample, this would not be sufficient to allow for AAV serotype identification, but this could en able one to narrow down the po ssibilities to a smaller group of

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116 potential serotypes, such as either A AV-1/AAV-2, or AAV-4/AAV-5/ AAV-8, based on the migration of VP3 in a gel. An ideal assay fo r AAV serotype identification would allow for a single sample to be identified on the basis of the characteristics of the sample. Other desirable features of an assay for AAV capsid serotype identi fication (CSI) include the development of a relatively fast and simple assay that is able to be easily incorporated into current methods in a vector core or cGMP fac ility. Based on these features, we have developed an LC/MS/MS method for determining AAV serot ype identification. SDS PAGE gels of viral protein samples are routinely run and silver stai ned to assess the final v ector products for purity. Figure 5-2. Coomassie stained samples for ma ss spec identification of AAV serotype. AAV-1, AAV-2, AAV-4, AAV-5 and AAV-8. VP3 for diff erent serotypes migrate differently in a 10% Criterion SDS-PAGE minigel, with AAV-1 and AAV-2 VP3 migrating faster than AAV-4, AAV-5 or AAV-8. The mi nigel format was used for this assay instead of the larger 15 cm gel format whic h has better resolution because vector core and GMP facilities routinely run gels of this size after production and purification of AAV vectors to demonstrate purity of the final product. Many silver stain products inte rfere with mass spec applica tions; therefore, Coomassie bluestaining was utilized for this assay. Initially, the method was evaluated using VP1, VP2 and VP3 of AAV-1, AAV-2 and AAV-5 to assess for feas ibility. Upon correct se rotype identification for all three serotypes, the assay was expanded to include AAV-4 and AAV-8. VP3 bands shown AAV1 AAV2 AAV4 AAV5 AAV8 AAV8 VP3 VP3 VP2 VP1

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117 in Figure 5-2 and Figure 5-3, were cut from th e gel, and LC/MS/MS was performed. The SDSPAGE gel functions as a separati on step for the viral proteins; therefore, samples of varying levels of purity were analyzed. Samples of AAV-4 and AAV-5 that were less pure were run in parallel with pure AAV-4 and AAV-5, as well as additional samples of AAV-8. After isolating the VP1, VP2 and VP3 protein bands from the gel, samples were coded and evaluated blindly by MS. Because there is considerable overlap wi th the AAV viral proteins VP1, VP2, and VP3, and since VP3 is present in the largest amount in in tact capsids, VP3 was utilized for the blind study. The N-terminus of VP3 of different serotypes wa s predicted to provide a unique fragment after in gel trypsin digestion that might be useful when analyzed by mass spectrometry; however, previously it has been shown that th is region is acetylated [127]. Mass spec data was analyzed using Mascot and X! Tandem. Mascot was set to search the NCBI database and limited to viral proteins. Th e proteomics software Scaffold was used to validate MS/MS based peptide and protein identifi cations. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm. The software Scaffold provides a user friendly interface for the evaluation of samples and is shown in Figure 5-4. The unknown sample codes

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118 Figure 5-3. Coomassie stained samples for ma ss spec identification. Samples of AAV-4 and AAV-5 that were less pure were run in parallel with purified AAV-4 and AAV-5, as well as additional samples of AAV-8 to dete rmine if purity is an important parameter for this assay. are listed across the top of the table. The column on the left hand side are the theoretical proteins, the accession number, and the protein molecular mass. Numbers highlighted in green indicate the number of unique fragments with a probabi lity of over 95% upon which the AAV serotype identification was based. The samples that were less pure, LL L (AAV-4), MMM (AAV-4), QQ (AAV-5 VP2), QQQ (AAV-5 VP3), RR (AAV-5 VP2), RRR (AAV-5 VP3) had detectable levels of contaminating proteins, such as cellular and he lper virus proteins us ed in production. In the initial experiment to determine feasibility of this assay, LC/MS/MS was performed on VP1, VP2 and VP3 of three AAV serotypes, AAV-1, AAV2 and AAV-5 (impure). This method can be used to identify contaminants; for example, the AAV Rep78 was identified in one impure prep since it migrated with similar mo lecular weight as VP2 (72 kDa). This assay may be useful for verifying that new purification methods have elimin ated proteins that are similar in size to the AAV4 AAV4 AAV4 AAV5 AAV5 AAV5 AAV8 AAV8 VP3 VP3

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119 Figure 5-4. Mass spec data analyzed using th e software program Scaffold enabled una mbiguous serotype identification for 13/17 samples (76%) and the correct serotype assignment was made for 17/17 samples (100%) based on the serotype with the largest number of unique fragments. The viral proteins, accession numbers, and protei n mass are the result of the database search. The unknown samples are listed as th eir alphabetic code, and the values in the table are the number of unique trypsin fragments. This value was used to correctly assign the serotype. Codes: AAA AAV-1; BBB AAV-8; CCC AAV-8; EEE AAV-4; GGG AAV-5; HHH AAV-2; KKK AAV-4; LLL AAV-4; MMM AAV-4; PPP AAV-1; QQ AAV-5; QQQ AAV-5; RR AAV5; RRR AAV-5; SSS AAV-8; XXX AAV-8; ZZZ AAV-5

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120 AAV viral proteins which would not be distinguishable in an SD S page gel to assess purity. In Chapter 4, we showed that proteolysis of AAV-2 VP1 results in a degradation product, VP1T, that is similar in size to VP2, which were dete cted using available antibodies for AAV-2. Fewer antibodies are available for other serotypes, a nd the A69 antibody that recognizes the N-terminus VP2 is unique for AAV serotype 2. For AAV-2, th e site of proteolytic cleavage mapped to a critical region in the capsid required for hepari n binding and as a result, capsids with a cleavage in the GH loop are removed during heparin chroma tography; however, if the cleavage had not been in a critical region, or for other serotype s such as AAV-8 that may be less stable, this technique will be useful for det ecting degradation products of VP1 that are similar in size to VP2. Another goal of this work was to determine if a capsid mutation has occurred, if it could be identified using this method. Because the ra w data utilized for mass spec is based on the fragmentation pattern of a known protein, this me thod will not be able to identify if a capsid mutation has occurred during produ ction. For example, a mutation in AAV-4 that resulted in the change of a basic amino acid at amino acid 544 to an acidic amino acid would no longer have a trypsin cleavage site at this pos ition and the resulting fragment would be much larger than expected. However, in the database search, this larger fragment would not be present in the database. Therefore, for this region of the AAV capsi d, there is a lack of co verage in the MS data for these amino acids. Figure 5-5 is LC/MS/MS data for wt AAV-4 a nd AAV-4 R544E mutant which shows partial coverage for wt AAV-4 in the region of the mutation which allows verification that R544 is a basic re sidue in the wild type virus, and a lack of coverage in R544E, which is due to a known mutation in the starting ma terial used in this assay. Coverage or the number of amino acids detected after mass spec anal ysis is rarely 100%, so the lack of coverage

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121 itself is not sufficient to assume that a muta tion has occurred. However, for AAV capsids like AAV-2, many alanine scanning capsid mutants ha ve been generated. These sequences are currently not in the da tabase. If a modified AAV capsid with a known mutation is added to the database, there is the potential for identifying that this sequence as been incorporated into the capsid and in the correct region based on this method, provided that th e data obtained shows coverage in that specific region; however, a lack of coverage would not verify that the capsid does not contain that mutation. This might be useful for A AV-2 capsids that have been engineered to target specific tissu e based on insertion at amino acid 587. For the AAV-4 mutant capsid used in this st udy, the result of the mu tation at the trypsin cleavage site is a very large fragment, which is too big to be detected in the mass spectrometry studies performed here. MS relies on matchi ng the fragment sizes ob tained to predicted fragments of known sequences in th e database. This large fragment if it were detectable would be unexpected when compared to the known seque nce of AAV-4. First attempts at trying to identify this mutation in AAV-4 involved buildin g a database that included the AAV-4 sequence with the mutation. Unfortunately, due to the us e of trypsin, which relies on a basic residue and the primary sequence of AAV-4, there isn t another basic residue within a reasonable distance and the resulting fragment was too large to detect. In the wt AAV-4 sample, we detect the fragment flanking the mutation, but this is not detectable in the mutant AAV-4 sample. Although lack of coverage could be from acetyla tion or post-translationa l modification, or poor ionization of the fragment, as well as other reas ons, coverage for wt AAV-4 in that region and a consistant lack of coverage fo r mutant AAV-4 sample may suggest that there is a mutation. If the mutation in this sample was a change in an ami no acid that was not a basi c residue which trypsin

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122 Figure 5-5. Mass spectrometry data for K 544E AAV-4 mutant and wt AAV4. A. For the K544E AAV-4 mutant, the mutation results in a lack of trypsin digestion at amino acid 544, which results in a much larger fragment th an expected (red box). There was a lack of coverage for the amino acids in this region as a result of the mutation for both K544E samples tested (LLL and MMM). B. For wt AAV-4 there is covera ge in a portion of this region, which ve rifies that for the wt AAV-4 control, amino acid 544 is a basic residue and was cut by trypsin (red box). This fragment was detected for both samples of wt AAV-4 (sample EEE, and KKK).

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123 relies on for cleavage (K or R), this would not su bstantially change the nu mber of amino acids in the fragment that would result in a fragment too large to detect The software Mascot which was utilized to analyze the mass spec data can potentia lly detect this mutation. This experiment will be repeated utilizing a different protease that doesnt rely on basic residues and the software Mascot will be used determine if the fragment containing the mutation can be identified. A database has been built that includes all of the viral proteins for all of the known serotypes. For the studies presented in this chap ter, the MS data was searched thru the NCBI database, which was limited to ALL viral protei ns. We have built a database that includes only the AAV sequences, as well as the mutant sequen ce for AAV-4. This is currently being further developed and this will be useful for customized gene therapy vectors in the future that have been modified. These sequences can be deposited into the database, and clinical grade preps can be tested and verified. Several investigators ha ve shown that single amino acid mutations in the AAV capsid can effect tropism [128]. For AAV-2, mutation of several amino acids has been shown to result in vector vector targeting to cardiac tissue. A vect or like this could potentially be utilized in clinical trials and AAV Capsid Se rotype Identification ( AAV-CSI) would be useful for validating that the therapeutic gene has been packaged into the correct capsid. Materials and Methods Viruses and cell lines. rAAV2-GFP vector viri ons were produced by transient transfection of HEK293 cells, cells were harvested, then ly sed with 0.5% deoxycholate (DOC), incubated for 30 minutes at 37C with 50 U/ml of benzona se, and purified on sequential CsCl density gradients. Cesium fractions were dialyzed and heparin chromatography was performed as a concentration step [111] rAAV1-GFP, rAAV4-GFP, rAAV5-GFP and rAAV8-GFP vector virions were purified by fr eeze/thaw and CsCl gradients followed by Q-sepharose chromatography. Infectivity of the AAV vectors were assayed on C12 cells in the presence of

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124 adenovirus as previously desc ribed [92]. Virions were dialyzed into 50mM Tris-Cl pH 8.0 containing 100mM NaCl using a 10,000 MWCO me mbrane (Pierce), aliquoted, and stored frozen at -20C or -80C. Protein gel electrophoresis SDS-PAGE. Samples were boiled for 3 minutes at 100 C, centrifuged briefly and loaded on a 10% polyacrylamide gel. SDS-PAGE polyacrylamide gel was performed at 125 V for 90 minutes [129]. SDS-PAGE gel staining protoc ol A 1% stock solution of Coomassie blue R-250 was prepared in dH2O. Gels were stained in a solution c onsisting of 12.5% Stain stock, 10% acetic acid and 50% methanol for one hour. Gels were de tained in destain solu tion consisting of 10% acetic acid and 50% methanol overnight. AAV bands were cut from the gel and gel slices were stored in destain until the samples were prepped for mass spec. Mass Spec In-gel digestion protocol Gel slices were washed to remove SDS. Gel slices were washed with 50% acetonitrile in dd water and vortexed 2 times for 15 minutes eac h. Wash solution was removed and discarded. Gel slices were covered with neat acetonitrile until the ge l is dehydrated. The liquid was removed after 5 10 minutes. The gel was rehydra ted in just enough ABC to cover the gel for 5 minutes (20 to 50 l). An equal volume of acetonitrile wa s added to give a 1:1 ratio of acetonitrile to ABC and vortexed for 15 minutes. Wash solution was removed and the gel was dried down in a speedvac for 10 15 minutes. Next reduction and alkylation of protein in the gel was performed. The gel piece was rehydrated in 100 l of 45 mM DTT at 55C for 30 minutes. The gel was submerged in the reduc ing buffer. The buffer was discarded after incubation. Tubes were chilled to room temper ature. The liquid was removed and replaced quickly with 100 l of freshly made 100 mM iodoacetamide, and incubated in the dark for 30

PAGE 125

125 minutes at room temperature. The buffer was removed. The gel was washed 3 times with 100 l 50% ACN/50 mM ABC with agitation each for 15 minutes. The gel wash washed until it was colorless. The gel pieces were dried in a speed vac until they were completely dry, 10 15 minutes. Next, the proteins were digested with trypsin. Promega Trypsin was prepared at a concentration of 12.5 ng/ l reconstituded in 50 mM ABC, pH 8.4, with 5 mM CaCl2. Enzyme and buffer were kept cold in an ice bucket and the ratio of enzyme to protein was approximately 1:20. The dried gel piece was chilled on ice, a nd the gel pieces were covered with ice cold trypsin digestion buffer. The tube was kept on ice for 45 minutes. The enzyme solution was removed and replaced with 5 to 20 l of 50 mM ABC and 5 mM Ca Cl2 without enzyme. Tubes were incubated overnight at 37C in a water bath The supernatant was pipetted into a clean tube. Stop the reaction with 5.0% glacia l acetic acid to a final concen tration of 0.5% acetic acid. Mass spec protocol Capillary rpHPLC separation of protein di gests was performed on a 15 cm x 75 um i.d. PepMap C18 column (LC Packings, San Franci sco, CA) in combination with an Ultimate Capillary HPLC System (LC Packings, San Fran cisco, CA) operated at a flow rate of 200 nL/min. Inline mass spectrometric analysis of the column eluate was accomplished by a hybrid quadrupole time-of-flight instrument (QSTAR, A pplied Biosystems, Foster City, CA) equipped with a nanoelectrospray source. Fragment ion data generated by Information Dependent Acquisition (IDA) via the QSTAR were searched against the NCBI nr sequence database using the Mascot (Matrix Science, London, UK) database search engine. Probabil ity-based MOWSE scores above the default significant value were considered for protein identification in addition to validation by manual interpretation of the tandem MS data.

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126 Database Searching Tandem mass spectra were extracte d, charge state deconvoluted and deisotoped by BioWorks version 2.0. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.0.01) and X! Tandem (www.thegpm.org; version 2006.04.01.2). X! Tandem was set up to search a subset of the NCBInr_20061201 data base also assuming trypsin. Mascot was set up to search the NCBInr_20070202 database (selected for Viruses, unknown version, 375509 entries) assuming the di gestion enzyme trypsin. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.30 Da and a parent ion tolerance of 0.30 Da. Iodoacetamide derivative of cysteine was specified in Mascot and X! Tandem as a fixed modification. S-carbamoylmethylcysteine cyclization (N-terminus) of the n-terminus, deamidation of asparagine and glutamine and oxid ation of methionine were specified in Mascot and X! Tandem as variable modifications. Criteria For Protein Identification Scaffold (version Scaffold-01_06_13, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptid e identifications were accepted if they could be established at greater than 90.0% probability as specified by the Peptide Prophe t algorithm [130]. Protein identifications were accepted if they could be es tablished at greater than 95.0% probability and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm [131]. Proteins that cont ained similar peptides and coul d not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

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127 APPENDIX AAV CAPSID ALIGNMENT PileUp MSF: 778 Type: P Check: 1667 .. Name: AAV_VR195_ABA71699 Len: 778 Check: 7040 Weight: 0 Name: AAV_VR355_ABA71701 Len: 778 Check: 6641 Weight: 0 Name: AAV1_NP_049542 Len: 778 Check: 7598 Weight: 0 Name: AAV1_VP1_AAD27757 Len: 778 Check: 7598 Weight: 0 Name: VP1_isolate_hu48_AAS99296 Len: 778 Check: 7947 Weight: 0 Name: AAV6_VP1_AAB95450 Len: 778 Check: 7905 Weight: 0 Name: VP1_isolate_hu43_AAS99291 Len: 778 Check: 7388 Weight: 0 Name: VP1_isolate_hu44_AAS99292 Len: 778 Check: 8584 Weight: 0 Name: VP1_isolate_hu46_AAS99294 Len: 778 Check: 8181 Weight: 0 Name: AAV10_AAT46337 Len: 778 Check: 8318 Weight: 0 Name: VP1_isolate_rh40_AAS99244 Len: 778 Check: 8189 Weight: 0 Name: VP1_isolate_hu37_AAS99285 Len: 778 Check: 9370 Weight: 0 Name: VP1_isolate_hu42_AAS99290 Len: 778 Check: 9751 Weight: 0 Name: VP1_isolate_hu40_AAS99288 Len: 778 Check: 31 Weight: 0 Name: VP1_isolate_hu67_AAS99312 Len: 778 Check: 8662 Weight: 0 Name: VP1_isolate_rh38_AAS99243 Len: 778 Check: 9071 Weight: 0 Name: VP1_isolate_hu41_AAS99289 Len: 778 Check: 9596 Weight: 0 Name: VP1_isolate_hu66_AAS99311 Len: 778 Check: 7948 Weight: 0 Name: VP1_isolate_hu17_AAS99267 Len: 778 Check: 9358 Weight: 0 Name: VP1_isolate_hu6_AAS99306 Len: 778 Check: 8523 Weight: 0 Name: VP1_isolate_rh25_AAS99242 Len: 778 Check: 9311 Weight: 0 Name: VP1_isolate_hu39_AAS99286 Len: 778 Check: 596 Weight: 0 Name: VP1_isolate_rh49_AAS99247 Len: 778 Check: 9799 Weight: 0 Name: VP1_isolate_rh50_AAS99248 Len: 778 Check: 1643 Weight: 0 Name: VP1_isolate_rh51_AAS99249 Len: 778 Check: 1980 Weight: 0 Name: VP1_isolate_rh52_AAS99250 Len: 778 Check: 2067 Weight: 0 Name: VP1_isolate_rh64_AAS99259 Len: 778 Check: 1835 Weight: 0 Name: VP1_isolate_rh53_AAS99251 Len: 778 Check: 1695 Weight: 0 Name: VP1_isolate_rh61_AAS99257 Len: 778 Check: 1423 Weight: 0 Name: VP1_isolate_rh58_AAS99255 Len: 778 Check: 455 Weight: 0 Name: VP1_isolate_rh57_AAS99254 Len: 778 Check: 832 Weight: 0

PAGE 128

128 Name: AAV8_VP1_AAN03857 Len: 778 Check: 8558 Weight: 0 Name: AAV8_YP_077180 Len: 778 Check: 8558 Weight: 0 Name: VP1_isolate_rh43_AAS99245 Len: 778 Check: 8051 Weight: 0 Name: VP1_isolate_pi1_AAS99238 Len: 778 Check: 3364 Weight: 0 Name: VP1_isolate_pi3_AAS99240 Len: 778 Check: 3123 Weight: 0 Name: VP1_isolate_pi2_AAS99239 Len: 778 Check: 3263 Weight: 0 Name: VP1_isolate_rh1_AAS99241 Len: 778 Check: 5271 Weight: 0 Name: AAV7_VP1_AAN03855 Len: 778 Check: 7315 Weight: 0 Name: AAV7_YP_077178 Len: 778 Check: 7315 Weight: 0 Name: VP1_isolate_rh48_AAS99246 Len: 778 Check: 7391 Weight: 0 Name: VP1_isolate_rh62_AAS99258 Len: 778 Check: 6254 Weight: 0 Name: VP1_isolate_rh55_AAS99253 Len: 778 Check: 8552 Weight: 0 Name: VP1_isolate_rh54_AAS99252 Len: 778 Check: 8226 Weight: 0 Name: VP1_isolate_rh60_AAS99256 Len: 778 Check: 6021 Weight: 0 Name: AAV9_VP1_AAS99264 Len: 778 Check: 6375 Weight: 0 Name: VP1_isolate_hu31_AAS99281 Len: 778 Check: 6990 Weight: 0 Name: VP1_isolate_hu32_AAS99282 Len: 778 Check: 7109 Weight: 0 Name: AAV11_AAT46339 Len: 778 Check: 6971 Weight: 0 Name: AAV4_NP_044927 Len: 778 Check: 3824 Weight: 0 Name: AAV4_VP1_AAC58045 Len: 778 Check: 3824 Weight: 0 Name: BOVINE_AAV_AAR26465 Len: 778 Check: 5658 Weight: 0 Name: BOVINE_AAV_YP_024971 Len: 778 Check: 5658 Weight: 0 Name: AAV5_VP1_AAD13756 Len: 778 Check: 8027 Weight: 0 Name: AAV5_YP_068409 Len: 778 Check: 8027 Weight: 0 Name: CAPRINE_AAV1_AAU84890 Len: 778 Check: 7023 Weight: 0 Name: GOAT_AAV_VP1_ABC69726 Len: 778 Check: 7023 Weight: 0 Name: RAT_AAV1_AAZ79676 Len: 778 Check: 7290 Weight: 0 Name: MOUSE_AAV1_AAZ79672 Len: 778 Check: 4579 Weight: 0 Name: AVIAN_AAV_ATCC_VR865_AAO32087 Len: 778 Check: 3058 Weight: 0 Name: AVIAN_AAV_ATCC_VR865_AAT48613 Len: 778 Check: 3058 Weight: 0 Name: AVIAN_AAV_ATCC_VR865_NP_852781 Len: 778 Check: 3058 Weight: 0 Name: AVIAN_AAV_Strain_DA1_AAT48615 Len: 778 Check: 453 Weight: 0 Name: AVIAN_AAV_Strain_DA1_YP_077183 Len: 778 Check: 453 Weight: 0 Name: DUCK_AAV_Strain_FM_AAA83225 Len: 778 Check: 3002 Weight: 0 Name: MUSCOVY_DUCK_PARVOVIRUS_YP_068412 Len: 778 Check: 233 Weight: 0 Name: MUSCOVY_DUCK_VP1_YP_068411 Len: 778 Check: 3002 Weight: 0 Name: MUSCOVY_DUCK_PARVOVIRUS_YP_068413 Len: 778 Check: 5241 Weight: 0 Name: Goose_AAV_VP1_AAA83230 Len: 778 Check: 6211 Weight: 0 Name: GOOSE_AAV_VP1_NP_043515 Len: 778 Check: 6211 Weight: 0 Name: AAV3_NP_043941 Len: 778 Check: 69 Weight: 0 Name: AAV3_Strain_H_AAC55049 Len: 778 Check: 69 Weight: 0

PAGE 129

129 Name: AAV3B_VP1_AAB95452 Len: 778 Check: 1343 Weight: 0 Name: VP1_isolate_hu1_AAS99260 Len: 778 Check: 1391 Weight: 0 Name: VP1_isolate_hu4_AAS99287 Len: 778 Check: 1303 Weight: 0 Name: VP1_isolate_hu2_AAS99270 Len: 778 Check: 1141 Weight: 0 Name: VP1_isolate_hu3_AAS99280 Len: 778 Check: 2673 Weight: 0 Name: VP1_isolate_hu60_AAS99307 Len: 778 Check: 1875 Weight: 0 Name: VP1_isolate_hu61_AAS99308 Len: 778 Check: 1690 Weight: 0 Name: VP1_isolate_hu25_AAS99276 Len: 778 Check: 772 Weight: 0 Name: VP1_isolate_hu15_AAS99265 Len: 778 Check: 1483 Weight: 0 Name: VP1_isolate_hu16_AAS99266 Len: 778 Check: 2044 Weight: 0 Name: VP1_isolate_hu18_AAS99268 Len: 778 Check: 2981 Weight: 0 Name: VP1_isolate_hu7_AAS99313 Len: 778 Check: 996 Weight: 0 Name: VP1_isolate_hu10_AAS99261 Len: 778 Check: 2978 Weight: 0 Name: VP1_isolate_hu11_AAS99262 Len: 778 Check: 2837 Weight: 0 Name: VP1_isolate_hu9_AAS99314 Len: 778 Check: 1894 Weight: 0 Name: VP1_isolate_hu53_AAS99300 Len: 778 Check: 1757 Weight: 0 Name: VP1_isolate_hu55_AAS99302 Len: 778 Check: 57 Weight: 0 Name: VP1_isolate_hu54_AAS99301 Len: 778 Check: 9885 Weight: 0 Name: VP1_isolate_huS17_AAU05370 Len: 778 Check: 1727 Weight: 0 Name: AAV2_VP1_AAC03780 Len: 778 Check: 3108 Weight: 0 Name: VP1_isolate_hu34_AAS99283 Len: 778 Check: 2718 Weight: 0 Name: VP1_isolate_hu35_AAS99284 Len: 778 Check: 2744 Weight: 0 Name: VP1_islolate_hu51_AAS99298 Len: 778 Check: 3098 Weight: 0 Name: VP1_isolate_hu52_AAS99299 Len: 778 Check: 4413 Weight: 0 Name: VP1_isolate_hu47_AAS99295 Len: 778 Check: 4162 Weight: 0 Name: VP1_isolate_hu45_AAS99293 Len: 778 Check: 4008 Weight: 0 Name: VP1_isolate_hu58_AAS99305 Len: 778 Check: 2430 Weight: 0 Name: VP1_isolate_hu49_AAS99297 Len: 778 Check: 3468 Weight: 0 Name: VP1_isolate_hu56_AAS99303 Len: 778 Check: 4209 Weight: 0 Name: VP1_isolate_hu57_AAS99304 Len: 778 Check: 1345 Weight: 0 Name: VP1_isolate_hu28_AAS99278 Len: 778 Check: 3099 Weight: 0 Name: VP1_isolate_hu29_AAS99279 Len: 778 Check: 2731 Weight: 0 Name: VP1_isolate_huT70_AAU05364 Len: 778 Check: 3586 Weight: 0 Name: VP1_isolate_hu13_AAS99263 Len: 778 Check: 1295 Weight: 0 Name: VP1_isolate_hu63_AAS99309 Len: 778 Check: 2301 Weight: 0 Name: VP1_isolate_hu64_AAS99310 Len: 778 Check: 2688 Weight: 0 Name: VP1_isolate_huT40_AAU05362 Len: 778 Check: 1971 Weight: 0 Name: VP1_isolate_huLG15_AAU05371 Len: 778 Check: 590 Weight: 0 Name: VP1_isolate_huT17_AAU05358 Len: 778 Check: 1851 Weight: 0 Name: VP1_isolate_huT41_AAU05372 Len: 778 Check: 2922 Weight: 0 Name: VP1_isolate_huT71_AAU05366 Len: 778 Check: 2922 Weight: 0

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130 Name: VP1_isolate_huT88_AAU05368 Len: 778 Check: 1757 Weight: 0 Name: VP1_isolate_huT32_AAU05360 Len: 778 Check: 2563 Weight: 0 Name: VP1_isolate_hu27_AAS99277 Len: 778 Check: 2017 Weight: 0 Name: VP1_isolate_hu19_AAS99269 Len: 778 Check: 2261 Weight: 0 Name: VP1_isolate_hu20_AAS99271 Len: 778 Check: 1010 Weight: 0 Name: VP1_isolate_hu21_AAS99272 Len: 778 Check: 1674 Weight: 0 Name: VP1_isolate_hu24_AAS99275 Len: 778 Check: 1674 Weight: 0 Name: VP1_isolate_hu22_AAS99273 Len: 778 Check: 2008 Weight: 0 Name: VP1_isolate_hu23_AAS99274 Len: 778 Check: 1124 Weight: 0 // 1 50 AAV_VR195_ABA71699 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... AAV_VR355_ABA71701 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... AAV1_NP_049542 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... AAV1_VP1_AAD27757 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu48_AAS99296 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... AAV6_VP1_AAB95450 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu43_AAS99291 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu44_AAS99292 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLRPGPPPPK PAERHK.... VP1_isolate_hu46_AAS99294 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... AAV10_AAT46337 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh40_AAS99244 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu37_AAS99285 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu42_AAS99290 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu40_AAS99288 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu67_AAS99312 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh38_AAS99243 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu41_AAS99289 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu66_AAS99311 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu17_AAS99267 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu6_AAS99306 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh25_AAS99242 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_hu39_AAS99286 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh49_AAS99247 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh50_AAS99248 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh51_AAS99249 ....MVADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh52_AAS99250 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh64_AAS99259 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh53_AAS99251 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ....

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131 VP1_isolate_rh61_AAS99257 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh58_AAS99255 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh57_AAS99254 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGAPKPK ANQQKQ.... AAV8_VP1_AAN03857 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGAPKPK ANQQKQ.... AAV8_YP_077180 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGAPKPK ANQQKQ.... VP1_isolate_rh43_AAS99245 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_pi1_AAS99238 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGAPQPK ANQQKQ.... VP1_isolate_pi3_AAS99240 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGAPQPK ANQQKQ.... VP1_isolate_pi2_AAS99239 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGAPQPK ANQQKQ.... VP1_isolate_rh1_AAS99241 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGAPKPK ANQQKQ.... AAV7_VP1_AAN03855 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... AAV7_YP_077178 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh48_AAS99246 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh62_AAS99258 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh55_AAS99253 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh54_AAS99252 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... VP1_isolate_rh60_AAS99256 ....MAADGY LPDWLEDN.. .LSEGIHEWW DPKPGAPKPK ANQQKQ.... AAV9_VP1_AAS99264 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGAPQPK ANQQHQ.... VP1_isolate_hu31_AAS99281 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu32_AAS99282 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... AAV11_AAT46339 ....MAADGY LPDWLEDN.. .LSEGIREWW DLKPGAPKPK ANQQKQ.... AAV4_NP_044927 .....MTDGY LPDWLEDN.. .LSEGVREWW ALQPGAPKPK ANQQHQ.... AAV4_VP1_AAC58045 .....MTDGY LPDWLEDN.. .LSEGVREWW ALQPGAPKPK ANQQHQ.... BOVINE_AAV_AAR26465 ....MSFVDH PPDWLES... .IGDGFREFL GLEAGPPKPK ANQQKQ.... BOVINE_AAV_YP_024971 ....MSFVDH PPDWLES... .IGDGFREFL GLEAGPPKPK ANQQKQ.... AAV5_VP1_AAD13756 ........MS FVDHPPDWLE EVGEGLREFL GLEAGPPKPK PNQQ...... AAV5_YP_068409 ........MS FVDHPPDWLE EVGEGLREFL GLEAGPPKPK PNQQ...... CAPRINE_AAV1_AAU84890 ........MS FVDHPPDWLE EVGEGLREFL GLEAGPPKPK PNQQ...... GOAT_AAV_VP1_ABC69726 ........MS FVDHPPDWLE EVGEGLREFL GLEAGPPKPK PNQQ...... RAT_AAV1_AAZ79676 ........MS FFDWIG...R KYANGAAEFW DLEPGPPKPK ..KA...... MOUSE_AAV1_AAZ79672 ........MS FFDWLGKQ.. .YAQGAAEFW DLKSGPPAPK KARKDG.... AVIAN_AAV_ATCC_VR865_AAO32087 MSLISDAIPD WLERLVKK.. .GVNAAADFY HLESGPPRPK ANQQTQ.... AVIAN_AAV_ATCC_VR865_AAT48613 MSLISDAIPD WLERLVKK.. .GVNAAADFY HLESGPPRPK ANQQTQ.... AVIAN_AAV_ATCC_VR865_NP_852781 MSLISDAIPD WLERLVKK.. .GVNAAADFY HLESGPPRPK ANQQTQ.... AVIAN_AAV_Strain_DA1_AAT48615 MSLISDAIPD WLERLVKK.. .GVNAAADFY HLESGPPHPK ANQQTQ.... AVIAN_AAV_Strain_DA1_YP_077183 MSLISDAIPD WLERLVKK.. .GVNAAADFY HLESGPPHPK ANQQTQ.... DUCK_AAV_Strain_FM_AAA83225 .......MST FLEKFEDW.. .YETAAASWR HLKAGAPKPK SNQQSQSVST MUSCOVY_DUCK_PARVOVIRUS_YP_068412 .......... .......... .......... .......... .......... MUSCOVY_DUCK_VP1_YP_068411 .......MST FLEKFEDW.. .YETAAASWR HLKAGAPKPK SNQQSQSVST MUSCOVY_DUCK_PARVOVIRUS_YP_068413 .......... .......... .......... .......... .......... Goose_AAV_VP1_AAA83230 .......MST FLDSFEEW.. .YETAAASWR NLKAGAPQPK PNQQSQSVSP

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132 GOOSE_AAV_VP1_NP_043515 .......MST FLDSFEEW.. .YETAAASWR NLKAGAPQPK PNQQSQSVSP AAV3_NP_043941 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGVPQPK ANQQHQ.... AAV3_Strain_H_AAC55049 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGVPQPK ANQQHQ.... AAV3B_VP1_AAB95452 ....MAADGY LPDWLEDN.. .LSEGIREWW ALKPGVPQPK ANQQHQ.... VP1_isolate_hu1_AAS99260 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu4_AAS99287 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu2_AAS99270 ....MAADGY PPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu3_AAS99280 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu60_AAS99307 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu61_AAS99308 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu25_AAS99276 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu15_AAS99265 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu16_AAS99266 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu18_AAS99268 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu7_AAS99313 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu10_AAS99261 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK LAERHQ.... VP1_isolate_hu11_AAS99262 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHQ.... VP1_isolate_hu9_AAS99314 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHQ.... VP1_isolate_hu53_AAS99300 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu55_AAS99302 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu54_AAS99301 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_huS17_AAU05370 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... AAV2_VP1_AAC03780 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu34_AAS99283 ....MAADGY LPDWLEDT.. .LSEGIRQRW KLKPGPPPPE PAERHK.... VP1_isolate_hu35_AAS99284 ....MAADGY LPDWLEDT.. .LSEGIRQRW KLKPGPPPPE PAERHK.... VP1_islolate_hu51_AAS99298 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu52_AAS99299 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu47_AAS99295 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHR.... VP1_isolate_hu45_AAS99293 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHR.... VP1_isolate_hu58_AAS99305 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu49_AAS99297 ....MAADGY LPDWLKDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu56_AAS99303 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu57_AAS99304 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPP.K PAERHK.... VP1_isolate_hu28_AAS99278 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu29_AAS99279 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_huT70_AAU05364 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu13_AAS99263 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu63_AAS99309 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu64_AAS99310 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_huT40_AAU05362 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_huLG15_AAU05371 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK....

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133 VP1_isolate_huT17_AAU05358 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_huT41_AAU05372 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_huT71_AAU05366 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_huT88_AAU05368 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_huT32_AAU05360 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu27_AAS99277 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu19_AAS99269 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu20_AAS99271 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu21_AAS99272 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu24_AAS99275 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu22_AAS99273 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... VP1_isolate_hu23_AAS99274 ....MAADGY LPDWLEDT.. .LSEGIRQWW KLKPGPPPPK PAERHK.... 51 100 AAV_VR195_ABA71699 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ AAV_VR355_ABA71701 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ AAV1_NP_049542 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ AAV1_VP1_AAD27757 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu48_AAS99296 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ AAV6_VP1_AAB95450 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu43_AAS99291 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu44_AAS99292 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu46_AAS99294 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ AAV10_AAT46337 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh40_AAS99244 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu37_AAS99285 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu42_AAS99290 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu40_AAS99288 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu67_AAS99312 .......DDG RGLVLLGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh38_AAS99243 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu41_AAS99289 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu66_AAS99311 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu17_AAS99267 .......DDG RGLVLPGCKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu6_AAS99306 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh25_AAS99242 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu39_AAS99286 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh49_AAS99247 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh50_AAS99248 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh51_AAS99249 .......GDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh52_AAS99250 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh64_AAS99259 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ

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134 VP1_isolate_rh53_AAS99251 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh61_AAS99257 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh58_AAS99255 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh57_AAS99254 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ AAV8_VP1_AAN03857 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ AAV8_YP_077180 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh43_AAS99245 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_pi1_AAS99238 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDQQ VP1_isolate_pi3_AAS99240 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDQQ VP1_isolate_pi2_AAS99239 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDQQ VP1_isolate_rh1_AAS99241 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHGKAYDQQ AAV7_VP1_AAN03855 .......DNG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ AAV7_YP_077178 .......DNG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh48_AAS99246 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh62_AAS99258 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh55_AAS99253 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh54_AAS99252 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_rh60_AAS99256 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ AAV9_VP1_AAS99264 .......DNA RGLVLPGYKY LGPGNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu31_AAS99281 .......DDS RGLVLPGYKY LGPGNGLDKG EPVNAADAAA LEHDKAYDQQ VP1_isolate_hu32_AAS99282 .......DDS RGLVLPGYKY LGPGNGLDKG EPVNAADAAA LEHDKAYDQQ AAV11_AAT46339 .......DDG RGLVLPGYKY LGPFNGLDKG EPVNAADAAA LEHDKAYDQQ AAV4_NP_044927 .......DNA RGLVLPGYKY LGPGNGLDKG EPVNAADAAA LEHDKAYDQQ AAV4_VP1_AAC58045 .......DNA RGLVLPGYKY LGPGNGLDKG EPVNAADAAA LEHDKAYDQQ BOVINE_AAV_AAR26465 .......DNA RGLVLPGYKY LGPGNGLDKG DPVNFADEVA REHDLSYQKQ BOVINE_AAV_YP_024971 .......DNA RGLVLPGYKY LGPGNGLDKG DPVNFADEVA REHDLSYQKQ AAV5_VP1_AAD13756 .....HQDQA RGLVLPGYNY LGPGNGLDRG EPVNRADEVA REHDISYNEQ AAV5_YP_068409 .....HQDQA RGLVLPGYNY LGPGNGLDRG EPVNRADEVA REHDISYNEQ CAPRINE_AAV1_AAU84890 .....HQDQA RGLVLPGYNY LGPGNGLDRG EPVNRADEVA REHDISYNEQ GOAT_AAV_VP1_ABC69726 .....HQDQA RGLVLPGYNY LGPGNGLDRG EPVNRADEVA REHDISYNEQ RAT_AAV1_AAZ79676 .....RVDDS AGFNFPGHKY LGPGNGLDRG EPVDADDAAA QKHDQEYQAL MOUSE_AAV1_AAZ79672 .........S AGFNFPGHKY LGPGNSLDRG DPVDADDAAA QKHDQSYQEQ AVIAN_AAV_ATCC_VR865_AAO32087 ..ESLEKDDS RGLVFPGYNY LGPFNGLDKG EPVNEADAAA LEHDKAYDLE AVIAN_AAV_ATCC_VR865_AAT48613 ..ESLEKDDS RGLVFPGYNY LGPFNGLDKG EPVNEADAAA LEHDKAYDLE AVIAN_AAV_ATCC_VR865_NP_852781 ..ESLEKDDS RGLVFPGYNY LGPFNGLDKG EPVNEADAAA LEHDKAYDLE AVIAN_AAV_Strain_DA1_AAT48615 ..ESPEKDDS RGLVFPGYKY LGPFNGLDKG KPVNEADAAA LEHDKAYDLE AVIAN_AAV_Strain_DA1_YP_077183 ..ESPEKDDS RGLVFPGYKY LGPFNGLDKG KPVNEADAAA LEHDKAYDLE DUCK_AAV_Strain_FM_AAA83225 DRKPQRKDNN RGFVLPGYKY VGPGNGLDKG PPVNKADSVA LEHDKAYDQQ MUSCOVY_DUCK_PARVOVIRUS_YP_068412 .......... .......... .......... .......... .......... MUSCOVY_DUCK_VP1_YP_068411 DRKPQRKDNN RGFVLPGYKY VGPGNGLDKG PPVNKADSVA LEHDKAYDQQ MUSCOVY_DUCK_PARVOVIRUS_YP_068413 .......... .......... .......... .......... ..........

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135 Goose_AAV_VP1_AAA83230 DREPERKDNN RGFVLPGYKY LGPGNGLDKG PPVNKADSVA LEHDKAYDQQ GOOSE_AAV_VP1_NP_043515 DREPERKDNN RGFVLPGYKY LGPGNGLDKG PPVNKADSVA LEHDKAYDQQ AAV3_NP_043941 .......DNR RGLVLPGYKY LGPGNGLDKG EPVNEADAAA LEHDKAYDQQ AAV3_Strain_H_AAC55049 .......DNR RGLVLPGYKY LGPGNGLDKG EPVNEADAAA LEHDKAYDQQ AAV3B_VP1_AAB95452 .......DNR RGLVLPGYKY LGPGNGLDKG EPVNEADAAA LEHDKAYDQQ VP1_isolate_hu1_AAS99260 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu4_AAS99287 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu2_AAS99270 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu3_AAS99280 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu60_AAS99307 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu61_AAS99308 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu25_AAS99276 .......DGS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu15_AAS99265 .......DDS RGLVLPGYKY LGPFNGLYKG EPVDEADAAA LEHDKAYDRQ VP1_isolate_hu16_AAS99266 .......DDS RGLVLPGYKY LGPFNGLYKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu18_AAS99268 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu7_AAS99313 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu10_AAS99261 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu11_AAS99262 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu9_AAS99314 .......DNS RGLVLPGYKY LGPSNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu53_AAS99300 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu55_AAS99302 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu54_AAS99301 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_huS17_AAU05370 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ AAV2_VP1_AAC03780 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu34_AAS99283 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu35_AAS99284 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_islolate_hu51_AAS99298 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu52_AAS99299 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu47_AAS99295 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu45_AAS99293 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu58_AAS99305 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu49_AAS99297 .......DDS GGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEYDKAYDRQ VP1_isolate_hu56_AAS99303 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu57_AAS99304 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu28_AAS99278 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu29_AAS99279 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_huT70_AAU05364 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu13_AAS99263 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu63_AAS99309 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu64_AAS99310 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_huT40_AAU05362 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ

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136 VP1_isolate_huLG15_AAU05371 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_huT17_AAU05358 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_huT41_AAU05372 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_huT71_AAU05366 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_huT88_AAU05368 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_huT32_AAU05360 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu27_AAS99277 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu19_AAS99269 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu20_AAS99271 .......DDS RGLVLPGYRY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu21_AAS99272 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu24_AAS99275 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu22_AAS99273 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ VP1_isolate_hu23_AAS99274 .......DDS RGLVLPGYKY LGPFNGLDKG EPVNEADAAA LEHDKAYDRQ 101 150 AAV_VR195_ABA71699 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPFG.L AAV_VR355_ABA71701 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPFG.L AAV1_NP_049542 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV1_VP1_AAD27757 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu48_AAS99296 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV6_VP1_AAB95450 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPFG.L VP1_isolate_hu43_AAS99291 LKAGDNPYPR YNHADAEFQE RLQEDTPFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu44_AAS99292 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu46_AAS99294 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV10_AAT46337 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh40_AAS99244 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu37_AAS99285 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu42_AAS99290 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu40_AAS99288 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu67_AAS99312 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh38_AAS99243 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu41_AAS99289 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.P VP1_isolate_hu66_AAS99311 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu17_AAS99267 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu6_AAS99306 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh25_AAS99242 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu39_AAS99286 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh49_AAS99247 LKAGDNPHLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh50_AAS99248 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh51_AAS99249 LKAGDNPYLR YNHADAELQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh52_AAS99250 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L

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137 VP1_isolate_rh64_AAS99259 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh53_AAS99251 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh61_AAS99257 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh58_AAS99255 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh57_AAS99254 LQAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV8_VP1_AAN03857 LQAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV8_YP_077180 LQAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh43_AAS99245 LEAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_pi1_AAS99238 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_pi3_AAS99240 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_pi2_AAS99239 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh1_AAS99241 LQAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV7_VP1_AAN03855 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV7_YP_077178 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh48_AAS99246 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh62_AAS99258 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh55_AAS99253 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh54_AAS99252 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_rh60_AAS99256 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV9_VP1_AAS99264 LKAGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRLLEPLG.L VP1_isolate_hu31_AAS99281 LKAGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRLLEPLG.L VP1_isolate_hu32_AAS99282 LKAGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRLLEPLG.L AAV11_AAT46339 LKAGDNPYLR YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV4_NP_044927 LKAGDNPYLK YNHADAEFQQ RLQGDTSFGG NLGRAVFQAK KRVLEPLG.L AAV4_VP1_AAC58045 LKAGDNPYLK YNHADAEFQQ RLQGDTSFGG NLGRAVFQAK KRVLEPLG.L BOVINE_AAV_AAR26465 LEAGDNPYLK YNHADAEFQE KLASDTSFGG NLGKAVFQAK KRILEPLG.L BOVINE_AAV_YP_024971 LEAGDNPYLK YNHADAEFQE KLASDTSFGG NLGKAVFQAK KRILEPLG.L AAV5_VP1_AAD13756 LEAGDNPYLK YNHADAEFQE KLADDTSFGG NLGKAVFQAK KRVLEPFG.L AAV5_YP_068409 LEAGDNPYLK YNHADAEFQE KLADDTSFGG NLGKAVFQAK KRVLEPFG.L CAPRINE_AAV1_AAU84890 LEAGDNPYLK YNHADAEFQE KLADDTSFGG NLGKAVFQAK KRVLEPFG.L GOAT_AAV_VP1_ABC69726 LEAGDNPYLK YNHADAEFQE KLADDTSFGG NLGKAVFQAK KRVLEPFG.L RAT_AAV1_AAZ79676 LESGENPYLT FNHADRQLQK DLAEDQSFEG NLARGLFEAK KLAAQVVG.V MOUSE_AAV1_AAZ79672 LEAGDNPYLK YNHADREFQE ALKDDTSFEG NLARGLFEAK KLVAEPLG.L AVIAN_AAV_ATCC_VR865_AAO32087 IKDGHNPYFE YNEADRRFQE RLKDDTSFGG NLGKAIFQAK KRVLEPFG.L AVIAN_AAV_ATCC_VR865_AAT48613 IKDGHNPYFE YNEADRRFQE RLKDDTSFGG NLGKAIFQAK KRVLEPFG.L AVIAN_AAV_ATCC_VR865_NP_852781 IKDGHNPYFE YNEADRRFQE RLKDDTSFGG NLGKAIFQAK KRVLEPFG.L AVIAN_AAV_Strain_DA1_AAT48615 LKDGHNPYFE YNEADRRFQE RLKDDTSFGG NLGKAIFQAK KRVLEPFG.L AVIAN_AAV_Strain_DA1_YP_077183 LKDGHNPYFE YNEADRRFQE RLKDDTSFGG NLGKAIFQAK KRVLEPFG.L DUCK_AAV_Strain_FM_AAA83225 LKAGDNPYIK FKHADQEFID NLQGDTSFGG NLGKAVFQAK KRILEPLG.L MUSCOVY_DUCK_PARVOVIRUS_YP_068412 .......... .......... .......... .......... .......... MUSCOVY_DUCK_VP1_YP_068411 LKAGDNPYIK FKHADQEFID NLQGDTSFGG NLGKAVFQAK KRILEPLG.L

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138MUSCOVY_DUCK_PARVOVIRUS_YP_068413 .......... .......... .......... .......... .......... Goose_AAV_VP1_AAA83230 LKAGDNPYIK FNHADQDFID SLQDDQSFGG NLGKAVFQAK KRILEPFG.L GOOSE_AAV_VP1_NP_043515 LKAGDNPYIK FNHADQDFID SLQDDQSFGG NLGKAVFQAK KRILEPFG.L AAV3_NP_043941 LKAGDNPYLK YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRILEPLG.L AAV3_Strain_H_AAC55049 LKAGDNPYLK YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRILEPLG.L AAV3B_VP1_AAB95452 LKAGDNPYLK YNHADAEFQE RLQEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_hu1_AAS99260 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu4_AAS99287 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu2_AAS99270 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu3_AAS99280 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLRPG VP1_isolate_hu60_AAS99307 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu61_AAS99308 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu25_AAS99276 LNSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu15_AAS99265 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu16_AAS99266 LDSGDNPYLK YNHAGAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu18_AAS99268 LESGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu7_AAS99313 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu10_AAS99261 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu11_AAS99262 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu9_AAS99314 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu53_AAS99300 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu55_AAS99302 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu54_AAS99301 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_huS17_AAU05370 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L AAV2_VP1_AAC03780 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu34_AAS99283 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu35_AAS99284 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_islolate_hu51_AAS99298 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu52_AAS99299 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu47_AAS99295 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu45_AAS99293 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu58_AAS99305 LDSGDNPYLK YDHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu49_AAS99297 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu56_AAS99303 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu57_AAS99304 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu28_AAS99278 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLS.L VP1_isolate_hu29_AAS99279 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_huT70_AAU05364 LESGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu13_AAS99263 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu63_AAS99309 LDSGDNPYPK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_hu64_AAS99310 LDGGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L

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139 VP1_isolate_huT40_AAU05362 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRVLEPLG.L VP1_isolate_huLG15_AAU05371 LDSGDNPYLK YNHADAKFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_huT17_AAU05358 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_huT41_AAU05372 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_huT71_AAU05366 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_huT88_AAU05368 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_huT32_AAU05360 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_hu27_AAS99277 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_hu19_AAS99269 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_hu20_AAS99271 LDSGDNPYLK YNHVDAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_hu21_AAS99272 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_hu24_AAS99275 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_hu22_AAS99273 LDSGDNPYLK YNHADAEFQE RLKGDTSFGG NLGRAVFQAK KRILEPLG.L VP1_isolate_hu23_AAS99274 LDSGDNPYLK YNHADAEFQE RLKEDTSFGG NLGRAVFQAK KRILEPLG.L 151 200 AAV_VR195_ABA71699 VEEGAKTAPG KK.RPVEQSP QE.PDSSSGI GKTG.QQPAK KRLNFGQT.. AAV_VR355_ABA71701 VEEGAKTAPG KK.RPVEQSP QE.PDSSSGI GKSG.QQPAK KRLNFGQT.. AAV1_NP_049542 VEEGAKTAPG KK.RPVEQSP QE.PDSSSGI GKTG.QQPAK KRLNFGQT.. AAV1_VP1_AAD27757 VEEGAKTAPG KK.RPVEQSP QE.PDSSSGI GKTG.QQPAK KRLNFGQT.. VP1_isolate_hu48_AAS99296 VEEGAKTAPG KK.RPVEQSP QE.PDSSSGI GKTG.QQPAK KRLNFGQT.. AAV6_VP1_AAB95450 VEEGAKTAPG KK.RPVEQSP QE.PDSSSGI GKTG.QQPAK KRLNFGQT.. VP1_isolate_hu43_AAS99291 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAK KRLNFGQT.. VP1_isolate_hu44_AAS99292 VEEGAETAPG KK.RPVEQSP QG.PDSSSGI GKTG.QQPAK KRLNFGQT.. VP1_isolate_hu46_AAS99294 VEEGAKTAPG KK.RPVEQSP QE.PDSPSGI GKTG.QQPAK KRLNFGQT.. AAV10_AAT46337 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAK KRLNFGQT.. VP1_isolate_rh40_AAS99244 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAK KRLNFGQT.. VP1_isolate_hu37_AAS99285 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAK KRLNFGQT.. VP1_isolate_hu42_AAS99290 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAK KRLNFGQT.. VP1_isolate_hu40_AAS99288 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAK KRLSFGQT.. VP1_isolate_hu67_AAS99312 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAK KRLNFGQT.. VP1_isolate_rh38_AAS99243 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QRPAK KRLNFGQT.. VP1_isolate_hu41_AAS99289 VEEAAKTAPG KK.RPVEPPP QRSPDSSTGI GKKG.QQPAK KRLNFGQT.. VP1_isolate_hu66_AAS99311 VEEAAKTAPG KK.RPVEPSP QRSPDSSAGI GKKG.QQPAK KRLNFGQT.. VP1_isolate_hu17_AAS99267 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKTG.QQPAK KRLNFGQT.. VP1_isolate_hu6_AAS99306 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKTG.QQPAK KRLNFGQT.. VP1_isolate_rh25_AAS99242 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKTG.QQPAK KRLNFGQT.. VP1_isolate_hu39_AAS99286 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGRT.. VP1_isolate_rh49_AAS99247 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh50_AAS99248 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAG KRLNFGQT.. VP1_isolate_rh51_AAS99249 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT..

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140 VP1_isolate_rh52_AAS99250 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh64_AAS99259 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh53_AAS99251 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh61_AAS99257 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh58_AAS99255 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh57_AAS99254 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. AAV8_VP1_AAN03857 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. AAV8_YP_077180 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh43_AAS99245 VEEGAKTAPG KK.RPVEQSP QE.PDSSSGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_pi1_AAS99238 VEEGAKTAPG KK.RPVEP.. ....DSSSGI GKSG.QQPAK KRLNFGQT.. VP1_isolate_pi3_AAS99240 VEEGAKTAPG KK.RPVEP.. ....DSSSGI GKSG.QQPAK KRLNFGPT.. VP1_isolate_pi2_AAS99239 VEEGAKTAPG KK.RPVEP.. ....DSSSGI GKSG.RQPAK KRLNFGQT.. VP1_isolate_rh1_AAS99241 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. AAV7_VP1_AAN03855 VEEGAKTAPA KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. AAV7_YP_077178 VEEGAKTAPA KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh48_AAS99246 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh62_AAS99258 AEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh55_AAS99253 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh54_AAS99252 VEEAAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. VP1_isolate_rh60_AAS99256 VEEGAKTAPG KK.RPVEPSP QRSPDSSTGI GKKG.QQPAR KRLNFGQT.. AAV9_VP1_AAS99264 VEEAAKTAPG KK.RPVEQSP QE.PDSSAGI GKSG.AQPAK KRLNFGQT.. VP1_isolate_hu31_AAS99281 VEEAAKTAPG KK.RPVEQSP QE.PDSSAGI GKSG.SQPAK KKLNFGQT.. VP1_isolate_hu32_AAS99282 VEEAAKTAPG KK.RPVEQSP QE.PDSSAGI GKSG.SQPAK KKLNFGQT.. AAV11_AAT46339 VEEGAKTAPG KK.RPLE.SP QE.PDSSSGI GKKG.KQPAR KRLNFEED.. AAV4_NP_044927 VEQAGETAPG KK.RPLIESP QQ.PDSSTGI GKKG.KQPAK KKLVFEDE.. AAV4_VP1_AAC58045 VEQAGETAPG KK.RPLIESP QQ.PDSSTGI GKKG.KQPAK KKLVFEDE.. BOVINE_AAV_AAR26465 VETPDKTAPA AKKRPLEQSP QE.PDSSSGV GKKG.KQPAR KRLNFDDE.. BOVINE_AAV_YP_024971 VETPDKTAPA AKKRPLEQSP QE.PDSSSGV GKKG.KQPAR KRLNFDDE.. AAV5_VP1_AAD13756 VEEGAKTAPT GKRID..... DHFPKRKK.. .......... ARTEEDSKPS AAV5_YP_068409 VEEGAKTAPT GKRID..... DHFPKRKK.. .......... ARTEEDSKPS CAPRINE_AAV1_AAU84890 VEEGAKTAPT GKRID..... DHFPKRKK.. .......... ARTEEDSKPS GOAT_AAV_VP1_ABC69726 VEEGAKTAPT GKRID..... DHFPKRKK.. .......... ARTEEDSKPS RAT_AAV1_AAZ79676 EEPELAPPVK RPHSP..... EKTPENQKGQ PRPDPRTPAK KRLEFSDQPG MOUSE_AAV1_AAZ79672 VEPELAPPSG RK.RPVQ... .....SSQES GYSS.SQDKR PNLDVDEE.. AVIAN_AAV_ATCC_VR865_AAO32087 VED.SKTAPT GDKRKGEDEP .RLPDTSSQT PKKN.KKPRK ERPSGGAE.. AVIAN_AAV_ATCC_VR865_AAT48613 VED.SKTAPT GDKRKGEDEP .RLPDTSSQT PKKN.KKPRK ERPSGGAE.. AVIAN_AAV_ATCC_VR865_NP_852781 VED.SKTAPT GDKRKGEDEP .RLPDTSSQT PKKN.KKPRK ERPSGGAE.. AVIAN_AAV_Strain_DA1_AAT48615 IEQPDNTAGT GEKR.....P .ERVDDFFPK KKKA.KTEQG KAPAQTGE.. AVIAN_AAV_Strain_DA1_YP_077183 IEQPDNTAGT GEKR.....P .ERVDDFFPK KKKA.KTEQG KAPAQTGE.. DUCK_AAV_Strain_FM_AAA83225 VEEPVNMAPA KKSS...... .GKLTDHDPI VKKP.KLSEE NSPSP..... MUSCOVY_DUCK_PARVOVIRUS_YP_068412 ......MAPA KKSS...... .GKLTDHDPI VKKP.KLSEE NSPSP.....

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141 MUSCOVY_DUCK_VP1_YP_068411 VEEPVNMAPA KKSS...... .GKLTDHDPI VKKP.KLSEE NSPSP..... MUSCOVY_DUCK_PARVOVIRUS_YP_068413 .......... .......... .......... .......... .......... Goose_AAV_VP1_AAA83230 VEDPVNTAPA KKNT...... .GKLTDHYPV VKKP.KLTEE VSAGG..... GOOSE_AAV_VP1_NP_043515 VEDPVNTAPA KKNT...... .GKLTDHYPV VKKP.KLTEE VSAGG..... AAV3_NP_043941 VEEAAKTAPG KK.GAVDQSP QE.PDSSSGV GKSG.KQPAR KRLNFGQT.. AAV3_Strain_H_AAC55049 VEEAAKTAPG KK.GAVDQSP QE.PDSSSGV GKSG.KQPAR KRLNFGQT.. AAV3B_VP1_AAB95452 VEEAAKTAPG KK.RPVDQSP QE.PDSSSGV GKSG.KQPAR KRLNFGQT.. VP1_isolate_hu1_AAS99260 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu4_AAS99287 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu2_AAS99270 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QRPAR KRLNFGQT.. VP1_isolate_hu3_AAS99280 LRKPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu60_AAS99307 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu61_AAS99308 VEEPVKTAPG KK.RPVEHPP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu25_AAS99276 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu15_AAS99265 VGEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.NQPAR KRLNFGQT.. VP1_isolate_hu16_AAS99266 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.NQPAR KRLNFGQT.. VP1_isolate_hu18_AAS99268 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu7_AAS99313 VEGPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu10_AAS99261 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.HQPAR KRLNFGQT.. VP1_isolate_hu11_AAS99262 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.HQPAR KRLNFGQT.. VP1_isolate_hu9_AAS99314 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.HQPAR KRLNFGQT.. VP1_isolate_hu53_AAS99300 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu55_AAS99302 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu54_AAS99301 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_huS17_AAU05370 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKSG.QQPAR KRLNFGQT.. AAV2_VP1_AAC03780 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu34_AAS99283 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu35_AAS99284 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_islolate_hu51_AAS99298 VGEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu52_AAS99299 VGEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu47_AAS99295 VGEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu45_AAS99293 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu58_AAS99305 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.NQPAR KRLNFGQT.. VP1_isolate_hu49_AAS99297 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu56_AAS99303 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.NQPAR KRLNFGQT.. VP1_isolate_hu57_AAS99304 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKAG.NQPAR KRLNFGQT.. VP1_isolate_hu28_AAS99278 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKSG.NQPAR KRLNFGQT.. VP1_isolate_hu29_AAS99279 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKSG.NQPAR KRLNFGQT.. VP1_isolate_huT70_AAU05364 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKSG.NQPAR KRLNFGQT.. VP1_isolate_hu13_AAS99263 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu63_AAS99309 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT..

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142 VP1_isolate_hu64_AAS99310 VEEPVKTAPG KK.RPVEHSL AE.PDSSSGT GKAG.QQPAR RRLNFGQT.. VP1_isolate_huT40_AAU05362 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.NQPAR KRLNFGQT.. VP1_isolate_huLG15_AAU05371 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKSG.QQPAR KRLNFGQT.. VP1_isolate_huT17_AAU05358 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKSG.QQPAR KRLNFGQT.. VP1_isolate_huT41_AAU05372 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKSG.QQPAR KRLNFGQT.. VP1_isolate_huT71_AAU05366 VEEPVKTAPG KK.RPVEHSP VE.PDSSSGT GKSG.QQPAR KRLNFGQT.. VP1_isolate_huT88_AAU05368 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKSG.QQPAR KRLNFGQT.. VP1_isolate_huT32_AAU05360 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKSG.QQPAR KRLNFGQT.. VP1_isolate_hu27_AAS99277 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu19_AAS99269 VEEPVKTAPG EK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu20_AAS99271 VEEPVKAAPG EK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu21_AAS99272 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu24_AAS99275 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu22_AAS99273 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. VP1_isolate_hu23_AAS99274 VEEPVKTAPG KK.RPVEHSP AE.PDSSSGT GKAG.QQPAR KRLNFGQT.. 201 250 AAV_VR195_ABA71699 GDSESVPDPQ PLG...EPPA TPAAVGPTTM ASGGGAPMAD NNEGADGVGN AAV_VR355_ABA71701 GDSESVPDPQ PLG...EPPA TPAALGPTTM ASGGGAPMAD NNEGADGVGN AAV1_NP_049542 GDSESVPDPQ PLG...EPPA TPAAVGPTTM ASGGGAPMAD NNEGADGVGN AAV1_VP1_AAD27757 GDSESVPDPQ PLG...EPPA TPAAVGPTTM ASGGGAPMAD NNEGADGVGN VP1_isolate_hu48_AAS99296 GDSESVPDPQ PLG...EPPA TPAAVGPTTM ASGGGAPMAD NNEGADGVGN AAV6_VP1_AAB95450 GDSESVPDPQ PLG...EPPA TPAAVGPTTM ASGGGAPMAD NNEGADGVGN VP1_isolate_hu43_AAS99291 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGN VP1_isolate_hu44_AAS99292 GDSESVPDPQ PLG...EPPA TPAAVGPTTM ASGGGAPMAD NNEGADGVGN VP1_isolate_hu46_AAS99294 GDSESVPDPQ PLG...EPPA TPAAVGPTTM ASGGGAPMAD NNEGADGVGN AAV10_AAT46337 GESESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh40_AAS99244 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_hu37_AAS99285 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_hu42_AAS99290 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_hu40_AAS99288 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_hu67_AAS99312 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh38_AAS99243 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_hu41_AAS99289 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_hu66_AAS99311 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_hu17_AAS99267 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_hu6_AAS99306 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh25_AAS99242 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_hu39_AAS99286 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh49_AAS99247 GDSESVPDPQ LIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh50_AAS99248 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS

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143 VP1_isolate_rh51_AAS99249 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh52_AAS99250 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh64_AAS99259 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh53_AAS99251 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh61_AAS99257 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh58_AAS99255 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh57_AAS99254 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS AAV8_VP1_AAN03857 GDSESVPDPQ PLG...EPPA APSGVGPNTM AAGGGAPMAD NNEGADGVGS AAV8_YP_077180 GDSESVPDPQ PLG...EPPA APSGVGPNTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh43_AAS99245 GDSESVPDPQ PLG...EPPA APSGVGPNTM AAGGGAPMAD NNEGADGVGS VP1_isolate_pi1_AAS99238 GDSESVPDPQ PLS...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGN VP1_isolate_pi3_AAS99240 GDSESVPDPQ PLS...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGN VP1_isolate_pi2_AAS99239 GDSESVPDPQ PLS...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGN VP1_isolate_rh1_AAS99241 GDSESVPDPQ PLG...EPPA APSGVGPNTM AAGGGAPMAD NNEGADGVGN AAV7_VP1_AAN03855 GDSESVPDPQ PLG...EPPA APSSVGSGTV AAGGGAPMAD NNEGADGVGN AAV7_YP_077178 GDSESVPDPQ PLG...EPPA APSSVGSGTV AAGGGAPMAD NNEGADGVGN VP1_isolate_rh48_AAS99246 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNKGADGVGN VP1_isolate_rh62_AAS99258 GDSESVPDPQ PIG...EPPA GPSGLGSGTM AAGGGAPMAD NNKGADGVGN VP1_isolate_rh55_AAS99253 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS VP1_isolate_rh54_AAS99252 GDSESVPDPQ PLG...EPPA GPSGLGSGTM AAGGGAPMAD NNEGADGVGN VP1_isolate_rh60_AAS99256 GDSESVPDPQ PIG...EPPA APSSVGSGTM AAGGGAPMAD NNEGADGVGS AAV9_VP1_AAS99264 GDTESVPDPQ PIG...EPPA APSGVGSLTM ASGGGAPVAD NNEGADGVGS VP1_isolate_hu31_AAS99281 GDTESVPDPQ PIG...EPPA APSGVGSLTM ASGGGAPVAD NNEGADGVGS VP1_isolate_hu32_AAS99282 GDTESVPDPQ PIG...EPPA APSGVGSLTM ASGGGAPVAD NNEGADGVGS AAV11_AAT46339 TGAGDGPP.. ......EGSD TSAMSSDIEM RAAPGGNAVD AGQGSDGVGN AAV4_NP_044927 TGAGDGPP.. ......EGST SGAMSDDSEM RAAAGGAAVE GGQGADGVGN AAV4_VP1_AAC58045 TGAGDGPP.. ......EGST SGAMSDDSEM RAAAGGAAVE GGQGADGVGN BOVINE_AAV_AAR26465 PGAGDGPPP. ......EGPS SGAMSTETEM RAAAGGNGGD AGQGAEGVGN BOVINE_AAV_YP_024971 PGAGDGPPP. ......EGPS SGAMSTETEM RAAAGGNGGD AGQGAEGVGN AAV5_VP1_AAD13756 TSSDAEAGPS GSQQLQIPAQ PASSLGADTM SAGGGGPLGD NNQGADGVGN AAV5_YP_068409 TSSDAEAGPS GSQQLQIPAQ PASSLGADTM SAGGGGPLGD NNQGADGVGN CAPRINE_AAV1_AAU84890 TSSDAEAGPS GSQQLQIPAQ PASSLGADTM SAGGGGPLGD NNQGADGVGN GOAT_AAV_VP1_ABC69726 TSSDAEAGPS GSQQLQIPAQ PASSLGADTM SAGGGGPLGD NNQGADGVGN RAT_AAV1_AAZ79676 SSADLPASSQ QSQ....PPA GVPGVVPGTM SAGGGAPVDD AQQGADGVGN MOUSE_AAV1_AAZ79672 DREFAAAAAE TET...GSAP PTGNLGPGTM AGGGSAPIDD GSYGADGVGN AVIAN_AAV_ATCC_VR865_AAO32087 DPGEGTSSNA GAA...APAS S...VGSSIM AEGGGGPVGD AGQGADGVGN AVIAN_AAV_ATCC_VR865_AAT48613 DPGEGTSSNA GAA...APAS S...VGSSIM AEGGGGPVGD AGQGADGVGN AVIAN_AAV_ATCC_VR865_NP_852781 DPGEGTSSNA GAA...APAS S...VGSSIM AEGGGGPVGD AGQGADGVGN AVIAN_AAV_Strain_DA1_AAT48615 DPGEGTSSNA GSS...APSS ....VGSSVM AEGGGGPMGD AGQGADGVGN AVIAN_AAV_Strain_DA1_YP_077183 DPGEGTSSNA GSS...APSS ....VGSSVM AEGGGGPMGD AGQGADGVGN DUCK_AAV_Strain_FM_AAA83225 .SNSGGEASA AAT...EGSE P...VAAPNM AEGGSGAMGD SAGGADGVGN

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144MUSCOVY_DUCK_PARVOVIRUS_YP_068412 .SNSGGEASA AAT...EGSE P...VAAPNM AEGGSGAMGD SAGGADGVGN MUSCOVY_DUCK_VP1_YP_068411 .SNSGGEASA AAT...EGSE P...VAAPNM AEGGSGAMGD SAGGADGVGN MUSCOVY_DUCK_PARVOVIRUS_YP_068413 .......... .......... .........M AEGGSGAMGD SAGGADGVGN Goose_AAV_VP1_AAA83230 .GSSAVQDGG ATA...EGTE P...VAASEM AEGGGGAMGD SSGGADGVGN GOOSE_AAV_VP1_NP_043515 .GSSAVQDGG ATA...EGTE P...VAASEM AEGGGGAMGD SSGGADGVGN AAV3_NP_043941 GDSESVPDPQ PLG...EPPA APTSLGSNTM ASGGGAPMAD NNEGADGVGN AAV3_Strain_H_AAC55049 GDSESVPDPQ PLG...EPPA APTSLGSNTM ASGGGAPMAD NNEGADGVGN AAV3B_VP1_AAB95452 GDSESVPDPQ PLG...EPPA APTSLGSNTM ASGGGAPMAD NNEGADGVGN VP1_isolate_hu1_AAS99260 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu4_AAS99287 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu2_AAS99270 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu3_AAS99280 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu60_AAS99307 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu61_AAS99308 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu25_AAS99276 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu15_AAS99265 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPVAD NNEGADGVGN VP1_isolate_hu16_AAS99266 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPVAD NNEGADGVGN VP1_isolate_hu18_AAS99268 GDADSVPDPQ PLG...QPPA APSGLGSTTM ASGSGAPVAD NNEGADGVGN VP1_isolate_hu7_AAS99313 GDADSVPDPQ PLG...QPPA APSGLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu10_AAS99261 GDADSVPDPQ PLG...QPPA APTSLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu11_AAS99262 GDADSVPDPQ PLG...QPPA APTSLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu9_AAS99314 GDADSVPDPQ PLG...QPPA APTSLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu53_AAS99300 GDADSVPDPQ PLR...QPPA APTSLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu55_AAS99302 GDADSVPDPQ PLG...QPPA APTSLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu54_AAS99301 GDADSVPDPQ PLG...QPPA APTSLGSTTM ATGSGAPMAD NNEGADGVGN VP1_isolate_huS17_AAU05370 GDSDSVPDPQ PLG...EPPA APTSLGSTTM ASGGGAPVAD NNEGADGVGN AAV2_VP1_AAC03780 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu34_AAS99283 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu35_AAS99284 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_islolate_hu51_AAS99298 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu52_AAS99299 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu47_AAS99295 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu45_AAS99293 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu58_AAS99305 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNDGADGVGN VP1_isolate_hu49_AAS99297 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu56_AAS99303 GDADSVPDPQ PLG...QPPA SPSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu57_AAS99304 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu28_AAS99278 GDSDSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu29_AAS99279 GDSDSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_huT70_AAU05364 GDSDSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu13_AAS99263 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN

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145 VP1_isolate_hu63_AAS99309 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_hu64_AAS99310 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_huT40_AAU05362 GDADSVPDPQ PLG...QPPA APSGLGTNTM ATGSGAPMAD NNEGADGVGN VP1_isolate_huLG15_AAU05371 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_huT17_AAU05358 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_huT41_AAU05372 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_huT71_AAU05366 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_huT88_AAU05368 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_huT32_AAU05360 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_hu27_AAS99277 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_hu19_AAS99269 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_hu20_AAS99271 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_hu21_AAS99272 GDADSVPDPR PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_hu24_AAS99275 GDADSVPDPR PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_hu22_AAS99273 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN VP1_isolate_hu23_AAS99274 GDADSVPDPQ PLG...QPPA APSGLGTNTM ASGSGAPMAD NNEGADGVGN 251 300 AAV_VR195_ABA71699 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSA ST.GASNDNH AAV_VR355_ABA71701 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSA ST.GASNDNH AAV1_NP_049542 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSA ST.GASNDNH AAV1_VP1_AAD27757 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSA ST.GASNDNH VP1_isolate_hu48_AAS99296 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISST ST.GASNDNH AAV6_VP1_AAB95450 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSA ST.GASNDNH VP1_isolate_hu43_AAS99291 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSA ST.GASNDNH VP1_isolate_hu44_AAS99292 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSA ST.GASNDNH VP1_isolate_hu46_AAS99294 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSA ST.GASNDNH AAV10_AAT46337 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh40_AAS99244 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_hu37_AAS99285 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_hu42_AAS99290 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_hu40_AAS99288 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_hu67_AAS99312 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh38_AAS99243 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_hu41_AAS99289 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_hu66_AAS99311 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_hu17_AAS99267 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_hu6_AAS99306 SSGNWHCDSA WLGDRVITTS TRPWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh25_AAS99242 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_hu39_AAS99286 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh49_AAS99247 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT

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146 VP1_isolate_rh50_AAS99248 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh51_AAS99249 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh52_AAS99250 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh64_AAS99259 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh53_AAS99251 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh61_AAS99257 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh58_AAS99255 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT VP1_isolate_rh57_AAS99254 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQTSNG TSGGSTNDNT AAV8_VP1_AAN03857 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGATNDNT AAV8_YP_077180 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGATNDNT VP1_isolate_rh43_AAS99245 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGATNDNT VP1_isolate_pi1_AAS99238 VSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSSNDNT VP1_isolate_pi3_AAS99240 VSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSSNDNT VP1_isolate_pi2_AAS99239 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSSNDNT VP1_isolate_rh1_AAS99241 SSGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISNG TSGGSTNDNT AAV7_VP1_AAN03855 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSE T.AGSTNDNT AAV7_YP_077178 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSE T.AGSTNDNT VP1_isolate_rh48_AAS99246 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSQ S.AGSTNDNV VP1_isolate_rh62_AAS99258 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSQ S.AGSTNDNV VP1_isolate_rh55_AAS99253 SSGNWHCDST RLGDRVITTS TRTWALPTYN NHLYKQISSQ S.AGSTNDNV VP1_isolate_rh54_AAS99252 ASGNWHCDST WLGDRVITTS TRTWALPTYN NHLYKQISSQ S.AGSTNDNV VP1_isolate_rh60_AAS99256 SSGNWHCDST WLGDRVITTS TRTWALPTHN NHLYKQISNG TSGGSTNDNV AAV9_VP1_AAS99264 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISNS TSGGSSNDNA VP1_isolate_hu31_AAS99281 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISNS TSGGSSNDNA VP1_isolate_hu32_AAS99282 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISNS TSGGSSNDNA AAV11_AAT46339 ASGDWHCDST WSEGKVTTTS TRTWVLPTYN NHLYLRLGTT S.....SSNT AAV4_NP_044927 ASGDWHCDST WSEGHVTTTS TRTWVLPTYN NHLYKRLGES L.....QSNT AAV4_VP1_AAC58045 ASGDWHCDST WSEGHVTTTS TRTWVLPTYN NHLYKRLGES L.....QSNT BOVINE_AAV_AAR26465 ASGDWHCDST WSESHVTTTS TRTWVLPTYN NHLYLRLGSS N.....ASDT BOVINE_AAV_YP_024971 ASGDWHCDST WSESHVTTTS TRTWVLPTYN NHLYLRLGSS N.....ASDT AAV5_VP1_AAD13756 ASGDWHCDST WMGDRVVTKS TRTWVLPSYN NHQYREIKSG S.VDGSNANA AAV5_YP_068409 ASGDWHCDST WMGDRVVTKS TRTWVLPSYN NHQYREIKSG S.VDGSNANA CAPRINE_AAV1_AAU84890 ASGDWHCDST WMGDRVVTKS TRTWVLPSYN NHQYREIKSG S.VDGSNANA GOAT_AAV_VP1_ABC69726 ASGDWHCDST WMGDRVVTKS TRTWVLPSYN NHQYREIKSG S.VDGSNANA RAT_AAV1_AAZ79676 ASGDWHCDSK WLGNRVLTRS TRTWVLPSYN NHLYKQISDA SGVHSLPGSR MOUSE_AAV1_AAZ79672 ASGDWHCDST WLDNCVITRT TRTWNLPTYN NHIYKRLNGT T....SGDQS AVIAN_AAV_ATCC_VR865_AAO32087 SSGNWHCDSQ WLENGVVTRT TRTWVLPSYN NHLYKRIQGP S..GGDNNNK AVIAN_AAV_ATCC_VR865_AAT48613 SSGNWHCDSQ WLENGVVTRT TRTWVLPSYN NHLYKRIQGP S..GGDNNNK AVIAN_AAV_ATCC_VR865_NP_852781 SSGNWHCDSQ WLENGVVTRT TRTWVLPSYN NHLYKRIQGP S..GGDNNNK AVIAN_AAV_Strain_DA1_AAT48615 SSGNWHCDSQ WLDNGVVTRT TRTWVLPSYN NHLYKRIQGP G..GTDPNNK AVIAN_AAV_Strain_DA1_YP_077183 SSGNWHCDSQ WLDNGVVTRT TRTWVLPSYN NHLYKRIQGP G..GTDPNNK

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147 DUCK_AAV_Strain_FM_AAA83225 ASGNWHCDSQ WLGDTVITKT TRTWVLPSYN NHMYQAITSG T..NPDSNTQ MUSCOVY_DUCK_PARVOVIRUS_YP_068412 ASGNWHCDSQ WLGDTVITKT TRTWVLPSYN NHMYQAITSG T..NPDSNTQ MUSCOVY_DUCK_VP1_YP_068411 ASGNWHCDSQ WLGDTVITKT TRTWVLPSYN NHMYQAITSG T..NPDSNTQ MUSCOVY_DUCK_PARVOVIRUS_YP_068413 ASGNWHCDSQ WLGDTVITKT TRTWVLPSYN NHMYQAITSG T..NPDSNTQ Goose_AAV_VP1_AAA83230 ASGNWHCDSQ WMGNTVITKT TRTWVLPSYN NHIYKAITSG T..SQDANVQ GOOSE_AAV_VP1_NP_043515 ASGNWHCDSQ WMGNTVITKT TRTWVLPSYN NHIYKAITSG T..SQDANVQ AAV3_NP_043941 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH AAV3_Strain_H_AAC55049 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH AAV3B_VP1_AAB95452 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu1_AAS99260 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu4_AAS99287 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu2_AAS99270 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu3_AAS99280 SSGNWHCDSQ WLDDRVIATS TRTWALPTYN NHLYKQISSQ S..GACNDNH VP1_isolate_hu60_AAS99307 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu61_AAS99308 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu25_AAS99276 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu15_AAS99265 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu16_AAS99266 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu18_AAS99268 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu7_AAS99313 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu10_AAS99261 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu11_AAS99262 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu9_AAS99314 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu53_AAS99300 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu55_AAS99302 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu54_AAS99301 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_huS17_AAU05370 SSGNWHCDSQ WLGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH AAV2_VP1_AAC03780 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu34_AAS99283 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu35_AAS99284 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_islolate_hu51_AAS99298 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu52_AAS99299 SSGNRHCDST WMGDRVITTS TRTWALPTYN NHLYRQISSQ S..GASNDNH VP1_isolate_hu47_AAS99295 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDSH VP1_isolate_hu45_AAS99293 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu58_AAS99305 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu49_AAS99297 SSGSWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu56_AAS99303 SSGNWHCDST WMGDRVVTTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu57_AAS99304 SSGDWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu28_AAS99278 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu29_AAS99279 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_huT70_AAU05364 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH

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148 VP1_isolate_hu13_AAS99263 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu63_AAS99309 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu64_AAS99310 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYRQISSQ S..GASNDNH VP1_isolate_huT40_AAU05362 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_huLG15_AAU05371 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_huT17_AAU05358 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_huT41_AAU05372 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_huT71_AAU05366 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_huT88_AAU05368 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_huT32_AAU05360 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu27_AAS99277 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu19_AAS99269 SSGNWYCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu20_AAS99271 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu21_AAS99272 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu24_AAS99275 SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu22_AAS99273 SSGNWHCDST WMGGRVITTS TRTWALPTYN NHLYKQISSQ S..GASNDNH VP1_isolate_hu23_AAS99274 SSGNWHCDST WMGDRVITTS TRTWALPTCN NHLYKQISSQ S..GASNDNH 301 350 AAV_VR195_ABA71699 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK AAV_VR355_ABA71701 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK AAV1_NP_049542 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK AAV1_VP1_AAD27757 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu48_AAS99296 YFGYGTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVE AAV6_VP1_AAB95450 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu43_AAS99291 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu44_AAS99292 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu46_AAS99294 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK AAV10_AAT46337 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh40_AAS99244 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu37_AAS99285 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu42_AAS99290 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu40_AAS99288 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu67_AAS99312 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh38_AAS99243 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKPFNIQVK VP1_isolate_hu41_AAS99289 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu66_AAS99311 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu17_AAS99267 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu6_AAS99306 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_rh25_AAS99242 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu39_AAS99286 YFGYSTPWGY LDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK

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149 VP1_isolate_rh49_AAS99247 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh50_AAS99248 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh51_AAS99249 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh52_AAS99250 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh64_AAS99259 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh53_AAS99251 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh61_AAS99257 YFGYSTPWGY FDFNRFHCHF SPRDWQRPIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh58_AAS99255 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh57_AAS99254 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK AAV8_VP1_AAN03857 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK AAV8_YP_077180 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_rh43_AAS99245 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_pi1_AAS99238 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL NFKLFNIQVK VP1_isolate_pi3_AAS99240 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL NFKLFNIQVK VP1_isolate_pi2_AAS99239 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_rh1_AAS99241 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK AAV7_VP1_AAN03855 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL RFKLFNIQVK AAV7_YP_077178 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL RFKLFNIQVK VP1_isolate_rh48_AAS99246 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN SNWGFRPKKL NFKLFNIQVK VP1_isolate_rh62_AAS99258 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL NFKLFNIQVK VP1_isolate_rh55_AAS99253 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL NFKLFNIQVK VP1_isolate_rh54_AAS99252 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL NFKLFNIQVK VP1_isolate_rh60_AAS99256 YFGYSTPWGY FDFNRFHCHF SPRDRQRLIN NNWGFRPKRL SFKLFNIQVK AAV9_VP1_AAS99264 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu31_AAS99281 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu32_AAS99282 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK AAV11_AAT46339 YNGFSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGLRPKAM RVKIFNIQVK AAV4_NP_044927 YNGFSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGMRPKAM RVKIFNIQVK AAV4_VP1_AAC58045 YNGFSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGMRPKAM RVKIFNIQVK BOVINE_AAV_AAR26465 FNGFSTPWGY FDFNRFHCHF SPRDWQRLIN NHWGLRPKSM QVRIFNIQVK BOVINE_AAV_YP_024971 FNGFSTPWGY FDFNRFHCHF SPRDWQRLIN NHWGLRPKSM QVRIFNIQVK AAV5_VP1_AAD13756 YFGYSTPWGY FDFNRFHSHW SPRDWQRLIN NYWGFRPRSL RVKIFNIQVK AAV5_YP_068409 YFGYSTPWGY FDFNRFHSHW SPRDWQRLIN NYWGFRPRSL RVKIFNIQVK CAPRINE_AAV1_AAU84890 YFGYSTPWGY FDFNRFHSHW SPRDWQRLIN NYWGFRPRSL RVKIFNIQVK GOAT_AAV_VP1_ABC69726 YFGYSTPWGY FDFNRFHSHW SPRDWQRLIN NYWGFRPRSL RVKIFNIQVK RAT_AAV1_AAZ79676 YFGYSTPWGY FDFNRFHCHF SPRDWQRLVN NHWGFRPKRL RVKLFNIQVK MOUSE_AAV1_AAZ79672 YFGFSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGLRPKSL RFKIFNIQVK AVIAN_AAV_ATCC_VR865_AAO32087 FFGFSTPWGY FDYNRFHCHF SPRDWQRLIN NNWGIRPKAM RFRLFNIQVK AVIAN_AAV_ATCC_VR865_AAT48613 FFGFSTPWGY FDYNRFHCHF SPRDWQRLIN NNWGIRPKAM RFRLFNIQVK AVIAN_AAV_ATCC_VR865_NP_852781 FFGFSTPWGY FDYNRFHCHF SPRDWQRLIN NNWGIRPKAM RFRLFNIQVK AVIAN_AAV_Strain_DA1_AAT48615 FFGFSTPWGY FDYNRFHCHF SPRDWQRLIN NNWGIRPKAM RFRLFNIQVK

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150 AVIAN_AAV_Strain_DA1_YP_077183 FFGFSTPWGY FDYNRFHCHF SPRDWQRLIN NNWGIRPKAM RFRLFNIQVK DUCK_AAV_Strain_FM_AAA83225 YAGYSTPWGY FDFNRFHCHF SPRDWQRLIN NHWGIRPKAL KFKIFNVQVK MUSCOVY_DUCK_PARVOVIRUS_YP_068412 YAGYSTPWGY FDFNRFHCHF SPRDWQRLIN NHWGIRPKAL KFKIFNVQVK MUSCOVY_DUCK_VP1_YP_068411 YAGYSTPWGY FDFNRFHCHF SPRDWQRLIN NHWGIRPKAL KFKIFNVQVK MUSCOVY_DUCK_PARVOVIRUS_YP_068413 YAGYSTPWGY FDFNRFHCHF SPRDWQRLIN NHWGIRPKAL KFKIFNVQVK Goose_AAV_VP1_AAA83230 YAGYSTPWGY FDFNRFHCHF SPRDWQRLIN NHWGIRPKSL KFKIFNVQVK GOOSE_AAV_VP1_NP_043515 YAGYSTPWGY FDFNRFHCHF SPRDWQRLIN NHWGIRPKSL KFKIFNVQVK AAV3_NP_043941 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL SFKLFNIQVR AAV3_Strain_H_AAC55049 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL SFKLFNIQVR AAV3B_VP1_AAB95452 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKKL SFKLFNIQVK VP1_isolate_hu1_AAS99260 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu4_AAS99287 YFGYSTPWGY FDFNRFHCHF SPRDWQRLVN NNRGFRPKRL NFKLFNIQVK VP1_isolate_hu2_AAS99270 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu3_AAS99280 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN SNWGFRPKRL NFKLFNIQVK VP1_isolate_hu60_AAS99307 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu61_AAS99308 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu25_AAS99276 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu15_AAS99265 YFGYSTPWGY FDFNRFHCHF SPRDRQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu16_AAS99266 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu18_AAS99268 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NSWGFRPKRL NFKLFNIQVK VP1_isolate_hu7_AAS99313 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu10_AAS99261 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu11_AAS99262 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu9_AAS99314 YFGCSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu53_AAS99300 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu55_AAS99302 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu54_AAS99301 YFGYSTPWGY FDFNRFHCRF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_huS17_AAU05370 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK AAV2_VP1_AAC03780 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu34_AAS99283 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu35_AAS99284 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_islolate_hu51_AAS99298 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu52_AAS99299 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu47_AAS99295 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu45_AAS99293 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu58_AAS99305 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVR VP1_isolate_hu49_AAS99297 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu56_AAS99303 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu57_AAS99304 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NLKLFNIQVK VP1_isolate_hu28_AAS99278 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu29_AAS99279 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK

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151 VP1_isolate_huT70_AAU05364 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu13_AAS99263 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu63_AAS99309 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVK VP1_isolate_hu64_AAS99310 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGSRPKRL NFKLFNIQVK VP1_isolate_huT40_AAU05362 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_huLG15_AAU05371 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL GFKLFNIQVK VP1_isolate_huT17_AAU05358 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_huT41_AAU05372 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_huT71_AAU05366 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_huT88_AAU05368 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_huT32_AAU05360 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu27_AAS99277 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu19_AAS99269 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu20_AAS99271 YFGYSTPWGH FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu21_AAS99272 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu24_AAS99275 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu22_AAS99273 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK VP1_isolate_hu23_AAS99274 YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL SFKLFNIQVK 351 400 AAV_VR195_ABA71699 EVTTSDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM AAV_VR355_ABA71701 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM AAV1_NP_049542 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM AAV1_VP1_AAD27757 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu48_AAS99296 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM AAV6_VP1_AAB95450 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu43_AAS99291 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu44_AAS99292 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu46_AAS99294 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGR LPPFPADVFM AAV10_AAT46337 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh40_AAS99244 EVTQDEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu37_AAS99285 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu42_AAS99290 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu40_AAS99288 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu67_AAS99312 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh38_AAS99243 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu41_AAS99289 EVTQNEGTKT VANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu66_AAS99311 EVTQNEGTET IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu17_AAS99267 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC PPPFPADVFM VP1_isolate_hu6_AAS99306 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC PPPFPADVFM VP1_isolate_rh25_AAS99242 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC PPPFPADVFM

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152 VP1_isolate_hu39_AAS99286 EVTQNEGTKT IANNLASTIQ VFTDSEYQPP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh49_AAS99247 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh50_AAS99248 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh51_AAS99249 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC QPPFPADVFM VP1_isolate_rh52_AAS99250 EVTQNEGTKT IANSLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh64_AAS99259 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh53_AAS99251 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh61_AAS99257 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh58_AAS99255 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh57_AAS99254 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM AAV8_VP1_AAN03857 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM AAV8_YP_077180 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh43_AAS99245 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_pi1_AAS99238 EVTQNEGTKT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_pi3_AAS99240 EVTQNEGTKT TANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_pi2_AAS99239 EVTQNEGTKT IANNLTSTVQ VFTDSKYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh1_AAS99241 EVTTNEGTKT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM AAV7_VP1_AAN03855 EVTTNDGVTT IANNLTSTIQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM AAV7_YP_077178 EVTTNDGVTT IANNLTSTIQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh48_AAS99246 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh62_AAS99258 EVTTGDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh55_AAS99253 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh54_AAS99252 EVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_rh60_AAS99256 EVTQNEGTKT IANNLTSTIQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM AAV9_VP1_AAS99264 EVTDNNGVKT IANNLTSTVQ VFTDSDYQLP YVLGSAHEGC LPPFPADVFM VP1_isolate_hu31_AAS99281 EVTDNNGVKT IANNLTSTVQ VFTDSDYQLP YVLGSAHEGC LPPFPADVFM VP1_isolate_hu32_AAS99282 EVTDNNGVKT IANNLTSTVQ VFTDSDYQLP YVLGSAHEGC LPPFPADVFM AAV11_AAT46339 EVTTSNGETT VANNLTSTVQ IFADSSYELP YVMDAGQEGS LPPFPNDVFM AAV4_NP_044927 EVTTSNGETT VANNLTSTVQ IFADSSYELP YVMDAGQEGS LPPFPNDVFM AAV4_VP1_AAC58045 EVTTSNGETT VANNLTSTVQ IFADSSYELP YVMDAGQEGS LPPFPNDVFM BOVINE_AAV_AAR26465 EVTTSNGETT VSNNLTSTVQ IFADSTYELP YVMDAGQEGS LPPFPNDVFM BOVINE_AAV_YP_024971 EVTTSNGETT VSNNLTSTVQ IFADSTYELP YVMDAGQEGS LPPFPNDVFM AAV5_VP1_AAD13756 EVTVQDSTTT IANNLTSTVQ VFTDDDYQLP YVVGNGTEGC LPAFPPQVFT AAV5_YP_068409 EVTVQDSTTT IANNLTSTVQ VFTDDDYQLP YVVGNGTEGC LPAFPPQVFT CAPRINE_AAV1_AAU84890 EVTVQDSTTT IANNLTSTVQ VFTDDDYQLP YVVGNGTEGC LPAFPPQVFT GOAT_AAV_VP1_ABC69726 EVTVQDSTTT IANNLTSTVQ VFTDDDYQLP YVVGNGTEGC LPAFPPQVFT RAT_AAV1_AAZ79676 EVTTTDSTTT VSNNLTSTVQ VFTDDEYQLP YVCGNATEGC LPPFPPDVFT MOUSE_AAV1_AAZ79672 EVTTQDSTKI ISNNLTSTVQ VFADTEYQLP YVIGSAHEGC LPPFPADVFM AVIAN_AAV_ATCC_VR865_AAO32087 EVTVQDFNTT IGNNLTSTVQ VFADKDYQLP YVLGSATEGT FPPFPADIYT AVIAN_AAV_ATCC_VR865_AAT48613 EVTVQDFNTT IGNNLTSTVQ VFADKDYQLP YVLGSATEGT FPPFPADIYT AVIAN_AAV_ATCC_VR865_NP_852781 EVTVQDFNTT IGNNLTSTVQ VFADKDYQLP YVLGSATEGT FPPFPADIYT

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153 AVIAN_AAV_Strain_DA1_AAT48615 EVTVQDSNTT IANNLTSTVQ VFADKDYQLP YVLGSATEGT FPPFPADIYT AVIAN_AAV_Strain_DA1_YP_077183 EVTVQDSNTT IANNLTSTVQ VFADKDYQLP YVLGSATEGT FPPFPADIYT DUCK_AAV_Strain_FM_AAA83225 EVTTQDQTKT IANNLTSTIQ IFTDNEHQLP YVLGSATEGT MPPFPSDVYA MUSCOVY_DUCK_PARVOVIRUS_YP_068412 EVTTQDQTKT IANNLTSTIQ IFTDNEHQLP YVLGSATEGT MPPFPSDVYA MUSCOVY_DUCK_VP1_YP_068411 EVTTQDQTKT IANNLTSTIQ IFTDNEHQLP YVLGSATEGT MPPFPSDVYA MUSCOVY_DUCK_PARVOVIRUS_YP_068413 EVTTQDQTKT IANNLTSTIQ IFTDNEHQLP YVLGSATEGT MPPFPSDVYA Goose_AAV_VP1_AAA83230 EVTTQDQTKT IANNLTSTIQ VFTDDEHQLP YVLGSATEGT MPPFPSDVYA GOOSE_AAV_VP1_NP_043515 EVTTQDQTKT IANNLTSTIQ VFTDDEHQLP YVLGSATEGT MPPFPSDVYA AAV3_NP_043941 GVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM AAV3_Strain_H_AAC55049 GVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM AAV3B_VP1_AAB95452 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu1_AAS99260 EVTQNGGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu4_AAS99287 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu2_AAS99270 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu3_AAS99280 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVPGSAHQGC LPPFPADVFM VP1_isolate_hu60_AAS99307 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu61_AAS99308 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu25_AAS99276 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu15_AAS99265 EVTQNDGTTT IANNLTSTVQ VFTDSGYQLP YVLGLAHQGC LPPFPADVFM VP1_isolate_hu16_AAS99266 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu18_AAS99268 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu7_AAS99313 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu10_AAS99261 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFT VP1_isolate_hu11_AAS99262 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu9_AAS99314 EVTQNDGTTT IANNLTSTVQ VFTDSEYPLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu53_AAS99300 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu55_AAS99302 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu54_AAS99301 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_huS17_AAU05370 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM AAV2_VP1_AAC03780 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu34_AAS99283 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu35_AAS99284 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_islolate_hu51_AAS99298 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu52_AAS99299 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu47_AAS99295 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu45_AAS99293 EVTQNDGTTT IANNLTSTVQ VFTDSGYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu58_AAS99305 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu49_AAS99297 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVPGSAHQGC LPPFPADVFM VP1_isolate_hu56_AAS99303 EVTQNDGTTT IANNLTSTVQ VFTDLEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu57_AAS99304 EVTQNDGTTT IANNLTSTVQ VFTDLEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu28_AAS99278 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM

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154 VP1_isolate_hu29_AAS99279 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_huT70_AAU05364 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu13_AAS99263 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu63_AAS99309 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu64_AAS99310 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_huT40_AAU05362 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_huLG15_AAU05371 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_huT17_AAU05358 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_huT41_AAU05372 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_huT71_AAU05366 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_huT88_AAU05368 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_huT32_AAU05360 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu27_AAS99277 EVTQNDGTTT IANNLTSTVQ VFTDSGYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu19_AAS99269 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu20_AAS99271 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu21_AAS99272 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu24_AAS99275 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu22_AAS99273 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM VP1_isolate_hu23_AAS99274 EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC LPPFPADVFM 401 450 AAV_VR195_ABA71699 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEEV AAV_VR355_ABA71701 IPQYGYLTLN NG...SKAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEEV AAV1_NP_049542 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEEV AAV1_VP1_AAD27757 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEEV VP1_isolate_hu48_AAS99296 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEEV AAV6_VP1_AAB95450 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu43_AAS99291 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEEV VP1_isolate_hu44_AAS99292 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEEV VP1_isolate_hu46_AAS99294 IPQYGYLTLN NG...SQAVG RSSSYCLEYF PSQMLRTGNN FTFSYTFEEV AAV10_AAT46337 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FEFSYTFEDV VP1_isolate_rh40_AAS99244 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FEFSYTFEDV VP1_isolate_hu37_AAS99285 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FEFSYTFEDV VP1_isolate_hu42_AAS99290 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FEFSYTFEDV VP1_isolate_hu40_AAS99288 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN SEFSYTFEDV VP1_isolate_hu67_AAS99312 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FEFSYTFEDV VP1_isolate_rh38_AAS99243 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FEFSYTFEDV VP1_isolate_hu41_AAS99289 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FEFSYTFEDV VP1_isolate_hu66_AAS99311 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FEFSYTFEDV VP1_isolate_hu17_AAS99267 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMRRTGNN FEFSYQFEDV VP1_isolate_hu6_AAS99306 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMRRTGNN FEFSYQFEDV

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155 VP1_isolate_rh25_AAS99242 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMRRTGNN FEFSYQFEDV VP1_isolate_hu39_AAS99286 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV VP1_isolate_rh49_AAS99247 IPQYGNLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV VP1_isolate_rh50_AAS99248 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV VP1_isolate_rh51_AAS99249 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV VP1_isolate_rh52_AAS99250 IPQYGYLTPN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV VP1_isolate_rh64_AAS99259 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV VP1_isolate_rh53_AAS99251 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV VP1_isolate_rh61_AAS99257 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYPFEDV VP1_isolate_rh58_AAS99255 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV VP1_isolate_rh57_AAS99254 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV AAV8_VP1_AAN03857 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFTYTFEDV AAV8_YP_077180 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFTYTFEDV VP1_isolate_rh43_AAS99245 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFTYTFEDV VP1_isolate_pi1_AAS99238 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_pi3_AAS99240 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_pi2_AAS99239 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_rh1_AAS99241 VPQYGYLTLN NG...SQALG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV AAV7_VP1_AAN03855 IPQYGYLTLN NG...SQSVG RSSFYCLEYF PSQMLRTGNN FEFSYSFEDV AAV7_YP_077178 IPQYGYLTLN NG...SQSVG RSSFYCLEYF PSQMLRTGNN FEFSYSFEDV VP1_isolate_rh48_AAS99246 IPQYGYLTLN NG...SQSVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_rh62_AAS99258 IPQYGYLTLN ND...SQSVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_rh55_AAS99253 IPQYGYLTLN NG...SQSVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_rh54_AAS99252 IPQYGYLTLN NG...SQSVG RSSFYCLEYF PSQVLRTGNN FTFSYTFEDV VP1_isolate_rh60_AAS99256 IPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FSFSYTFEDV AAV9_VP1_AAS99264 IPQYGYLTLN DG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV VP1_isolate_hu31_AAS99281 IPQYGYLTLN DG...GQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV VP1_isolate_hu32_AAS99282 IPQYGYLTLN DG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV AAV11_AAT46339 VPQYGYCGIV TG.ENQNQTD RNAFYCLEYF PSQMLRTGNN FEMAYNFEKV AAV4_NP_044927 VPQYGYCGLV TGNTSQQQTD RNAFYCLEYF PSQMLRTGNN FEITYSFEKV AAV4_VP1_AAC58045 VPQYGYCGLV TGNTSQQQTD RNAFYCLEYF PSQMLRTGNN FEITYSFEKV BOVINE_AAV_AAR26465 VPQYGYCGLV TGGSSQNQTD RNAFYCLEYF PSQMLRTGNN FEMVYKFENV BOVINE_AAV_YP_024971 VPQYGYCGLV TGGSSQNQTD RNAFYCLEYF PSQMLRTGNN FEMVYKFENV AAV5_VP1_AAD13756 LPQYGYATLN RD.NTENPTE RSSFFCLEYF PSKMLRTGNN FEFTYNFEEV AAV5_YP_068409 LPQYGYATLN RD.NTENPTE RSSFFCLEYF PSKMLRTGNN FEFTYNFEEV CAPRINE_AAV1_AAU84890 LPQYGYATLN RD.NGDNPTE RSSFFCLEYF PSKMLRTGNN FEFTYSFEEV GOAT_AAV_VP1_ABC69726 LPQYGYATLN RD.NGDNPTE RSSFFCLEYF PSKMLRTGNN FEFTYSFEEV RAT_AAV1_AAZ79676 LPQYGYATLN DS.NNGNPTN RSSFFCLEYF PSKMLRTGNN FEFTFEFEDV MOUSE_AAV1_AAZ79672 LPQYGYCTRQ DG.NSNNPTP RSAFYCLEYF PSKMLRTGNS FEFTYNFEKV AVIAN_AAV_ATCC_VR865_AAO32087 IPQYGYCTLN YN.N..EAVD RSAFYCLDYF PSDMLRTGNN FEFTYTFEDV AVIAN_AAV_ATCC_VR865_AAT48613 IPQYGYCTLN YN.N..EAVD RSAFYCLDYF PSDMLRTGNN FEFTYTFEDV

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156 AVIAN_AAV_ATCC_VR865_NP_852781 IPQYGYCTLN YN.N..EAVD RSAFYCLDYF PSDMLRTGNN FEFTYTFEDV AVIAN_AAV_Strain_DA1_AAT48615 IPQYGYCTLN YN.N..EAVD RSAFYCLDYF PSDMLRTGNN FEFTYTFEDV AVIAN_AAV_Strain_DA1_YP_077183 IPQYGYCTLN YN.N..EAVD RSAFYCLDYF PSDMLRTGNN FEFTYTFEDV DUCK_AAV_Strain_FM_AAA83225 LPQYGYCTMH TNQSGARFND RSAFYCLEYF PSQMLRTGNN FEFSFEFEEV MUSCOVY_DUCK_PARVOVIRUS_YP_068412 LPQYGYCTMH TNQSGARFND RSAFYCLEYF PSQMLRTGNN FEFSFEFEEV MUSCOVY_DUCK_VP1_YP_068411 LPQYGYCTMH TNQSGARFND RSAFYCLEYF PSQMLRTGNN FEFSFEFEEV MUSCOVY_DUCK_PARVOVIRUS_YP_068413 LPQYGYCTMH TNQSGARFND RSAFYCLEYF PSQMLRTGNN FEFSFEFEEV Goose_AAV_VP1_AAA83230 LPQYGYCTMH TNQNGARFND RSAFYCLEYF PSQMLRTGNN FEFTFDFEEV GOOSE_AAV_VP1_NP_043515 LPQYGYCTMH TNQNGARFND RSAFYCLEYF PSQMLRTGNN FEFTFDFEEV AAV3_NP_043941 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV AAV3_Strain_H_AAC55049 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV AAV3B_VP1_AAB95452 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu1_AAS99260 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu4_AAS99287 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu2_AAS99270 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu3_AAS99280 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu60_AAS99307 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu61_AAS99308 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu25_AAS99276 VPQYGYLTLN NG...SQAVG RSPFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu15_AAS99265 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu16_AAS99266 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu18_AAS99268 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu7_AAS99313 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu10_AAS99261 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN LTFSYTFEDV VP1_isolate_hu11_AAS99262 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu9_AAS99314 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu53_AAS99300 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu55_AAS99302 VPQYGYLTLN NG...SQAVG RSSFYCLECF PSQMLRTGNN FQFSYTFEDV VP1_isolate_hu54_AAS99301 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV VP1_isolate_huS17_AAU05370 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV AAV2_VP1_AAC03780 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu34_AAS99283 VPQYGYLTLN NE...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu35_AAS99284 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_islolate_hu51_AAS99298 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu52_AAS99299 VPQYGYLTLS NG...SQAVG RSSFYCPEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu47_AAS99295 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu45_AAS99293 VPQYGYPTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu58_AAS99305 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu49_AAS99297 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu56_AAS99303 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu57_AAS99304 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV

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157 VP1_isolate_hu28_AAS99278 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu29_AAS99279 VPQYGYLTLN NG...SQAVG RSSFYCLGYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_huT70_AAU05364 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu13_AAS99263 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu63_AAS99309 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu64_AAS99310 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_huT40_AAU05362 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_huLG15_AAU05371 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_huT17_AAU05358 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_huT41_AAU05372 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_huT71_AAU05366 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_huT88_AAU05368 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_huT32_AAU05360 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu27_AAS99277 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu19_AAS99269 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu20_AAS99271 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu21_AAS99272 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu24_AAS99275 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV VP1_isolate_hu22_AAS99273 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQTLRTGNN FTFSYTFEDV VP1_isolate_hu23_AAS99274 VPQYGYLTLN NG...SQAVG RSSFYCLEYF PSQMLRTGNN FTFSYTFEDV 451 500 AAV_VR195_ABA71699 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.NQSGSAQN KDLLFSRGSP AAV_VR355_ABA71701 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.NQSGSAQN KDLLFSRGSP AAV1_NP_049542 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.NQSGSAQN KDLLFSRGSP AAV1_VP1_AAD27757 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.NQSGSAQN KDLLFSRGSP VP1_isolate_hu48_AAS99296 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.NQSGSAQN KDLLFSRGSP AAV6_VP1_AAB95450 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.NQSGSAQN KDLLFSRGSP VP1_isolate_hu43_AAS99291 PLHSSYAHSQ SLDRLMNPLI VQYLYYLNRT Q.NQSGSAQN KDLLFSRGSP VP1_isolate_hu44_AAS99292 PFHSSYAHSQ SLDRLMNPLI DQYLYYPNRT Q.NQSGSAQN KDLLFSRGSP VP1_isolate_hu46_AAS99294 PLHSSCAHSQ SLDRLMNPLI DQYLYYLNRT Q.NQSGSAQN RDLLFSRGSP AAV10_AAT46337 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TQGT QQLLFSQAGP VP1_isolate_rh40_AAS99244 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TQGT QQLLFSQAGP VP1_isolate_hu37_AAS99285 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TQGT QQLLFSQAGP VP1_isolate_hu42_AAS99290 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TQGT QQLLFSQAGP VP1_isolate_hu40_AAS99288 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TQGT QQLLFSQAGP VP1_isolate_hu67_AAS99312 PFHSGYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TQGT QQLLFSQAGP VP1_isolate_rh38_AAS99243 PFHSSYAHSQ SLDRLMNPLI DQYLHYLSRT QSTGG.TQGT QQLLFSQAGP VP1_isolate_hu41_AAS99289 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TQGT QQLLFSQAGP VP1_isolate_hu66_AAS99311 PFHSSCAHSQ SSDRLMNPLI DQYLYYLSRT RSTGG.TQGT QQLLFSQAGP VP1_isolate_hu17_AAS99267 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP

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158 VP1_isolate_hu6_AAS99306 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP VP1_isolate_rh25_AAS99242 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP VP1_isolate_hu39_AAS99286 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSRAGP VP1_isolate_rh49_AAS99247 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP VP1_isolate_rh50_AAS99248 PFHSSYAHSQ SLDRLMNPLV DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP VP1_isolate_rh51_AAS99249 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP VP1_isolate_rh52_AAS99250 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLSSQAGP VP1_isolate_rh64_AAS99259 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP VP1_isolate_rh53_AAS99251 PFHSSYVHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP VP1_isolate_rh61_AAS99257 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP VP1_isolate_rh58_AAS99255 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP VP1_isolate_rh57_AAS99254 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTGG.TAGT QQLLFSQAGP AAV8_VP1_AAN03857 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QTTGG.TANT QTLGFSQGGP AAV8_YP_077180 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QTTGG.TANT QTLGFSQGGP VP1_isolate_rh43_AAS99245 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QTTGG.TANT QTLGFSQGGP VP1_isolate_pi1_AAS99238 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QTNG..TNAT QTLLFAQAGP VP1_isolate_pi3_AAS99240 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QTNG..TNAT QTLLFAQAGP VP1_isolate_pi2_AAS99239 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QTNG..TNAT QTLLFAQAGP VP1_isolate_rh1_AAS99241 PFHSSYAHSQ SLDRLMNPLI DQYLYYLVRT QTTG..TGGT QTLAFSQAGP AAV7_VP1_AAN03855 PFHSSYAHSQ SLDRLMNPLI DQYLYYLART QSNPGGTAGN RELQFYQGGP AAV7_YP_077178 PFHSSYAHSQ SLDRLMNPLI DQYLYYLART QSNPGGTAGN RELQFYQGGP VP1_isolate_rh48_AAS99246 PFHSSYAHSQ SLDRLMNPLI DQYLYYLART QSNAGGTAGN RELQFYQGGP VP1_isolate_rh62_AAS99258 PFHSSYAHSQ SLDRLMNPLI DQYLYYLART QSNAGGTAGN RELQFYQGGP VP1_isolate_rh55_AAS99253 PFHSSYAHSQ SLDRLMNPLI DQYLYYLART QSNAGGTAGN RELQFYQGGP VP1_isolate_rh54_AAS99252 PFHSSYAHSQ SLDRLMNPLI DQYLYYLART QSNPGGTSGN RELQFYQGGP VP1_isolate_rh60_AAS99256 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT QSTEG.TAGT QQLLFSQAGP AAV9_VP1_AAS99264 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSG..QNQ QTLKFSVAGP VP1_isolate_hu31_AAS99281 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSG..QNQ QTLKFSVAGP VP1_isolate_hu32_AAS99282 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSG..QNQ QTLKFSVAGP AAV11_AAT46339 PFHSMYAHSQ SLDRLMNPLL DQYLWHLQST TSGETLNQGN AATTFGKIRS AAV4_NP_044927 PFHSMYAHSQ SLDRLMNPLI DQYLWGLQST TTGTTLNAGT ATTNFTKLRP AAV4_VP1_AAC58045 PFHSMYAHSQ SLDRLMNPLI DQYLWGLQST TTGTTLNAGT ATTNFTKLRP BOVINE_AAV_AAR26465 PFHSMYAHSQ SLDRLMNPLL DQYLWELQST TSGGTLNQGN SATNFAKLTK BOVINE_AAV_YP_024971 PFHSMYAHSQ SLDRLMNPLL DQYLWELQST TSGGTLNQGN SATNFAKLTK AAV5_VP1_AAD13756 PFHSSFAPSQ NLFKLANPLV DQYLYRFVST NNT....... GGVQFNKNLA AAV5_YP_068409 PFHSSFAPSQ NLFKLANPLV DQYLYRFVST NNT....... GGVQFNKNLA CAPRINE_AAV1_AAU84890 PFHCSFAPSQ NLFKLANPLV DQYLYRFVST SAT....... GAIQFQKNLA GOAT_AAV_VP1_ABC69726 PFHCSFAPSQ NLFKLANPLV DQYLYRFVST SAT....... GAIQFQKNLA RAT_AAV1_AAZ79676 PFHTGFAPCQ NLFKLSNPLV DQYLYRFQGT DNSSS..STP GVVKFEKCVA MOUSE_AAV1_AAZ79672 PFHSMWAHNQ SLDRLMNPLI DQYLYYLDVT SSTG...... ..FTYQKGVH AVIAN_AAV_ATCC_VR865_AAO32087 PFHSMFAHNQ TLDRLMNPLV DQYLWAFSSV SQAGS...SG RALHYSRATK

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159 AVIAN_AAV_ATCC_VR865_AAT48613 PFHSMFAHNQ TLDRLMNPLV DQYLWAFSSV SQAGS...SG RALHYSRATK AVIAN_AAV_ATCC_VR865_NP_852781 PFHSMFAHNQ TLDRLMNPLV DQYLWAFSSV SQAGS...SG RALHYSRATK AVIAN_AAV_Strain_DA1_AAT48615 PFHSMFAHNQ TLDRLMNPLV DQYLWAFSSV SQTGS...SG RALNYSRATK AVIAN_AAV_Strain_DA1_YP_077183 PFHSMFAHNQ TLDRLMNPLV DQYLWAFSSV SQTGS...SG RALNYSRATK DUCK_AAV_Strain_FM_AAA83225 PFHSMFAHSQ DLDRLMNPLL DQYLWNFSEV NGG....... RNAQFKKAVK MUSCOVY_DUCK_PARVOVIRUS_YP_068412 PFHSMFAHSQ DLDRLMNPLL DQYLWNFSEV NGG....... RNAQFKKAVK MUSCOVY_DUCK_VP1_YP_068411 PFHSMFAHSQ DLDRLMNPLL DQYLWNFSEV NGG....... RNAQFKKAVK MUSCOVY_DUCK_PARVOVIRUS_YP_068413 PFHSMFAHSQ DLDRLMNPLL DQYLWNFSEV NGG....... RNAQFKKAVK Goose_AAV_VP1_AAA83230 PFHSMFAHSQ DLDRLMNPLV DQYLWNFNEV DSS....... RNAQFKKAVK GOOSE_AAV_VP1_NP_043515 PFHSMFAHSQ DLDRLMNPLV DQYLWNFNEV DSS....... RNAQFKKAVK AAV3_NP_043941 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT QGTTSGTTNQ SRLLFSQAGP AAV3_Strain_H_AAC55049 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT QGTTSGTTNQ SRLLFSQAGP AAV3B_VP1_AAB95452 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT QGTTSGTTNQ SRLLFSQAGP VP1_isolate_hu1_AAS99260 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNKT Q.TNSGTLQQ SRLLFSQAGP VP1_isolate_hu4_AAS99287 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNKT Q.TNSGTLQQ SRLLFSQAGP VP1_isolate_hu2_AAS99270 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNKT Q.TNSGTLQQ SRLLFSQAGP VP1_isolate_hu3_AAS99280 PFHSSYAHCQ SLDRLMNPLI DQYLYYLNKT Q.TNSGTLQQ SRLLFSQAGP VP1_isolate_hu60_AAS99307 PFHSSYAHSQ SLDRLMNPLV DQYLYYLNKT Q.TNSGTLQQ SRLLFSQAGP VP1_isolate_hu61_AAS99308 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNKT Q.TNSGTLQQ SRLLFSQAGP VP1_isolate_hu25_AAS99276 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNKT Q.TNSGTLQQ SRLLFSQAGP VP1_isolate_hu15_AAS99265 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNKT Q.SNSGTLQQ SRLLFSQAGP VP1_isolate_hu16_AAS99266 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNKT Q.SNSGTLQQ SRLLFSQAGP VP1_isolate_hu18_AAS99268 PFHSSYAHSQ SLDRLLNPLI DQYLYYLNKT Q.SNSGTLQQ SRLLFSQAGP VP1_isolate_hu7_AAS99313 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNKT Q.SNSGTLQQ SRLLFSQAGP VP1_isolate_hu10_AAS99261 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.SNSGTLQQ SRLLFSQAGP VP1_isolate_hu11_AAS99262 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.SNSGTLQQ SRLLFSQAGP VP1_isolate_hu9_AAS99314 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.SNSGTLQQ SRLLFSQAGP VP1_isolate_hu53_AAS99300 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.TASGTQQ. SRLLFSQAGP VP1_isolate_hu55_AAS99302 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNRT Q.TASGTQQ. SRLLFSQAGP VP1_isolate_hu54_AAS99301 PFHSSYAHSQ GLDRLMNPLI DQYLYYLNRT Q.TASGTQQ. SRLLFSQAGP VP1_isolate_huS17_AAU05370 PFHSSYAHSQ SLDRLMNPLI DQYLYYLNKT Q.SASGTVQQ SRLLFSQAGP AAV2_VP1_AAC03780 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu34_AAS99283 PFHSSYAHSQ SLGRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu35_AAS99284 PFHSSYAHSQ SLGRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_islolate_hu51_AAS99298 PFHSGYAHSQ SLDRLMNPLI DQYLYYLSTT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu52_AAS99299 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSTT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu47_AAS99295 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSTT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu45_AAS99293 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSTT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu58_AAS99305 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu49_AAS99297 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu56_AAS99303 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA

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160 VP1_isolate_hu57_AAS99304 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu28_AAS99278 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu29_AAS99279 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_huT70_AAU05364 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu13_AAS99263 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu63_AAS99309 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_hu64_AAS99310 PFHSSYAHSQ SLDRLMNPLV DQYLYYLSRT N.TPSGTTTQ SRLQFSQAGA VP1_isolate_huT40_AAU05362 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_huLG15_AAU05371 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_huT17_AAU05358 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_huT41_AAU05372 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_huT71_AAU05366 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_huT88_AAU05368 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_huT32_AAU05360 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_hu27_AAS99277 PFHSSYAHGQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_hu19_AAS99269 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_hu20_AAS99271 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_hu21_AAS99272 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_hu24_AAS99275 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_hu22_AAS99273 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA VP1_isolate_hu23_AAS99274 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT N.TPSGTTTM SRLQFSQAGA 501 550 AAV_VR195_ABA71699 AGMSVQPKNW LPGPCYRQQR VSKTKTGNNN S.....NFTW TGASKYNLNG AAV_VR355_ABA71701 AGMSVQPKNW LPGPCYRQQR VSKTKTDNNN S.....NFTW TGASKYNLNG AAV1_NP_049542 AGMSVQPKNW LPGPCYRQQR VSKTKTDNNN S.....NFTW TGASKYNLNG AAV1_VP1_AAD27757 AGMSVQPKNW LPGPCYRQQR VSKTKTDNNN S.....NFTW TGASKYNLNG VP1_isolate_hu48_AAS99296 AGMSVQPKNW LPGPCYRQQR VSKTKTDNNN S.....NFTW TGASKYNLNG AAV6_VP1_AAB95450 AGMSVQPKNW LPGPCYRQQR VSKTKTDNNN S.....NFTW TGASKYNLNG VP1_isolate_hu43_AAS99291 AGMSVQPKNW LPGPCYRQQR VSKTKTDNNN S.....NFTW TGASKYNLNG VP1_isolate_hu44_AAS99292 AGMSVQPKNW LPGPCYRQQR VSKTKTDNNN S.....NFTW TGASKYNLNG VP1_isolate_hu46_AAS99294 AGMSVQPKNW LPGPCYRQQR VSKTKTDNNN S.....NFTW TGASKYNLNG AAV10_AAT46337 ANMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh40_AAS99244 ANMSARAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_hu37_AAS99285 ANMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_hu42_AAS99290 ANMSAQAKNW LPGPCYRQQR VSTTLSQSNN S.....NFAW TGATKYHLNG VP1_isolate_hu40_AAS99288 ANMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_hu67_AAS99312 ANMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh38_AAS99243 ANMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_hu41_AAS99289 ANMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_hu66_AAS99311 ANMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG

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161 VP1_isolate_hu17_AAS99267 NNMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_hu6_AAS99306 NNMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh25_AAS99242 NNMSAQAKNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_hu39_AAS99286 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh49_AAS99247 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh50_AAS99248 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh51_AAS99249 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh52_AAS99250 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh64_AAS99259 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh53_AAS99251 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh61_AAS99257 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh58_AAS99255 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh57_AAS99254 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG AAV8_VP1_AAN03857 NTMANQAKNW LPGPCYRQQR VSTTTGQNNN S.....NFAW TAGTKYHLNG AAV8_YP_077180 NTMANQAKNW LPGPCYRQQR VSTTTGQNNN S.....NFAW TAGTKYHLNG VP1_isolate_rh43_AAS99245 NTMANQAKNW LPGPCYRQQR VSTTTGQNNN S.....NFAW TAGTKYHLNG VP1_isolate_pi1_AAS99238 QNMSAQAKNW LPGPCYRQQR VSTTVSQNNN S.....NFTW TGATKYHLNG VP1_isolate_pi3_AAS99240 QNMSAQAKNW LPGPCYRQQR VSTAVSQNNN S.....NFTW TGATKYHLNG VP1_isolate_pi2_AAS99239 QNMSAQAKNW LPGPCYRQQR VSTTVSQNNN S.....NFTW TGATKYHLNG VP1_isolate_rh1_AAS99241 SSMANQARNW VPGPCYRQQR VSTTTNQNNN S.....NFAW TGAAKFKLNG AAV7_VP1_AAN03855 STMAEQAKNW LPGPCFRQQR VSKTLDQNNN S.....NFAW TGATKYHLNG AAV7_YP_077178 STMAEQAKNW LPGPCFRQQR VSKTLDQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh48_AAS99246 TTMAEQAKNW LPGPCFRQQR VSKTLDQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh62_AAS99258 TTMAEQAKNW LPGPCFRQQR VSKTLDQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh55_AAS99253 TTMAEQAKNW LPGPCFRQRR VSKTLDQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh54_AAS99252 STMAEQAKNW LPGPCFRQQR VSKTLDQNNN S.....NFAW TGATKYHLNG VP1_isolate_rh60_AAS99256 SNMSAQARNW LPGPCYRQQR VSTTLSQNNN S.....NFAW TGATKYHLNG AAV9_VP1_AAS99264 SNMAVQGRNY IPGPSYRQQR VSTTVTQNNN S.....EFAW PGASSWALNG VP1_isolate_hu31_AAS99281 SNMAVQGRNY IPGPSYRQQR VSTTVTQNNN S.....EFAW PGASSWALNG VP1_isolate_hu32_AAS99282 SNMAVQGRNY IPGPSYRQQR VSTTVTQNNN S.....EFAW PGASSWALNG AAV11_AAT46339 GDFAFYRKNW LPGPCVKQQR FSKTASQNYK IPASGGNALL KYDTHYTLNN AAV4_NP_044927 TNFSNFKKNW LPGPSIKQQG FSKTANQNYK IPATGSDSLI KYETHSTLDG AAV4_VP1_AAC58045 TNFSNFKKNW LPGPSIKQQG FSKTANQNYK IPATGSDSLI KYETHSTLDG BOVINE_AAV_AAR26465 TNFSGYRKNW LPGPMMKQQR FSKTASQNYK IPQGRNNSLL HYETRTTLDG BOVINE_AAV_YP_024971 TNFSGYRKNW LPGPMMKQQR FSKTASQNYK IPQGRNNSLL HYETRTTLDG AAV5_VP1_AAD13756 GRYANTYKNW FPGPMGRTQG WNLGSG..VN RASVSAF... ATTNRMELEG AAV5_YP_068409 GRYANTYKNW FPGPMGRTQG WNLGSG..VN RASVSAF... ATTNRMELEG CAPRINE_AAV1_AAU84890 GRYANTYKNW FPGPMGRTQG WNTSSGSSTN RVSVNNF... SVSNRMNLEG GOAT_AAV_VP1_ABC69726 GRYANTYKNW FPGPMGRTQG WNTSSGSSTN RVSVNNF... SVSNRMNLEG RAT_AAV1_AAZ79676 HNYAQQYKNW LTGAFQRNQD YNYVSG.TSN YKGVVGS... NQNNLQRIEN MOUSE_AAV1_AAZ79672 TNLPEQERNW LPGPGIRNQA WFNSATGNNP LTG.....TW QYSNKYVLEN

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162 AVIAN_AAV_ATCC_VR865_AAO32087 TNMAAQYRNW LPGPFFRDQQ IFTGASNITK N...NVFSVW EKGKQWELDN AVIAN_AAV_ATCC_VR865_AAT48613 TNMAAQYRNW LPGPFFRDQQ IFTGASNITK N...NVFSVW EKGKQWELDN AVIAN_AAV_ATCC_VR865_NP_852781 TNMAAQYRNW LPGPFFRDQQ IFTGASNITK N...NVFSVW EKGKQWELDN AVIAN_AAV_Strain_DA1_AAT48615 TNMATQYRNW LPGPFVRDQQ IFTGASNITQ N...NVFNVW DKGKQWVIDN AVIAN_AAV_Strain_DA1_YP_077183 TNMATQYRNW LPGPFVRDQQ IFTGASNITQ N...NVFNVW DKGKQWVIDN DUCK_AAV_Strain_FM_AAA83225 GAFGAMGRNW LPGPKLLDQR VRAYSGGTDN Y...ANWSIW SKGNKVFLKD MUSCOVY_DUCK_PARVOVIRUS_YP_068412 GAFGAMGRNW LPGPKLLDQR VRAYSGGTDN Y...ANWSIW SKGNKVFLKD MUSCOVY_DUCK_VP1_YP_068411 GAFGAMGRNW LPGPKLLDQR VRAYSGGTDN Y...ANWSIW SKGNKVFLKD MUSCOVY_DUCK_PARVOVIRUS_YP_068413 GAFGAMGRNW LPGPKLLDQR VRAYSGGTDN Y...ANWSIW SKGNKVFLKD Goose_AAV_VP1_AAA83230 GAYGTMGRNW LPGPKFLDQR VRAYTGGTDN Y...ANWNIW SNGNKVNLKD GOOSE_AAV_VP1_NP_043515 GAYGTMGRNW LPGPKFLDQR VRAYTGGTDN Y...ANWNIW SNGNKVNLKD AAV3_NP_043941 QSMSLQARNW LPGPCYRQQR LSKTANDNNN S.....NFPW TAASKYHLNG AAV3_Strain_H_AAC55049 QSMSLQARNW LPGPCYRQQR LSKTANDNNN S.....NFPW TAASKYHLNG AAV3B_VP1_AAB95452 QSMSLQARNW LPGPCYRQQR LSKTANDNNN S.....NFPW TAASKYHLNG VP1_isolate_hu1_AAS99260 TNMSLQAKNW LPGPCYRQQR LSKQANGNNN S.....NFPW TAATKYHLNG VP1_isolate_hu4_AAS99287 TNMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu2_AAS99270 TNMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu3_AAS99280 TNMSLQAKNW LPGPCYRQQR LSKQANDNNN C.....NFPW TAATKYHLNG VP1_isolate_hu60_AAS99307 TNMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu61_AAS99308 TNMSLQAKNR LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu25_AAS99276 TNMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu15_AAS99265 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu16_AAS99266 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu18_AAS99268 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu7_AAS99313 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu10_AAS99261 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu11_AAS99262 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYRLNG VP1_isolate_hu9_AAS99314 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TAATKYHLNG VP1_isolate_hu53_AAS99300 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TGATKYYLNG VP1_isolate_hu55_AAS99302 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TGATKYHLNG VP1_isolate_hu54_AAS99301 TSMSLQAKNW LPGPCYRQQR LSKQANDNNN S.....NFPW TGATKYHLNG VP1_isolate_huS17_AAU05370 TSMSLQAKNW LPGPCYRQQR LSKQANENNN S.....NFPW TAATKYHLNG AAV2_VP1_AAC03780 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu34_AAS99283 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu35_AAS99284 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_islolate_hu51_AAS99298 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu52_AAS99299 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu47_AAS99295 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu45_AAS99293 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu58_AAS99305 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW IGATKYHLNG VP1_isolate_hu49_AAS99297 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....EYSW TGATKYHLNG

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163 VP1_isolate_hu56_AAS99303 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu57_AAS99304 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN G.....EYSW TGATKYHLNG VP1_isolate_hu28_AAS99278 SDIQDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu29_AAS99279 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_huT70_AAU05364 SDIRDQSRNW LPGPCYRQQR VSKTPADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu13_AAS99263 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu63_AAS99309 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_hu64_AAS99310 SDIRDQSRNW LPGPCYRQQR VSKTSADNNN S.....EYSW TGATKYHLNG VP1_isolate_huT40_AAU05362 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_huLG15_AAU05371 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_huT17_AAU05358 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_huT41_AAU05372 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_huT71_AAU05366 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_huT88_AAU05368 SDIRDQSRNW LPGPSYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_huT32_AAU05360 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_hu27_AAS99277 SDVRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_hu19_AAS99269 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_hu20_AAS99271 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_hu21_AAS99272 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_hu24_AAS99275 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_hu22_AAS99273 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG VP1_isolate_hu23_AAS99274 SDIRDQSRNW LPGPCYRQQR VSKTAADNNN S.....DYSW TGATKYHLNG 551 600 AAV_VR195_ABA71699 HESIINPGTA MASHKDDEDK FFPMSGVMIF GKESA...GA SNTALDNVMI AAV_VR355_ABA71701 RESIINPGTA MASHKDDEDK FFPMSGVMIF GKESA...GA SNTALDNVMI AAV1_NP_049542 RESIINPGTA MASHKDDEDK FFPMSGVMIF GKESA...GA SNTALDNVMI AAV1_VP1_AAD27757 RESIINPGTA MASHKDDEDK FFPMSGVMIF GKESA...GA SNTALDNVMI VP1_isolate_hu48_AAS99296 RESIINPGTA VASHKDDEDK FFPMSGVMIF GKESA...GA SSTALDNVMI AAV6_VP1_AAB95450 RESIINPGTA MASHKDDKDK FFPMSGVMIF GKESA...GA SNTALDNVMI VP1_isolate_hu43_AAS99291 RESIINPGTA MASHKDDEDK FFPMSGVMIF GKESA...GA SNTALDNVMI VP1_isolate_hu44_AAS99292 RESIINPGTA MASHKDDEDK FFPMSGVMIF GKESA...GA SNTALDNVMI VP1_isolate_hu46_AAS99294 RESIINPGTA MASHKDDEDK FFPMSGVMIF GKESA...GA SNTALDNVMI AAV10_AAT46337 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GR DNVDYSSVML VP1_isolate_rh40_AAS99244 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GR DNVDYSSVML VP1_isolate_hu37_AAS99285 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GR DNVDYSSVML VP1_isolate_hu42_AAS99290 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GR DNVDYSSVML VP1_isolate_hu40_AAS99288 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GR DNVDYSSVML VP1_isolate_hu67_AAS99312 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GR DNVDYSSVML VP1_isolate_rh38_AAS99243 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GR DNVDYSSVML VP1_isolate_hu41_AAS99289 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GR DNVDYSSVML

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164 VP1_isolate_hu66_AAS99311 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GR DNVDYSSVML VP1_isolate_hu17_AAS99267 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GK DNVDYSSVML VP1_isolate_hu6_AAS99306 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GK DNVDYSSVML VP1_isolate_rh25_AAS99242 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GK DNVDYSSVML VP1_isolate_hu39_AAS99286 RDSLVNPGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNVDYSNVML VP1_isolate_rh49_AAS99247 RDSLVNPGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNMGYSNVML VP1_isolate_rh50_AAS99248 RDSLVNPGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNVDYSNVML VP1_isolate_rh51_AAS99249 RDSLVNPGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNVDYSNVML VP1_isolate_rh52_AAS99250 RDSLVNPGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNVDYSNVML VP1_isolate_rh64_AAS99259 RDSLVNPGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNVDYSNVML VP1_isolate_rh53_AAS99251 RDSLVNSGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNVDYSNVML VP1_isolate_rh61_AAS99257 RDSLVNPGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNVDYSNVML VP1_isolate_rh58_AAS99255 RDSLVNPGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNVDYSNVML VP1_isolate_rh57_AAS99254 RDSLVNPGVA MATNKDDEDR FFPSSGILMF GKQGA...GK DNVDYSNVML AAV8_VP1_AAN03857 RNSLANPGIA MATHKDDEER FFPSNGILIF GKQNA...AR DNADYSDVML AAV8_YP_077180 RNSLANPGIA MATHKDDEER FFPSNGILIF GKQNA...AR DNADYSDVML VP1_isolate_rh43_AAS99245 RNSLANPGIA MATHKDDEER FFPVTGSCFW .QQNA...AR DNADYSDVML VP1_isolate_pi1_AAS99238 RDSLVSPGVA MATHKDDEER FFPSSGVLMF GKQGA...GK DNVEYTNVML VP1_isolate_pi3_AAS99240 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GK DNVEYTNVML VP1_isolate_pi2_AAS99239 RDSLVNPGVA MATHKDDEER FFPSSGVLMF GKQGA...GK DNVEYTNVML VP1_isolate_rh1_AAS99241 RDSLMNPGVA MASHKDDEDR FFPSSGVLIF GKQGA...GN DGVDYSQVLI AAV7_VP1_AAN03855 RNSLVNPGVA MATHKDDEDR FFPSSGVLIF GKTGA...TN KTT.LENVLM AAV7_YP_077178 RNSLVNPGVA MATHKDDEDR FFPSSGVLIF GKTGA...TN KTT.LENVLM VP1_isolate_rh48_AAS99246 RNSLVNPGVA MATHKDDEER FFPSSGVLIF GKTGA...AN KTT.LENVLM VP1_isolate_rh62_AAS99258 RNSLVNPGVA MATHKDDEER FFPSSGVLIF GKTGA...AN KTT.LENVLM VP1_isolate_rh55_AAS99253 RNSLVNPGVA MATHKDDEER FFPSSGVLIF GKTGA...AN KTT.LENVLM VP1_isolate_rh54_AAS99252 RNSLVNPGVA MATHKDDEDR FFPSSGVLIF GKTGA...TN KTT.LENVLM VP1_isolate_rh60_AAS99256 RDSLVNPGVA MATHKDDEER FFPSSGVLIF GKTGA...AN KTT.LENVLM AAV9_VP1_AAS99264 RNSLMNPGPA MASHKEGEDR FFPLSGSLIF GKQGT...GR DNVDADKVMI VP1_isolate_hu31_AAS99281 RNSLMNPGPA MASHKEGEDR FFPLSGSLIF GKQGT...GR DNVDADKVMI VP1_isolate_hu32_AAS99282 RNSLMNPGPA MASHKEGEDR FFPLSGSLIF GKQGT...GR DNVDADKVMI AAV11_AAT46339 RWSNIAPGPP MATAGPSDGD FS.NAQLIFP GPSVT...GN TTTSANNLLF AAV4_NP_044927 RWSALTPGPP MATAGPADSK FS.NSQLIFA GPKQN...GN TATVPGTLIF AAV4_VP1_AAC58045 RWSALTPGPP MATAGPADSK FS.NSQLIFA GPKQN...GN TATVPGTLIF BOVINE_AAV_AAR26465 RWSNFAPGTA MATAANDATD FS.QAQLIFA GPNIT...GN TTTDANNLMF BOVINE_AAV_YP_024971 RWSNFAPGTA MATAANDATD FS.QAQLIFA GPNIT...GN TTTDANNLMF AAV5_VP1_AAD13756 ASYQVPPQPN GMTNNLQGSN TYALENTMIF NSQPANPGTT ATYLEGNMLI AAV5_YP_068409 ASYQVPPQPN GMTNNLQGSN TYALENTMIF NSQPANPGTT ATYLEGNMLI CAPRINE_AAV1_AAU84890 ASYQVNPQPN GMTNTLQGSN RYALENTMIF NAQNATPGTT SVYPEDNLLL GOAT_AAV_VP1_ABC69726 ASYQVNPQPN GMTNTLQGSN RYALENTMIF NAQNATPGTT SVYPEDNLLL RAT_AAV1_AAZ79676 VQFAIAPDVP SMCNHLEGTN MIALDNSLIF RDVSTAPGDT TQYNINQVIV

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165 MOUSE_AAV1_AAZ79672 RASKIAPGPA MG....IEST KFDGNGIIFS KEYIT...NV NTANPNQVNI AVIAN_AAV_ATCC_VR865_AAO32087 RTNLMQPGPA AATTFSGEPD RQAMQNTLAF SRTVYDQ.TT ATTDRNQILI AVIAN_AAV_ATCC_VR865_AAT48613 RTNLMQPGPA AATTFSGEPD RQAMQNTLAF SRTVYDQ.TT ATTDRNQILI AVIAN_AAV_ATCC_VR865_NP_852781 RTNLMQPGPA AATTFSGEPD RQAMQNTLAF SRTVYDQ.TT ATTDRNQILI AVIAN_AAV_Strain_DA1_AAT48615 RINMMQPGPA AATTFSGEPD RQAMQNTLAF SRTVYDQ.TT STTDRNQLLI AVIAN_AAV_Strain_DA1_YP_077183 RINMMQPGPA AATTFSGEPD RQAMQNTLAF SRTVYDQ.TT STTDRNQLLI DUCK_AAV_Strain_FM_AAA83225 REYLLQPGPV ATTHTEDQAS SVPAQNIIGI AKDPYRS.GS TLAGISDIMV MUSCOVY_DUCK_PARVOVIRUS_YP_068412 REYLLQPGPV ATTHTEDQAS SVPAQNIIGI AKDPYRS.GS TLAGISDIMV MUSCOVY_DUCK_VP1_YP_068411 REYLLQPGPV ATTHTEDQAS SVPAQNIIGI AKDPYRS.GS TLAGISDIMV MUSCOVY_DUCK_PARVOVIRUS_YP_068413 REYLLQPGPV ATTHTEDQAS SVPAQNIIGI AKDPYRS.GS TLAGISDIMV Goose_AAV_VP1_AAA83230 RQYLLQPGPV SATYTEGEAS SLPAQNILGI AKDPYRS.GS TTAGISDIMV GOOSE_AAV_VP1_NP_043515 RQYLLQPGPV SATYTEGEAS SLPAQNILGI AKDPYRS.GS TTAGISDIMV AAV3_NP_043941 RDSLVNPGPA MASHKDDEEK FFPMHGNLIF GKEGT...TA SNAELDNVMI AAV3_Strain_H_AAC55049 RDSLVNPGPA MASHKDDEEK FFPMHGNLIF GKEGT...TA SNAELDNVMI AAV3B_VP1_AAB95452 RDSLVNPGPA MASHKDDEEK FFPMHGNLIF GKEGT...TA SNAELDNVMI VP1_isolate_hu1_AAS99260 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLENVMI VP1_isolate_hu4_AAS99287 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLENVMI VP1_isolate_hu2_AAS99270 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLENVMI VP1_isolate_hu3_AAS99280 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLENVMI VP1_isolate_hu60_AAS99307 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLENVMI VP1_isolate_hu61_AAS99308 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLENVMI VP1_isolate_hu25_AAS99276 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLENVMI VP1_isolate_hu15_AAS99265 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLDNVMI VP1_isolate_hu16_AAS99266 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLDNVMI VP1_isolate_hu18_AAS99268 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLDNVMI VP1_isolate_hu7_AAS99313 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLDNVMI VP1_isolate_hu10_AAS99261 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLEHVMI VP1_isolate_hu11_AAS99262 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLEHVMI VP1_isolate_hu9_AAS99314 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKQGT...NA NDADLEHVMI VP1_isolate_hu53_AAS99300 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKEGT...NA TNAELENVMI VP1_isolate_hu55_AAS99302 RDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKEGT...NA TNAELENVMI VP1_isolate_hu54_AAS99301 GDSLVNPGPA MASHKDDEEK FFPMHGTLIF GKEGT...NA TNAELENVMI VP1_isolate_huS17_AAU05370 RDSLVNPGPP MASHKDDEEK FFPMHGTLIF GKEGT...NA NNAELENVMI AAV2_VP1_AAC03780 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu34_AAS99283 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu35_AAS99284 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_islolate_hu51_AAS99298 RDSLVNPGPA MASHKDNEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu52_AAS99299 RDSLVNPGPA MASHKDNEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu47_AAS99295 RDSLVNPGPA MASHKDNEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu45_AAS99293 RDSLVNPGPA VASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu58_AAS99305 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI

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166 VP1_isolate_hu49_AAS99297 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu56_AAS99303 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu57_AAS99304 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu28_AAS99278 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu29_AAS99279 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGP...EK TNVDIEKVMI VP1_isolate_huT70_AAU05364 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu13_AAS99263 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQGS...EK TNVDIEKVMI VP1_isolate_hu63_AAS99309 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_hu64_AAS99310 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_huT40_AAU05362 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_huLG15_AAU05371 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDTEKVMI VP1_isolate_huT17_AAU05358 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_huT41_AAU05372 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_huT71_AAU05366 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_huT88_AAU05368 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_huT32_AAU05360 RDSLVNPGPA MASHKDDEEK FFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_hu27_AAS99277 RDSLVNPGPA MASHKDDEEK YFPQSGVLVF GKQDS...GK TNVDIEKVMI VP1_isolate_hu19_AAS99269 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_hu20_AAS99271 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_hu21_AAS99272 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_hu24_AAS99275 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_hu22_AAS99273 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI VP1_isolate_hu23_AAS99274 RDSLVNPGPA MASHKDDEEK YFPQSGVLIF GKQDS...GK TNVDIEKVMI 601 650 AAV_VR195_ABA71699 TDEEEIKATN PVATERFGTV AVNFQSSSTH PATGDVHVMG ALPGMVWQDR AAV_VR355_ABA71701 TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHVMG ALPGMVWQDR AAV1_NP_049542 TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG ALPGMVWQDR AAV1_VP1_AAD27757 TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG ALPGMVWQDR VP1_isolate_hu48_AAS99296 TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG ALPGMVWQDR AAV6_VP1_AAB95450 TDEEEIKATN PVATERFGTV AVNLQSSSTD PATGDVHVMG ALPGMVWQDR VP1_isolate_hu43_AAS99291 TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG ALPGMVWQDR VP1_isolate_hu44_AAS99292 TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG ALPGMVWQGR VP1_isolate_hu46_AAS99294 TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG ALPGMVWQDR AAV10_AAT46337 TSEEEIKTTN PVATEQYGVV ADNLQQANTG PIVGNVNSQG ALPGMVWQNR VP1_isolate_rh40_AAS99244 TSEEEIKTTN PVATEQYGVV ADNLQQANTG PIVGNVNSQG ALPGMVWQNR VP1_isolate_hu37_AAS99285 TSEEEIKTTN PVATEQYGVV ADNLQQTNTG PIVGNVNSQG ALPGMVWQNR VP1_isolate_hu42_AAS99290 TSEEEIKTTN PVATEQYGVV ADNLQQTNTG PIVGNVNSQG ALPGMVWQNR VP1_isolate_hu40_AAS99288 TSEEEIKTTN PVATEQYGVV ADNLQQTNTG PIVGNVNSQG ALPGMVWQNR VP1_isolate_hu67_AAS99312 TSEEEIKTTN PVATEQYGVV ADNLQQTNTG PIVGNVNSQG ALPGMVWQNR VP1_isolate_rh38_AAS99243 TSEEEIKTTN PVATEQYGVV ADNLQQTNTG PIVGNVNSQG ALPGMVWQNR

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167 VP1_isolate_hu41_AAS99289 TSEEEIKTTN PVATEQYGVV ADNLQQTNTG PIVGNVNSQG ALPGMVWQNR VP1_isolate_hu66_AAS99311 TSEEEIKTTN PVATEQYGVV ADNLQQTNTG PIVGNVNSQG ALPGMVWQNR VP1_isolate_hu17_AAS99267 TSEEEIKTTN PVATEQYGVV ADNLQQQNAA PIVGAVNSQG ALPGMVWQNR VP1_isolate_hu6_AAS99306 TSEEEIKTTN PVATEQYGVV ADNLQQQNAA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh25_AAS99242 TSEEEIKTTN PVATEQYGVV ADNLQQQNAA PIVGAVNSQG ALPGMVWQNR VP1_isolate_hu39_AAS99286 TSEEEIKTTN PVATEQYGVV ADNLQQQNTA PTVGAVNSQG ALPGMVWQNR VP1_isolate_rh49_AAS99247 TSEEEIKTTN PVATEQYGVV ADNLQQQNTA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh50_AAS99248 TSEEEIKTTN PVATEQYGVV ADNLQQQNTA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh51_AAS99249 TSEEEIKTTN PVATEQYGVV ADNLQQQNTA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh52_AAS99250 TSEEEIKTTN PVATEQYGVV ADNLQQQNTA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh64_AAS99259 TSEEEIKTTN PVATEQYGVV ADNLQQQNTA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh53_AAS99251 TSEEEIKTTN PVATEQYGVV ADNLQQQNTA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh61_AAS99257 TSEEEIKTTN PVATEQYGVV ADNLQQQDTA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh58_AAS99255 TSEEEIKTTN PVATEQYGVV ADNLQQQNTA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh57_AAS99254 TSEEEIKTTN PVATEQYGVV ADNLQQQNTA PIVGAVNSQG ALPGMVWQNR AAV8_VP1_AAN03857 TSEEEIKTTN PVATEEYGIV ADNLQQQNTA PQIGTVNSQG ALPGMVWQNR AAV8_YP_077180 TSEEEIKTTN PVATEEYGIV ADNLQQQNTA PQIGTVNSQG ALPGMVWQNR VP1_isolate_rh43_AAS99245 TSEEEIKTTN PVATEEYGIV ADNLQQQNTA PQIGTVNSQG ALPGMVWQNR VP1_isolate_pi1_AAS99238 TSEEEIKTTN PVATEQYGVV ADNLQQTNSA PIVGAVNSQG ALPGMVWQNR VP1_isolate_pi3_AAS99240 TSEEEIKTTN PVATEQYGVV ADNLQQTNSA PIVGAVNSQG ALPGMVWQNR VP1_isolate_pi2_AAS99239 TSEEEIKTTN PVATEQYGVV ADNLQQTNSA PIVGAVNSQG ALPGMVWQNR VP1_isolate_rh1_AAS99241 TDEEEIKATN PVATEEYGAV AINNQAANTQ AQTGLVHNQG VIPGMVWQNR AAV7_VP1_AAN03855 TNEEEIRPTN PVATEEYGIV SSNLQAANTA AQTQVVNNQG ALPGMVWQNR AAV7_YP_077178 TNEEEIRPTN PVATEEYGIV SSNLQAANTA AQTQVVNNQG ALPGMVWQNR VP1_isolate_rh48_AAS99246 TNEEEIRPTN PVATEEYGTV SSNLQAANTA AQTQVVNNQG ALPGMVWQNR VP1_isolate_rh62_AAS99258 TNEEEIRPTN PVATEEYGTV SSNLQAANTA AQTQVVNNQG ALPGMVWQNR VP1_isolate_rh55_AAS99253 TNEEEIRPTN PVATEEYGTV SSNLQAANTA AQTQVVNNQG ALPGMVWQNR VP1_isolate_rh54_AAS99252 TNEEEIRPTN PVATEEYGIV SSNLQAANTA AQTQVVNNQG ALPGMVWQNR VP1_isolate_rh60_AAS99256 TNEEEIRPTN PVATEEYGTV SSNLQAANTA AQTQVVNNQG ALPGMVWQNR AAV9_VP1_AAS99264 TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG ILPGMVWQDR VP1_isolate_hu31_AAS99281 TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG ILPGMVWQDR VP1_isolate_hu32_AAS99282 TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG ILPGMVWQDR AAV11_AAT46339 TSEEEIAATN PRDTDMFGQI ADNNQNATTA PITGNVTAMG VLPGMVWQNR AAV4_NP_044927 TSEEELAATN ATDTDMWGNL PGGDQSNSNL PTVDRLTALG AVPGMVWQNR AAV4_VP1_AAC58045 TSEEELAATN ATDTDMWGNL PGGDQSNSNL PTVDRLTALG AVPGMVWQNR BOVINE_AAV_AAR26465 TSEDELRATN PRDTDLFGHL ATNQQNATTV PTVDDVDGVG VYPGMVWQDR BOVINE_AAV_YP_024971 TSEDELRATN PRDTDLFGHL ATNQQNATTV PTVDDVDGVG VYPGMVWQDR AAV5_VP1_AAD13756 TSESETQPVN RVAYNVGGQM ATNNQSSTTA PATGTYNLQE IVPGSVWMER AAV5_YP_068409 TSESETQPVN RVAYNVGGQM ATNNQSSTTA PATGTYNLQE IVPGSVWMER CAPRINE_AAV1_AAU84890 TSESETQPVN RVAYNTGGQM ATNAQNATTA PTVGTYNLQE VLPGSVWMER GOAT_AAV_VP1_ABC69726 TSESETQPVN RVAYNTGGQM ATNAQNATTA PTVGTYNLQE VLPGSVWMER

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168 RAT_AAV1_AAZ79676 TSEAETQSVN AYSGDTCGRI VNNSQNSGTN AGTTGINYKG TMPSSVWMDR MOUSE_AAV1_AAZ79672 TRETEINSTN PLAGGSLGAH ANNSQNTTTA PTLDHTNVMG VFPGSVWQDR AVIAN_AAV_ATCC_VR865_AAO32087 TNEDEIRPTN SVGIDAWGAV PTNNQSIVTP GTRAAVNNQG ALPGMVWQNR AVIAN_AAV_ATCC_VR865_AAT48613 TNEDEIRPTN SVGIDAWGAV PTNNQSIVTP GTRAAVNNQG ALPGMVWQNR AVIAN_AAV_ATCC_VR865_NP_852781 TNEDEIRPTN SVGIDAWGAV PTNNQSIVTP GTRAAVNNQG ALPGMVWQNR AVIAN_AAV_Strain_DA1_AAT48615 TNEDEIRPTN SVGIDTWGVV PNNNQSKVTA GTRAAINNQG ALPGMVWQNR AVIAN_AAV_Strain_DA1_YP_077183 TNEDEIRPTN SVGIDTWGVV PNNNQSKVTA GTRAAINNQG ALPGMVWQNR DUCK_AAV_Strain_FM_AAA83225 TDEQEIAPTN GVGWRPYGLT VTNEQNTTTA PTNAELEVLG ALPGMVWQNR MUSCOVY_DUCK_PARVOVIRUS_YP_068412 TDEQEIAPTN GVGWRPYGLT VTNEQNTTTA PTNAELEVLG ALPGMVWQNR MUSCOVY_DUCK_VP1_YP_068411 TDEQEIAPTN GVGWRPYGLT VTNEQNTTTA PTNAELEVLG ALPGMVWQNR MUSCOVY_DUCK_PARVOVIRUS_YP_068413 TDEQEIAPTN GVGWRPYGLT VTNEQNTTTA PTNAELEVLG ALPGMVWQNR Goose_AAV_VP1_AAA83230 TEEQEVAPTN GVGWKPYGRT VTNEQNTTTA PTSSDLDVLG ALPGMVWQNR GOOSE_AAV_VP1_NP_043515 TEEQEVAPTN GVGWKPYGRT VTNEQNTTTA PTSSDLDVLG ALPGMVWQNR AAV3_NP_043941 TDEEEIRTTN PVATEQYGTV ANNLQSSNTA PTTGTVNHQG ALPGMVWQDR AAV3_Strain_H_AAC55049 TDEEEIRTTN PVATEQYGTV ANNLQSSNTA PTTGTVNHQG ALPGMVWQDR AAV3B_VP1_AAB95452 TDEEEIRTTN PVATEQYGTV ANNLQSSNTA PTTRTVNDQG ALPGMVWQDR VP1_isolate_hu1_AAS99260 TDEEEIRATN PVATEQYGTV SNNLQNSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu4_AAS99287 TDEEEIRATN PVATEQYGTV SNNLQNSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu2_AAS99270 TDEEEIRATN PVATEQYGTV SNNLQNSNTG PTTGTVNRQG ALPGMVWQDR VP1_isolate_hu3_AAS99280 TDEEEIRPTN PVATEQYGTV SNNLQNSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu60_AAS99307 TDEEEIRTTN PVATEQYGTV SNNLQNSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu61_AAS99308 TDEEEIRTTN PVATEQYGTV SNNLQNSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu25_AAS99276 TDEEEIRTTN PVATEQYGTV SNNLQNSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu15_AAS99265 TDEEEIRTTN PVATEQYGYV SNNLQNSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu16_AAS99266 TDEEEIRTTN PVATEQYGYV SNNLQDSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu18_AAS99268 TDEEEIRTTN PVATEQYGYV SNNLQNSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu7_AAS99313 TDEEEIRTTN PVATEQYGYV SNNLQNSNTG PTTGTVNHQG ALPGMVWQDR VP1_isolate_hu10_AAS99261 TDEEEIRTTN PVATEQYGNV SNNLQNSNTG PTTENVNHQG ALPGMVWQDR VP1_isolate_hu11_AAS99262 TDEEEIRTTN PVATEQYGNV SNNLQNSNTG PTTENVNHQG ALPGMVWQDR VP1_isolate_hu9_AAS99314 TDEEEIRTTN PVATEQYGNV SNNLQNSNTG PTTENVNHQG ALPGMVWQDR VP1_isolate_hu53_AAS99300 TDEEEIRTTN PVATEQYGYV SNNLQNSNTA ASTETVNHQG ALPGMVWQDR VP1_isolate_hu55_AAS99302 TDEEEIRTTN PVATEQYGYV SNNLQNSNTA ASTETVNHQG ALPGMVWQDR VP1_isolate_hu54_AAS99301 TDEEEIRTTN PVATEQYGYV SNNLQNSNTA ASTETVNHQG ALPGMVWQDR VP1_isolate_huS17_AAU05370 TDEEEIRTTN PVATEQYGNV SNNLQNSNTA PSTGTVNHQG ALPGMVWQDR AAV2_VP1_AAC03780 TDEEEIRTTN PVATEQYGSV STNLQRGNRQ AATADVNTQG VLPGMVWQDR VP1_isolate_hu34_AAS99283 TDEEEIRTTN PVATEQYGSV STNLQRGNRQ AATADVNTQG VLPGMVWQDR VP1_isolate_hu35_AAS99284 TDEEEIRTTN PVATEQYGSV STNLQRGNRQ AATADVNTQG VLPGMVWQDR VP1_islolate_hu51_AAS99298 TDEEEIRTTN PVATEQYGSV STNLQRGNRQ AATADVNTQG VLPGMVWQDR VP1_isolate_hu52_AAS99299 TDEEEIRTTN PVATEQYGSV STNLQRGNRQ AATADVNTQG VLPGMVWQDR VP1_isolate_hu47_AAS99295 TDEEEIRTTN PVATEQYGSV STNLQRGNRQ AATADVNTQG VLPGMVWQDR VP1_isolate_hu45_AAS99293 TDEEEIRTTN PVATEQYGSV STNLQRGNRQ AATADVNTQG VLPGMVWQDR

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169 VP1_isolate_hu58_AAS99305 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ ASTADVNTQG VLPGMVWQDR VP1_isolate_hu49_AAS99297 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATADVNTQG VLPGMVWQDR VP1_isolate_hu56_AAS99303 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu57_AAS99304 TDEEEIRTTN PVATEQYGSV STNLQSGNTR AATSDVNTQG VLPGMVWQDR VP1_isolate_hu28_AAS99278 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATADVNTQG VLPGMVGQDR VP1_isolate_hu29_AAS99279 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATADVNTQG VLPGMVWQDR VP1_isolate_huT70_AAU05364 TDEEEIRTTN PVATEQYGTV STNLQSSNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu13_AAS99263 TDEEEIRTTN PVATEQYGSV STNLQGGNTQ AATADVNTQG VLPGMVWQDR VP1_isolate_hu63_AAS99309 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu64_AAS99310 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_huT40_AAU05362 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_huLG15_AAU05371 TDEEEIRTTN PVATEQYGAV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_huT17_AAU05358 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_huT41_AAU05372 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATADVNTQG VLPGMVWQDR VP1_isolate_huT71_AAU05366 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATADVNTQG VLPGMVWQDR VP1_isolate_huT88_AAU05368 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATADVNTQG VLPGMVWQDR VP1_isolate_huT32_AAU05360 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu27_AAS99277 TDEEEIRTTN PAATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu19_AAS99269 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu20_AAS99271 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu21_AAS99272 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu24_AAS99275 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu22_AAS99273 TDEEEIRTTN PVATEQYGSV STNLQSGNTQ AATSDVNTQG VLPGMVWQDR VP1_isolate_hu23_AAS99274 TDEEEIRTTN PVATEQYGSV STYLQSGNTQ AATSDVNTQG VLPGMVWQDR 651 700 AAV_VR195_ABA71699 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPPA AAV_VR355_ABA71701 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPPA AAV1_NP_049542 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPANPPA AAV1_VP1_AAD27757 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPANPPA VP1_isolate_hu48_AAS99296 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPANPPA AAV6_VP1_AAB95450 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPPA VP1_isolate_hu43_AAS99291 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPANPPA VP1_isolate_hu44_AAS99292 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPANPPA VP1_isolate_hu46_AAS99294 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPANPPA AAV10_AAT46337 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh40_AAS99244 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_hu37_AAS99285 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_hu42_AAS99290 DVYLQGPIWA KIPHTDGNFH PSPLMGGLGL KHPPPQILIK NTPVPADPPT VP1_isolate_hu40_AAS99288 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_hu67_AAS99312 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT

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170 VP1_isolate_rh38_AAS99243 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_hu41_AAS99289 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_hu66_AAS99311 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_hu17_AAS99267 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_hu6_AAS99306 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh25_AAS99242 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_hu39_AAS99286 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh49_AAS99247 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh50_AAS99248 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh51_AAS99249 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh52_AAS99250 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh64_AAS99259 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh53_AAS99251 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh61_AAS99257 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQVLIK NTPVPADPPT VP1_isolate_rh58_AAS99255 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK STPVPADPPT VP1_isolate_rh57_AAS99254 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT AAV8_VP1_AAN03857 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT AAV8_YP_077180 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_rh43_AAS99245 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT VP1_isolate_pi1_AAS99238 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPV VP1_isolate_pi3_AAS99240 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPV VP1_isolate_pi2_AAS99239 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPV VP1_isolate_rh1_AAS99241 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPL AAV7_VP1_AAN03855 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE AAV7_YP_077178 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE VP1_isolate_rh48_AAS99246 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE VP1_isolate_rh62_AAS99258 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE VP1_isolate_rh55_AAS99253 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE VP1_isolate_rh54_AAS99252 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPANPPE VP1_isolate_rh60_AAS99256 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHP..QILIK NTPVPANPPE AAV9_VP1_AAS99264 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT VP1_isolate_hu31_AAS99281 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT VP1_isolate_hu32_AAS99282 DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT AAV11_AAT46339 DIYYQGPIWA KIPHADGHFH PSPLIGGFGL KHPPPQIFIK NTPVPANPAT AAV4_NP_044927 DIYYQGPIWA KIPHTDGHFH PSPLIGGFGL KHPPPQIFIK NTPVPANPAT AAV4_VP1_AAC58045 DIYYQGPIWA KIPHTDGHFH PSPLIGGFGL KHPPPQIFIK NTPVPANPAT BOVINE_AAV_AAR26465 DIYYQGPIWA KIPHTDGHFH PSPLIGGFGL KSPPPQIFIK NTPVPANPAT BOVINE_AAV_YP_024971 DIYYQGPIWA KIPHTDGHFH PSPLIGGFGL KSPPPQIFIK NTPVPANPAT AAV5_VP1_AAD13756 DVYLQGPIWA KIPETGAHFH PSPAMGGFGL KHPPPMMLIK NTPVPGN.IT AAV5_YP_068409 DVYLQGPIWA KIPETGAHFH PSPAMGGFGL KHPPPMMLIK NTPVPGN.IT CAPRINE_AAV1_AAU84890 DVYLQGPIWA KIPETGAHFH PSPAMGGFGL KHPPPMMLIK NTPVPGN.IT

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171 GOAT_AAV_VP1_ABC69726 DVYLQGPIWA KIPETGAHFH PSPAMGGFGL KHPPPMMLIK NTPVPGN.IT RAT_AAV1_AAZ79676 DVYLQGPIWA KIPHTGAHFH PSPMMGGFGL RNPPPMMLIK NTPVPGN.VT MOUSE_AAV1_AAZ79672 DIYLQGQIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPADPPT AVIAN_AAV_ATCC_VR865_AAO32087 DIYPTGTHLA KIPDTDNHFH PSPLIGRFGC KHPPPQIFIK NTPVPANPSE AVIAN_AAV_ATCC_VR865_AAT48613 DIYPTGTHLA KIPDTDNHFH PSPLIGRFGC KHPPPQIFIK NTPVPANPSE AVIAN_AAV_ATCC_VR865_NP_852781 DIYPTGTHLA KIPDTDNHFH PSPLIGRFGC KHPPPQIFIK NTPVPANPSE AVIAN_AAV_Strain_DA1_AAT48615 DIYLQGPIWA KIPDTDNHFH PSPLIGGFGC KHPPPQIFIK NTPVPANPSE AVIAN_AAV_Strain_DA1_YP_077183 DIYLQGPIWA KIPDTDNHFH PSPLIGGFGC KHPPPQIFIK NTPVPANPSE DUCK_AAV_Strain_FM_AAA83225 DIYLQGPIWA KIPKTDGKPH PSPNLGGFGL HNPPPQVFIK NTPVPADPPL MUSCOVY_DUCK_PARVOVIRUS_YP_068412 DIYLQGPIWA KIPKTDGKPH PSPNLGGFGL HNPPPQVFIK NTPVPADPPL MUSCOVY_DUCK_VP1_YP_068411 DIYLQGPIWA KIPKTDGKPH PSPNLGGFGL HNPPPQVFIK NTPVPADPPL MUSCOVY_DUCK_PARVOVIRUS_YP_068413 DIYLQGPIWA KIPKTDGKPH PSPNLGGFGL HNPPPQVFIK NTPVPADPPL Goose_AAV_VP1_AAA83230 DIYLQGPIGA KIPKTDGKFH PSPNLGGFGL HNPPPQVFIK NTPVPADPPV GOOSE_AAV_VP1_NP_043515 DIYLQGPIGA KIPKTDGKFH PSPNLGGFGL HNPPPQVFIK NTPVPADPPV AAV3_NP_043941 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT AAV3_Strain_H_AAC55049 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT AAV3B_VP1_AAB95452 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu1_AAS99260 DVYLQGPIWA KIPHTDGHFH PSPLTGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu4_AAS99287 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu2_AAS99270 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu3_AAS99280 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK STPVPANPPT VP1_isolate_hu60_AAS99307 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu61_AAS99308 DVYLQGPIWA KIPHTDGHFH PSPLVGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu25_AAS99276 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu15_AAS99265 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu16_AAS99266 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu18_AAS99268 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu7_AAS99313 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu10_AAS99261 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu11_AAS99262 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu9_AAS99314 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu53_AAS99300 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu55_AAS99302 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_hu54_AAS99301 DVYLRGPIWA KIPHADGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT VP1_isolate_huS17_AAU05370 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT AAV2_VP1_AAC03780 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu34_AAS99283 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu35_AAS99284 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_islolate_hu51_AAS99298 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu52_AAS99299 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGP KHPPPQILIK NTPVPANPST VP1_isolate_hu47_AAS99295 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST

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172 VP1_isolate_hu45_AAS99293 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu58_AAS99305 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu49_AAS99297 DVHLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu56_AAS99303 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu57_AAS99304 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu28_AAS99278 DVYLQGPTWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu29_AAS99279 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_huT70_AAU05364 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu13_AAS99263 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu63_AAS99309 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu64_AAS99310 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_huT40_AAU05362 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_huLG15_AAU05371 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_huT17_AAU05358 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_huT41_AAU05372 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_huT71_AAU05366 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_huT88_AAU05368 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_huT32_AAU05360 DVYLQGPIWA KIPHTDGHFY PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu27_AAS99277 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu19_AAS99269 DVYLQGPIWA KIPHTDGHFH PSPLVGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu20_AAS99271 DVYLQGPIWA KIPHTDGHFH PSPPMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu21_AAS99272 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu24_AAS99275 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu22_AAS99273 DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST VP1_isolate_hu23_AAS99274 DVYLRGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPST 701 750 AAV_VR195_ABA71699 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN AAV_VR355_ABA71701 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN AAV1_NP_049542 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN AAV1_VP1_AAD27757 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VP1_isolate_hu48_AAS99296 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN AAV6_VP1_AAB95450 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VP1_isolate_hu43_AAS99291 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAS VP1_isolate_hu44_AAS99292 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VP1_isolate_hu46_AAS99294 EFSATKFASF ITQYSAGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN AAV10_AAT46337 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_rh40_AAS99244 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_hu37_AAS99285 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_hu42_AAS99290 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_hu40_AAS99288 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN

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173 VP1_isolate_hu67_AAS99312 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_rh38_AAS99243 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_hu41_AAS99289 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_hu66_AAS99311 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_hu17_AAS99267 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu6_AAS99306 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_rh25_AAS99242 TFSQAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_hu39_AAS99286 AFNQAKLNSF IAQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_rh49_AAS99247 AFNQAKLNSF ITQYGTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_rh50_AAS99248 AFNQAKLNSF ITQYSTGQVS VEIEWELQKE NSKRWSPEIQ YTSNYYKSTN VP1_isolate_rh51_AAS99249 AFNQAKLNSF ITQYSTGQVS VEIEWEPQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_rh52_AAS99250 AFNQAKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_rh64_AAS99259 AFNQAKLNSF ITQYSTGQVS VEIVWELQKE NSKRRNPEIQ YTSNYYKSTN VP1_isolate_rh53_AAS99251 AFNQAKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_rh61_AAS99257 AFNQAKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VP1_isolate_rh58_AAS99255 AFNQAKLNSF ITQYSTGQVS VEIEWELQKE NSKCWNPEIQ YTSNYYKSTN VP1_isolate_rh57_AAS99254 AFNQAKLNSF ITQYSTGQVS AEIEWELQKE NSKRWNPEIQ YTSNYYKSTN AAV8_VP1_AAN03857 TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTS AAV8_YP_077180 TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTS VP1_isolate_rh43_AAS99245 TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTS VP1_isolate_pi1_AAS99238 NFTDAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSAN VP1_isolate_pi3_AAS99240 NFTDAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSAN VP1_isolate_pi2_AAS99239 NFTDAKLASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSAN VP1_isolate_rh1_AAS99241 TFNQAKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN AAV7_VP1_AAN03855 VFTPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNFEKQTG AAV7_YP_077178 VFTPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNFEKQTG VP1_isolate_rh48_AAS99246 VFTPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNFDKQTG VP1_isolate_rh62_AAS99258 VFTPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNFDKQTG VP1_isolate_rh55_AAS99253 VFTPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNFDKQTG VP1_isolate_rh54_AAS99252 VFTPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNFDKQTG VP1_isolate_rh60_AAS99256 VFTPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNFDKQTG AAV9_VP1_AAS99264 AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VP1_isolate_hu31_AAS99281 AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VP1_isolate_hu32_AAS99282 AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN AAV11_AAT46339 TFTAARVDSF ITQYSTGQVA VQIEWEIEKE RSKRWNPEVQ FTSNYGNQSS AAV4_NP_044927 TFSSTPVNSF ITQYSTGQVS VQIDWEIQKE RSKRWNPEVQ FTSNYGQQNS AAV4_VP1_AAC58045 TFSSTPVNSF ITQYSTGQVS VQIDWEIQKE RSKRWNPEVQ FTSNYGQQNS BOVINE_AAV_AAR26465 TFSPARINSF ITQYSTGQVA VKIEWEIQKE RSKRWNPEVQ FTSNYGAQDS BOVINE_AAV_YP_024971 TFSPARINSF ITQYSTGQVA VKIEWEIQKE RSKRWNPEVQ FTSNYGAQDS AAV5_VP1_AAD13756 SFSDVPVSSF ITQYSTGQVT VEMEWELKKE NSKRWNPEIQ YTNNYNDPQF AAV5_YP_068409 SFSDVPVSSF ITQYSTGQVT VEMEWELKKE NSKRWNPEIQ YTNNYNDPQF

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174 CAPRINE_AAV1_AAU84890 SFSDVPVSSF ITQYSTGQVT VEMEWELKKE NSKRWNPEIQ YTNNYNDPQF GOAT_AAV_VP1_ABC69726 SFSDVPVSSF ITQYSTGQVT VEMEWELKKE NSKRWNPEIQ YTNNYNDPQF RAT_AAV1_AAZ79676 TFTEVKVNQF ITQYSTGQIT VDVEWELQKE NSKRWNPEIQ YTNNYSNNTF MOUSE_AAV1_AAZ79672 EFNANKISSF ITQYSTGQVT VEMEWELQKE TSKRWNPEIQ YSDDSSSTSG AVIAN_AAV_ATCC_VR865_AAO32087 TFQTAKVASF INQYSTGQCT VEIFWELKKE TSKRWNPEIQ FTSNFGNAAD AVIAN_AAV_ATCC_VR865_AAT48613 TFQTAKVASF INQYSTGQCT VEIFWELKKE TSKRWNPEIQ FTSNFGNAAD AVIAN_AAV_ATCC_VR865_NP_852781 TFQTAKVASF INQYSTGQCT VEIFWELKKE TSKRWNPEIQ FTSNFGNAAD AVIAN_AAV_Strain_DA1_AAT48615 TFQTAKVASF INQYSTGQCT VEIFWELKKE TSKRWNPEIQ FTSNFGNAAD AVIAN_AAV_Strain_DA1_YP_077183 TFQTAKVASF INQYSTGQCT VEIFWELKKE TSKRWNPEIQ FTSNFGNAAD DUCK_AAV_Strain_FM_AAA83225 EYVNQKWNSY ITQYSTGQCT VEMVWELRKE NSKRWNPEIQ FTSNFGNRTS MUSCOVY_DUCK_PARVOVIRUS_YP_068412 EYVNQKWNSY ITQYSTGQCT VEMVWELRKE NSKRWNPEIQ FTSNFGNRTS MUSCOVY_DUCK_VP1_YP_068411 EYVNQKWNSY ITQYSTGQCT VEMVWELRKE NSKRWNPEIQ FTSNFGNRTS MUSCOVY_DUCK_PARVOVIRUS_YP_068413 EYVNQKWNSY ITQYSTGQCT VEMVWELRKE NSKRWNPEIQ FTSNFGNRTS Goose_AAV_VP1_AAA83230 EYVHQKWNSY ITQYSTGQCT VEMVWELRKE NSKRWNPEIQ FTSNFSNRTS GOOSE_AAV_VP1_NP_043515 EYVHQKWNSY ITQYSTGQCT VEMVWELRKE NSKRWNPEIQ FTSNFSNRTS AAV3_NP_043941 TFSPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN AAV3_Strain_H_AAC55049 TFSPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN AAV3B_VP1_AAB95452 TFSPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu1_AAS99260 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu4_AAS99287 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu2_AAS99270 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu3_AAS99280 NFSSAKFASS ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu60_AAS99307 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu61_AAS99308 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu25_AAS99276 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu15_AAS99265 NFSSAKFASF ITQYSTGQVS VEIEWELQKE DSKRWNPEIQ YTSNYNKPVN VP1_isolate_hu16_AAS99266 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu18_AAS99268 NFSSSKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu7_AAS99313 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu10_AAS99261 NYSSAKFASF ITQYSTGQVS VEIEWELRKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu11_AAS99262 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu9_AAS99314 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu53_AAS99300 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu55_AAS99302 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu54_AAS99301 NFSSAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_huS17_AAU05370 NFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN AAV2_VP1_AAC03780 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu34_AAS99283 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu35_AAS99284 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_islolate_hu51_AAS99298 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu52_AAS99299 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN

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175 VP1_isolate_hu47_AAS99295 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu45_AAS99293 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu58_AAS99305 TFSAAKFASF ITQYSTGRVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu49_AAS99297 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu56_AAS99303 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu57_AAS99304 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu28_AAS99278 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu29_AAS99279 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_huT70_AAU05364 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu13_AAS99263 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu63_AAS99309 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu64_AAS99310 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_huT40_AAU05362 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_huLG15_AAU05371 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_huT17_AAU05358 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_huT41_AAU05372 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_huT71_AAU05366 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_huT88_AAU05368 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_huT32_AAU05360 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu27_AAS99277 TFSAAKFVSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu19_AAS99269 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu20_AAS99271 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu21_AAS99272 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu24_AAS99275 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu22_AAS99273 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VP1_isolate_hu23_AAS99274 TFSAAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN 751 778 AAV_VR195_ABA71699 ..VDFTVDNN GLYTEPRPIG TRYLTRPL AAV_VR355_ABA71701 ..VDFTVDNN GLYTEPRPIG TRYLTRPL AAV1_NP_049542 ..VDFTVDNN GLYTEPRPIG TRYLTRPL AAV1_VP1_AAD27757 ..VDFTVDNN GLYTEPRPIG TRYLTRPL VP1_isolate_hu48_AAS99296 ..VDFTVDNN GLYTEPRPIG TRYLTRPL AAV6_VP1_AAB95450 ..VDFTVDNN GLYTEPRPIG TRYLTRPL VP1_isolate_hu43_AAS99291 ..VDFTVDNN GLYTEPRPIG TRYLTRPL VP1_isolate_hu44_AAS99292 ..VDFTVDNN GLYTEPRPIG TRYLTRPL VP1_isolate_hu46_AAS99294 ..VDFTVDNN GLYTEPRPIG TRYLTRPL AAV10_AAT46337 ..VDFAVNTE GTYSEPRPIG TRYLTRNL VP1_isolate_rh40_AAS99244 ..VDFAVNSE GTYSEPRPIG TRYLTRNL VP1_isolate_hu37_AAS99285 ..VDFAVNTE GTYSEPRPIG TRYLTRNL VP1_isolate_hu42_AAS99290 ..VDFAVNTE GTYSEPRPIG TRYLTRNL

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176 VP1_isolate_hu40_AAS99288 ..VDFAVNTE GTYSEPRPIG TRYLTRNL VP1_isolate_hu67_AAS99312 ..VDFAVNTE GTYSEPRPIG TRYLTRNL VP1_isolate_rh38_AAS99243 ..VDFAVNTE GTYSEPRPIG TRYLTRNL VP1_isolate_hu41_AAS99289 ..VDFAVNTE GTYSEPRPIG TRYLTRNL VP1_isolate_hu66_AAS99311 ..VDFAVNTE GTYSEPRPIG TRYLTRNL VP1_isolate_hu17_AAS99267 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu6_AAS99306 ..VDFAVNTE GTYSEPRPIG TRYLTRNL VP1_isolate_rh25_AAS99242 ..VDFAVNTE GTYSEPRPIG TRYLTRNL VP1_isolate_hu39_AAS99286 ..ADFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_rh49_AAS99247 ..VDFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_rh50_AAS99248 ..VDFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_rh51_AAS99249 ..VDFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_rh52_AAS99250 ..VDFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_rh64_AAS99259 ..VDFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_rh53_AAS99251 ..VDFAVNTE GVYSEPRPIG TRYPTRNL VP1_isolate_rh61_AAS99257 ..VDFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_rh58_AAS99255 ..VDFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_rh57_AAS99254 ..VDFAVNTE GVYSEPRPIG TRYLTRNL AAV8_VP1_AAN03857 ..VDFAVNTE GVYSEPRPIG TRYLTRNL AAV8_YP_077180 ..VDFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_rh43_AAS99245 ..VDFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_pi1_AAS99238 ..VDFAVNAD GVYSEPRPIG TRYLTRNL VP1_isolate_pi3_AAS99240 ..VDFAVNAD GVYSEPRPIG TRYLTRNL VP1_isolate_pi2_AAS99239 ..VDFAVNAD GVYSEPRPIG TRYLTRNL VP1_isolate_rh1_AAS99241 ..VDFAVNTE GVYSEPRPIG TRYLTRNL AAV7_VP1_AAN03855 ..VDFAVDSQ GVYSEPRPIG TRYLTRNL AAV7_YP_077178 ..VDFAVDSQ GVYSEPRPIG TRYLTRNL VP1_isolate_rh48_AAS99246 ..VDFAVDSQ GVYSEPRPIG TRYLTRNL VP1_isolate_rh62_AAS99258 ..VDFAVDSQ GVYSEPRPIG TRYLTRNL VP1_isolate_rh55_AAS99253 ..VDFAVDSQ GVYSEPRPIG TRYLTRNL VP1_isolate_rh54_AAS99252 ..VDFAVDSQ GVYSEPRPIG TRYLTRNL VP1_isolate_rh60_AAS99256 ..VDFAVDSQ GVYSEPRPIG TRYLTRNL AAV9_VP1_AAS99264 ..VEFAVNTE GVYSEPRPIG TRYLTRNL VP1_isolate_hu31_AAS99281 ..VEFAVSTE GVYSEPRPIG TRYLTRNL VP1_isolate_hu32_AAS99282 ..VEFAVNTE GVYSEPRPIG TRYLTRNL AAV11_AAT46339 ..MLWAPDTT GKYTEPRVIG SRYLTNHL AAV4_NP_044927 ..LLWAPDAA GKYTEPRAIG TRYLTHHL AAV4_VP1_AAC58045 ..LLWAPDAA GKYTEPRAIG TRYLTHHL BOVINE_AAV_AAR26465 ..LLWAPDNA GAYKEPRAIG SRYLTNHL BOVINE_AAV_YP_024971 ..LLWAPDNA GAYKEPRAIG SRYLTNHL AAV5_VP1_AAD13756 ..VDFAPDST GEYRTTRPIG TRYLTRPL

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177 AAV5_YP_068409 ..VDFAPDST GEYRTTRPIG TRYLTRPL CAPRINE_AAV1_AAU84890 ..VDFAPDGS GEYRTTRAIG TRYLTRPL GOAT_AAV_VP1_ABC69726 ..VDFAPDGS GEYRTTRAIG TRYLTRPL RAT_AAV1_AAZ79676 ..VDFAPNAN GDYQMTRPIG TRYPTRPP MOUSE_AAV1_AAZ79672 SILHFAPDDV GNYKEFRSIG TRYLTRPL AVIAN_AAV_ATCC_VR865_AAO32087 ..IQFAVSDT GSYSEPRPIG TRYLTKPL AVIAN_AAV_ATCC_VR865_AAT48613 ..IQFAVSDT GSYSEPRPIG TRYLTKPL AVIAN_AAV_ATCC_VR865_NP_852781 ..IQFAVSDT GSYSEPRPIG TRYLTKPL AVIAN_AAV_Strain_DA1_AAT48615 ..IQFAVSDT GSYSEPRPIG TRYLTKPL AVIAN_AAV_Strain_DA1_YP_077183 ..IQFAVSDT GSYSEPRPIG TRYLTKPL DUCK_AAV_Strain_FM_AAA83225 ..TMFAPNET GGYVEDRLIG TRYLTQNL MUSCOVY_DUCK_PARVOVIRUS_YP_068412 ..TMFAPNET GGYVEDRLIG TRYLTQNL MUSCOVY_DUCK_VP1_YP_068411 ..TMFAPNET GGYVEDRLIG TRYLTQNL MUSCOVY_DUCK_PARVOVIRUS_YP_068413 ..TMFAPNET GGYVEDRLIG TRYLTQNL Goose_AAV_VP1_AAA83230 ..IMFAPNET GGYVEDRLIG TRYLTQNL GOOSE_AAV_VP1_NP_043515 ..IMFAPNET GGYVEDRLIG TRYLTQNL AAV3_NP_043941 ..VDFTVDTN GVYSEPRPIG TRYLTRNL AAV3_Strain_H_AAC55049 ..VDFTVDTN GVYSEPRPIG TRYLTRNL AAV3B_VP1_AAB95452 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu1_AAS99260 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu4_AAS99287 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu2_AAS99270 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu3_AAS99280 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu60_AAS99307 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu61_AAS99308 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu25_AAS99276 ..VDFTVDNN GVYSEPRPIG TRYLTRNL VP1_isolate_hu15_AAS99265 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu16_AAS99266 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu18_AAS99268 ..VDFTVDTN GVYSEPRPIG TRYPTRNL VP1_isolate_hu7_AAS99313 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu10_AAS99261 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu11_AAS99262 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu9_AAS99314 ..VDFTVDTN GVYSEPCPIG TRYLTRNL VP1_isolate_hu53_AAS99300 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu55_AAS99302 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu54_AAS99301 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_huS17_AAU05370 ..VDFTVDTN GVYSEPRPIG TRYLTRNL AAV2_VP1_AAC03780 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu34_AAS99283 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu35_AAS99284 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_islolate_hu51_AAS99298 ..VDFTVDTN GVYSEPRPIG TRYLTRNL

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178 VP1_isolate_hu52_AAS99299 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu47_AAS99295 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu45_AAS99293 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu58_AAS99305 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu49_AAS99297 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu56_AAS99303 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu57_AAS99304 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu28_AAS99278 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu29_AAS99279 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_huT70_AAU05364 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu13_AAS99263 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu63_AAS99309 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu64_AAS99310 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_huT40_AAU05362 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_huLG15_AAU05371 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_huT17_AAU05358 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_huT41_AAU05372 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_huT71_AAU05366 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_huT88_AAU05368 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_huT32_AAU05360 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu27_AAS99277 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu19_AAS99269 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu20_AAS99271 ..VDFTVDTN GVYSEPRPIG ARYLTRNL VP1_isolate_hu21_AAS99272 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu24_AAS99275 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu22_AAS99273 ..VDFTVDTN GVYSEPRPIG TRYLTRNL VP1_isolate_hu23_AAS99274 ..VDFTVDTN GVYSEPRPIG TRYLTRNL

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193 BIOGRAPHICAL SKETCH Kim Van Vliet was born in Cleveland, Ohio. She moved to Orlando, Florida and attended Lake Mary High School. After grad uation, she attended the Universi ty of Central Florida (UCF) where she earned her B.S. in Biology and her B.S. in Psychology. She moved to Boca Raton, Florida and studied at Florida Atlantic University (FAU). Kim earned her M.S. in Biological Sciences at FAU. Kim moved to Ga inesville, Florida to continue her studies at the University of Florida and earned her Ph.D. in Medical Sciences through the Department of Molecular Genetics and Microbiology. Upon completion of her Ph.D. program, Kim will continue to do research.