Group Title: Virology Journal 2006, 3:13
Title: A phosphorylation map of the bovine papillomavirus E1 helicase
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Title: A phosphorylation map of the bovine papillomavirus E1 helicase
Series Title: Virology Journal 2006, 3:13
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Creator: Lentz MR
Stevens SM
Raynes J
Elkhoury N
Publication Date: 38784
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Volume ID: VID00001
Source Institution: University of Florida
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Virology Journal


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BioMed Central


Research


A phosphorylation map of the bovine papillomavirus EI helicase
Michael R Lentz*1, Stanley M Stevens Jr2, Joshua Raynes' and
Nancy Elkhoury'


Address: 'Department of Biology, University of North Florida, 4567 St. Johns Bluff Rd., S., Jacksonville, FL 32224, USA and 2Proteomics Core,
Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610, USA
Email: Michael R Lentz* mlentz@unf.edu; Stanley M Stevens sstevens@biotech.ufl.edu; Joshua Raynes neuroleo@msn.com;
Nancy Elkhoury nde26@yahoo.com
* Corresponding author


Published: 08 March 2006
Virology journal2006, 3:13 doi: 10.1 186/1743-422X-3-13
This article is available from: http://www.virologyj.com/content/3/l/13


Received: 24 August 2005
Accepted: 08 March 2006


2006Lentz et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Papillomaviruses undergo a complex life cycle requiring regulated DNA replication.
The papillomavirus El helicase is essential for viral DNA replication and plays a key role in
controlling viral genome copy number. The El helicase is regulated at least in part by protein
phosphorylation, however no systematic approach to phosphate site mapping has been attempted.
We have utilized mass spectrometry of purified bovine papillomavirus El protein to identify and
characterize new sites of phosphorylation.
Results: Mass spectrometry and in silicon sequence analysis were used to identify phosphate sites
on the BPV E I protein and kinases that may recognize these sites. Five new and two previously
known phosphorylation sites were identified. A phosphate site map was created and used to
develop a general model for the role of phosphorylation in E I function.
Conclusion: Mass spectrometric analysis identified seven phosphorylated amino acids on the BPV
El protein. Taken with three previously identified sites, there are at least ten phosphoamino acids
on BPV El. A number of kinases were identified by sequence analysis that could potentially
phosphorylate El at the identified positions. Several of these kinases have known roles in regulating
cell cycle progression. A BPV El phosphate map and a discussion of the possible role of
phosphorylation in El function are presented.


Background
Papillomaviruses infect epithelial cells of cutaneous or
mucosal origin in a variety of vertebrate hosts. An infec-
tion is established in the basal layer of the epithelium, and
a complex viral life cycle is carried out, dependent on the
differentiation state of the host cell [ 1-3]. Upon entry into
a basal epithelial cell, the infecting genome is transiently
amplified to approximately 50 to 200 copies, establishing
a latent infection. As latently infected cells divide, the viral
genomes are replicated on average once per cell cycle to


maintain this low genome copy number [4,5]. Minimal
viral gene expression is observed during the latent period.
As progeny cells migrate towards the epithelial surface, a
differentiation pathway is triggered, leading to changes in
viral gene expression, genome amplification, and assem-
bly of progeny virions.

The papillomavirus genome must undergo three distinct
modes of DNA replication during the course of an infec-
tion: transient amplification immediately upon infection;


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1078.54


1837.987


2129.08
2383.92
a2
S2705.13

II ,1LJ L .... I ,2.i ,,


800 1000 1200 1400 1600 1800 2000 2200


2807


2 3312.31
..27 11


2400 2600 2800 3000 3200 3400 3600 3800 4000


m/z, amu


Figure I
MALDI-qTOF MS analysis of E I tryptic digest. Protein characterization by peptide mass fingerprinting allowed for over
50% sequence coverage of the El phosphoprotein. Letters "a" and "b" indicate two peaks of low signal intensity corresponding
to the phosphopeptides LDLIDEEEDpSEEDGDSMR and VLpTPLQVQGEGEGR, respectively.


regulated replication during latency to maintain a con-
stant copy number; and vegetative replication to amplify
copy number prior to virion assembly. Viral DNA replica-
tion is initiated by the El protein, a virally-encoded
nuclear phosphoprotein [6]. Along with the viral E2 pro-
tein, El identifies and binds the viral origin DNA
sequence, distorts and unwinds the parental double helix,
and recruits the host cell replication machinery by direct
interactions with host replication proteins [7-9]. El is an
ATP-dependent DNA helicase that unwinds DNA at the
viral replication fork, while other replication functions are
supplied by the host cell (reviewed in [101).

We and others have proposed that the complicated regu-
lation observed for papillomavirus DNA replication is
imposed by host cell regulatory mechanisms [11-16]. Cell


cycle progression and cellular differentiation are control-
led in part by phosphorylation of key target proteins by
cellular kinases (reviewed in [17-20]). Several labs are
investigating the role of El phosphorylation on bovine
papillomavirus DNA replication activity, and have pro-
vided strong evidence that viral DNA replication is regu-
lated by El phosphorylation [11,12,21-23]. A number of
individual phosphorylation sites on BPV El have been
identified by several groups, but no systematic effort to
identify all of the phosphorylated amino acid positions of
this protein has been undertaken. Here we report five pre-
viously unidentified phosphorylation sites and confirm
two known sites, identified by a combination of mass
spectrometry (MS) methods. With sites previously identi-
fied by other methods, this brings the total number of
phosphate positions on BPV El to ten. This El phosphate


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1482.74


1403.65


1671.79


1319.60


977.43


,I II I


I i lll


L~ILL J.l~~rlr-uyullrru--~rr.r~ylUulyUL Y-l-l --~IWY~LVIZ IIYLI~YWL~LC L1~lll YLrlrl -IUL~I _1 -I-u -..lrL-.l--r-..-


1


II Y I Ihml~nlI ULIhmI hlI _IU IIIIIIIm


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Time mm


0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
694.85 Time, mm
C


701.85

LDLIDEEEDpSEEDGDSMR
726.27, +34-'~
463.57 670.34
613.32 |
1089.39, +2
...... l ,,,... L .. .. ,. ... ...... .....L___... ..._....
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500
m/z, amu
Figure 2
Tandem mass spectrometric analysis of the EI tryptic digest. A. Base peak ion chromatogram obtained by HPLC/ESI-
qTOF MS and MS/MS analysis of the El tryptic digest. B. Selected ion retrieval for m/z 1088.8, which corresponds to the dou-
bly-charged tryptic phosphopeptide LDLIDEEEDpSEEDGDSMR. C. Full scan mass spectrum at RT 53.1 min showing the pres-
ence of several tryptic peptides including the doubly and triply-charged phosphopeptide LDLIDEEEDpSEEDGDSMR.


map will provide a new tool to more fully understand
viral replication and serve as a useful model for investigat-
ing regulation of viral and cellular DNA replication.

Results
Identification of El phosphopeptides by mass
spectrometry
Very little El protein is produced during the course of an
infection or in BPV transformed cells. In order to generate
quantities of purified El necessary for mass spectrometry
analysis, El protein was isolated and purified from insect
Sf9 cells infected with a recombinant baculovirus [23-25].
Samples of purified El were separated from protein con-
taminants by electrophoresis through polyacrylamide
gels, stained with coomassie brilliant blue, and the El
band cut from the gel. Protease digestions were performed


directly in the gel slice. Phosphopeptides generally exhibit
low ionization efficiencies which makes mass detection
difficult. Furthermore, stoichiometry of phosphorylation
can be relatively low, further complicating detection. A
combination of mass spectrometric-based methods was
therefore used to identify major phosphorylation sites on
the El protein. Matrix-assisted laser desorption/ioniza-
tion (MALDI) and electrospray ionization (ESI) quadru-
pole time-of-flight (qTOF) mass spectrometry were
employed since both ionization techniques have been
shown to provide complementary information from pep-
tide mass fingerprint (PMF) analysis [26,27]. Coupling
HPLC to ESI also increases observation of phosphopep-
tides by minimizing signal suppression from other more
abundant peptides. Fig. 1 is a MALDI-qTOF mass spec-
trum displaying the tryptic fragment profile obtained after


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y4 112+


II l I I I I I III 111


II I


I I1


Sba i i i

VLipTPLiQVQlG EG EG R
y 1 ... y y y7 y6 y..... y...
yll y12 yl1 yl0 y9 y8 y7 y6 y4 y2


Y1
II


y12
I


[lIII .I....ll I I illi I ..... .... .IJlh. lil.. I h I l lIllill Ill III 11111I11 IIII I II I II I
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
m/z, amu


imai l''' huh mYYI~III hi I J.1 Iy M


100 200 300 400 500 600 700 800 900 1000


b3 b4
L DLIID EEEIDpSIEEDGDS MiR
y14 y12 yll y10 y9 y8 y7 y6 y5 y4 y3 yl


10

y8 Y9 Y11 yl 2
1.111111 1111I1I


1100 1200 1300 1400 1500 1600 1700 1800


m/z, amu


Figure 3
MS/MS spectra of the phosphopeptides VLpTPLQVQGEGEGR and LDLIDEEEDpSEEDGDSMR. Low-energy
sequence ions (b and y-type ions) produced by collision-induced dissociation allowed for identification of several El phosphor-
ylation sites after searching the tandem MS data against the NCBI nr sequence database with the MASCOT algorithm. A. Spec-
trum for VLpTPLQVQGEGEGR. B. Spectrum for LDLIDEEEDpSEEDGDSMR. The b and y-type ions are indicated on the
peptide sequence and on the corresponding spectrum peak. A differential modification of 80 Da for serine and threonine was
included in the MASCOT search parameters.


in-gel digestion of the El phosphoprotein. From this anal-
ysis, low-abundance peptides could be observed with
enough signal intensity in order to sequence and poten-
tially identify sites of phosphorylation. For example, tryp-
tic peptides containing the phosphorylated residues S584
and T126 (m/z 2176 and 1562, respectively, identified on
Fig. 1) were present at low abundance, however, enough
sequence ions were produced upon collision-induced dis-
sociation (CID) to determine that the peptides were phos-
phorylated (data not shown). These and other peptides
were singled out for further analysis because their mass
corresponded to that of a potentially phosphorylated pep-
tide.


Since the whole tryptic digest had been placed on a single
MALDI target spot for the PMF analysis, signal suppres-
sion of other components within the mixture including
phosphorylated peptides can occur. For this reason, ESI
was utilized given the feasibility of coupling liquid chro-
matographic techniques to this particular ionization
source. Fig. 2 demonstrates a tandem mass spectrometric
analysis of the El tryptic digest. Fig. 2A is the base-peak
ion chromatogram obtained upon rpHPLC-qTOFMS and
MS/MS analysis of the El tryptic digest mixture. The com-
plexity of the digest mixture is apparent from the ion chro-
matogram, demonstrating the advantage of HPLC
separation prior to mass spectrometric analysis in terms of


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y2


1 1 1


b2 b3 y


y4


II 1,.I.,


y7


liiiU 11 II II


l. ,.1 ,ll


IIIC I I II


----~ -'"--~-'~''- "~'~""'' ~ "11 1i I 1 11~~


Virology Joumnal 2006, 3:13








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Table I: E I phosphate sites identified by MS analysis and site characteristics. Phospho-amino acid positions identified in this study are
shown in the left column, followed by the sequence of the surrounding amino acids. The phosphorylated position is highlighted in bold
type. The NetPhos2.0 and NetPhosK scores for each phospho-amino acid is shown. 1.0 is the maximum score, and 0.5 is the default
threshold for likely phosphorylation. The kinases predicted by the NetPhosK algorithm are shown. Where there is no NetPhosK score,
that position was not predicted and the kinases were identified by manual analysis and comparison to published consensus sequences
[44]. *, phosphate sites identified by MS and not previously known; **, phosphate sites identified by MS, confirming previously known
sites.


NetPhos Score


Predicted Kinase


NetPhosK Score


VESDRYDSQDEDFVD
VLGSSQNSSGSEASE
LGSSQNSSGSEASET
NSSGSEASETPVKRR
NEANRVLTPLQVQGE
AQTTLNESLQTEKFD
LIDEEEDSEEDGDSM


0.997
0.926
0.993
0.333
0.959
0.253
0.998


minimizing signal suppression. The peptide with m/z
1088.8 is predicted to correspond to the tryptic peptide
containing phosphoserine 584. Selected ion retrieval was
performed, and the data shown in fig. 2B. The full-scan
mass spectrum of this peak (Fig. 2C) reveals several pep-
tides corresponding to the doubly- and triply-charged
tryptic peptide in which serine 584 is phosphorylated.


CK2 ATM DNAPK RSK 0.64 0.58 0.56 0.52
CK2
CK2 0.59
CKI, CK2
p38MPK 0.52
DNAPK 0.53
CK2 CKI 0.69 0.53


After searching the tandem mass spectrometric data with
the Mascot database search algorithm, several sites of
phosphorylation including those observed from MALDI
analysis were identified. Fig. 3 displays MS/MS spectra of
the phosphopeptides LDLIDEEEDpSEEDGDSMR and
VLpTPLQVQGEGEGR obtained from tandem mass spec-
trometric analysis of the El tryptic digest. This data is rep-
resentative of data collected for other identified


ATPase/Helicase 27

DNA Binding 308
DNA Pola Binding 424
E2 Binding 250 340
Nuclear Localization

1 100 200 300 400 500 00
BPV El H1
Protein

ST126-P
S48-P 9- S305-P S584-P
S109-P
T102-P
S100-P
--95-P
--94-P
S90-P



Figure 4
Positional phosphate map and functional domains of the BPV E I protein. The 605 amino acid protein is represented
on the lower horizontal line. The position of each of the phosphorylated amino acids is shown below. Functional domains are
represented by the solid bars above. Functional domain boundaries in most cases were determined by deletion mutagenesis
analysis as described in [10] and references therein.


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Amino Acid


Sequence


Ser 48**
Ser 94*
Ser 95*
Ser 100*
Thr 126*
Ser 305*
Ser 584**


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phosphopeptides. "y" peaks correspond to ion fragments
derived from the carboxyl terminus of the tryptic peptide,
and "b" ions are generated from the amino terminal end.
In general, the low-energy fragment ions observed for
each MS/MS spectra covered enough of each peptide
sequence to identify the residue in which phosphoryla-
tion had occurred. A list of the total phosphorylation sites
identified by mass spectrometric analysis is provided in
table 1.

In silico sequence analysis of BPV El protein
Unknown phosphorylation sites on proteins can be pre-
dicted by newly developed algorithms. These programs
use neural networks to predict unknown phosphorylation
sites based on the sequence context of known sites in
phosphoproteins. The BPV El protein sequence was sub-
mitted to NetPhos 2.0 [28], and the results are included in
table 1. All of the sites identified in this study are predicted
by this program, however some are predicted only weakly.
There are nineteen other predicted serine or threonine
sites that have not been positively identified (data not
shown; see discussion), as well as seven tyrosines. It has
previously been shown that BPV El labelled in vivo with
32P phosphate does not contain label on tyrosine, as
shown by phosphoamino acid analysis [21,24]; therefore
the predicted tyrosine sites will not be further considered.
In order to identify the kinases most likely to target the
sites we identified, the BPV El sequence was analyzed by
manual sequence analysis and through NetPhosK 1.0,
which predicts the most probable kinases based on infor-
mation from evolutionarily conserved sites on known
phosphoproteins [29]. The cellular kinases predicted to
modify the phosphorylation sites were determined and
included in table 1. The complete list includes ATM, CDK,
CK1, CK2, DNAPK, p38MAPK, and RSK, however it is
possible that not all of these predicted kinases interact
with El. Several sites are potential targets of multiple
kinases with similar probabilities. Determining the rele-
vant kinases for El phosphorylation in a complete infec-
tion cycle in the natural host is not possible at this time.

Discussion
Using MS analysis, we have identified five new phos-
phoamino acid positions on insect cell derived BPV El
protein, and confirmed two others previously identified
through mutation analysis. Taken with other previously
published sites threoninee 102 [21], serine 90 [30], and
serine 109 [23]) we present a map of the major sites of
phosphate addition on this viral DNA helicase. This map
is shown in fig. 4. In total there are ten sites: series 48, 90,
94, 95, 100, 109, 303, and 584; and threonines 102 and
126. This data correlates well with previously published in
vivo labelling and phosphoamino acid analysis data, in
which phosphoserine accounts for approximately 90 per-


cent of the label, with phosphothreonine contributing the
remaining ten percent [21,24].

This and previous studies to identify in vivo El phosphate
sites have been carried out on protein derived from bacu-
lovirus infected insect cells. We acknowledge the potential
for variation from this cell line and the natural mamma-
lian host, however, there is currently no system in which
sufficient quantities of El protein can be generated from
mammalian cells for these mapping studies. When direct
comparison has been possible, it is observed that protein
phosphorylation patterns in mammalian and insect sys-
tems are very similar, varying primarily quantitatively
rather than qualitatively [31]. We are confident that the
sites described here are comparable to the map that would
be derived from El protein produced in mammalian cells.
A direct comparison is desirable, and efforts will continue
to develop a system for high-level El expression in a
mammalian cell line.

The map presented here does not take into account any
differences in the proportion of the protein sample that
has phosphate at a particular site versus those that do not.
We expect that phosphorylation/dephosphorylation will
vary with the cell cycle and/or through the viral life cycle.
Some sites may be only transiently phosphorylated, or
phosphorylated only in more differentiated cells, and may
therefore be missed in this screen. Late stage baculovirus
infected cells are predominantly in the G2 stage of the cell
cycle [32]. The phosphorylation pattern of our protein
sample may therefore vary either quantitatively or qualita-
tively from protein found in natural host cells infected
with BPV. Phosphate site analysis of El prepared from dif-
ferent cell cycle stages of synchronized cells would be
highly desirable, but is not possible at this time.

NetPhos2.0 predicts phosphorylation of 17 series or
threonines that have not been identified as phosphate
sites. This is not surprising since the algorithm used char-
acterizes the local amino acid sequence only, and does not
take into account three-dimensional structure, subcellular
localization, or other structural features [28]. By ignoring
these important structural and functional features, predic-
tion algorithms identify sites that may be unrealistic in the
cellular setting. Nevertheless, it is possible that our analy-
sis has missed one or more rare or transient sites.

Using the kinase prediction program NetPhosK, the list of
potential kinases targeting the known El phosphate sites
is large, including ATM, CDK, CK1, CK2, DNAPK,
p38MAPK, PKA, PKC, PKG, and RSK. It is unlikely that all
of the predicted enzymes interact with El. The prediction
algorithm compares the submitted amino acid sequence
to known sites [29]. It does not take into account cell type,
subcellular localization, protein function, or other poten-


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tially important features, however it defines a useful start-
ing point for further analysis. Based on El's role in viral
DNA replication, kinases known to be involved in cell
cycle progression or DNA metabolism seem most likely to
be involved in El modification. Five of the identified ser-
ines are in a consensus for the kinase CK2, and two others
are likely cyclin/Cdk sites. Two are predicted targets for
protein kinase C (PKC), and one by DNA-dependent pro-
tein kinase (DNAPK). Serines 90, 109, and 584 (CK2);
and threonine 102 (cyclin Cdk) were previously shown to
be phosphorylated by the predicted kinases in vitro
[21,23,24,30]. Serines 90 and 109 are in consensus
sequences for PKC. These sites were previously shown to
be phosphorylated by mutation analysis, but were not
identified in this MS screen. It is possible that the relevant
PKC isozymes(s) are less active in late stage baculovirus
infected insect cells. This enzyme consists of a family of at
least twelve isozymes implicated is a wide variety of cell
signalling pathways, including the G1/S cell cycle transi-
tion [33,34]. Other known functions include a role in reg-
ulating differentiation of epithelial tissue, and could
therefore couple viral DNA replication to the differentia-
tion state of the host cell [23,35-40].

CK2 is predicted to phosphorylate half of the sites identi-
fied on the El protein. CK2 is a ubiquitous enzyme whose
role in the cell is under investigation but is still poorly
defined. A wide range of identified CK2 substrates impli-
cates this kinase in a number of critical cell functions,
including cell cycle regulation, cell survival, and regula-
tion of gene expression [18,20,41-44]. A substantial pro-
portion of known CK2 substrates are viral in origin [44].
In previous experiments, two CK2 sites on BPV El seriess
48 and 584) were studied by mutation analysis. Mutation
of either site to alanine completely eliminated viral repli-
cation, while acidic substitutions restored replication
function [12,45]. The specific role of these and other CK2
sites in El function remains to be determined.

BPV DNA replication has been shown to require binding
of El by cyclin E-Cdk2 in a Xenopus extract system,
although the specific role of the cyclin/kinase was not
determined [11]. Phosphorylation of HPV-11 El by Cdk
was recently shown to regulate nuclear entry of the pro-
tein by masking a nuclear export signal, however this sig-
nal is not contained in the BPV El sequence [46,47]. There
are three potential sites for Cdk phosphorylation in BPV
El (threonines 102 and 126; serine 283); the specific
amino acid(s) required for replication in the Xenopus sys-
tem were not identified. Point mutations at threonine 102
or serine 283 do not significantly alter DNA replication in
a transient system [[21]; Lentz, unpublished results], and
serine 283 has not been shown to be a target for phospho-
rylation in this or other studies. A recent model proposes
that BPV El concentration controls viral DNA replication


in latently infected cells [11,13,14]. In this model, El is
targeted for degradation by the anaphase promoting com-
plex, and is stabilized at the G1/S transition by interaction
with cyclin E/Cdk2. Our data supports this model by iden-
tifying threonine 126 as a potential target of the Cdk activ-
ity. A functional analysis of threonine 126 may clarify the
role of Cdk in BPV DNA replication.

Of the ten phosphate sites, six are tightly clustered within
20 amino acids, between series 90 and 109. Two more lie
on either side of this cluster, at position 48 and 126. This
clustering is easily seen in fig. 4. It is notable that the
majority of the phosphate sites are concentrated on the
amino-terminal domain of the protein. This region of the
protein is the least conserved among the many El protein
sequences that have been determined to date, and has few
common functions among different El proteins [10]. The
only conserved functional domain identified in this
region of the protein is the nuclear localization signal.
This and other BPV El functional domains are identified
in fig. 4. In both BPV-1 El and HPV-11 El, the carboxyl-
terminal two-thirds can function in replication following
truncation of the amino-terminus, suggesting only a sup-
porting or regulatory role for the amino terminal domain
[48-50]. In BPV El, known functions of the amino termi-
nal domain include nuclear import, and interaction
domains for the viral E2 protein and cellular DNA
polymerase alpha [[10] and references therein]. More
recently, several crystal structures implicate the carboxyl-
terminal domain in several key El functions including El
dimerization, E1-DNA interactions, and E1-E2 interac-
tions. These structures include an origin assembly inter-
mediate between the helicase catalytic domain of HPV-18
El and the viral E2 protein [51]; a dimer of BPV El DNA
binding domains [52]; and the BPV El DNA binding
domain bound to origin DNA [53]. These structures dem-
onstrate that the amino-terminal region of El is not
required for these protein-protein or protein-DNA inter-
actions. The essential DNA binding, ATPase, and helicase
domains are all located in the carboxyl-terminal domain
where only two phosphate sites, series 305 and 584, are
located [10]. Serine 305 is located in the DNA binding
domain, but is not in a readily identifiable kinase motif,
and is poorly conserved among El proteins. Serine 584 is
near the end of the consensus D box of the ATPase
domain. Mutation of serine 584 to alanine abolishes DNA
replication in a transient assay, however bacterially
derived El protein functions as a helicase in vitro, so the
precise role of this phosphorylation event remains unclear
[12]. Our map supports a general model in which the
amino terminal domain of BPV-1 El regulates or
enhances El function, while the carboxyl domain pro-
vides essential DNA binding and enzymatic activities. We
hypothesize that phosphorylation within the amino ter-
minal domain contributes to regulation or enhancement


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of El function. Our phosphate site map will be useful for
directing future molecular analysis of the role ofphospho-
rylation in El function and viral DNA replication.

Conclusion
This report describes an analysis of phosphorylation sites
of the BPV El helicase by mass spectrometry methods.
Five previously unknown sites were identified, and two
previously known sites were confirmed. Taken with other
known sites, there are at least ten amino acids on El that
are phosphorylated. The position of the phosphate sites
on the protein and the kinases predicted to interact with
El support a model in which phosphorylation of El
enhances or regulates its activities during the complex
viral life cycle.

Methods
Protein expression and purification
El protein was synthesized in and purified from recom-
binant baculovirus infected insect cells as previously
described, with several modifications [23]. Briefly, Spodop-
tera frugiperda Sf9 cells were maintained as adherent cul-
tures in TNMFH medium supplemented with 10% (v/v)
fetal bovine serum (JRH Biosciences), penicillin, and
streptomycin. Generation of recombinant baculovirus
expressing FLAG-tagged BPV El protein under control of
the polyhedrin promoter was described previously [23].
Protein was prepared from ten, 10 cm dishes of adherent
Sf9 cells 48 hours post-infection (pi). 30 minutes prior to
harvest, cells were treated with 10 nM calyculin A, a PP1
and PP2A phosphatase inhibitor. Adherent cells were
scraped into the culture medium, and along with any
detached cells were pelleted and stored at -80 C.

Protein was extracted from salt-washed nuclei as
described [25]. El purification was carried out by passing
extracts over a column containing M2 anti-FLAG antibody
bound to sepharose beads (Sigma). After washing to
remove unbound proteins, the FLAG-E1 protein was
eluted with synthetic FLAG peptide according to the man-
ufacturers directions (Sigma). Fractions were analyzed by
polyacrylamide gel electrophoresis and El containing
fractions were pooled and concentrated by dialysis against
solid sucrose, followed by dialysis into El storage buffer
(50 mM Tris-HC1 (pH 7.8), 1 mM EDTA, 1 mM dithioth-
reitol (DTT), 12.5 mM MgCl2, 100 mM KC1, 0.3 mM
NaC1, 10% (v/v) glycerol). Purified El protein was elec-
trophoresed into 10% SDS polyacrylamide gels and
stained with coomassie brilliant blue. The El containing
gel fragments were isolated, digested in-gel with trypsin,
and the corresponding tryptic peptides were used directly
for mass spectrometry analysis.


Mass spectrometry analysis of purified El protein
Capillary reversed-phase (rp) HPLC separation of El pro-
tein digests was performed on a 15 cm x 75 |im i.d. Pep-
Map 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. A capillary trap with the same stationary
phase chemistry as the analytical column was used in
combination with the Switchos isocratic solvent delivery
pump in order to concentrate and desalt the sample prior
to LC/MS/MS analysis. Gradient flow rates between 200-
300 nl/min were obtained by splitting a flow of 180 tiL/
min supplied by the gradient HPLC pump. Solvent A was
0.1% acetic acid in 95% water / 5% acetonitrile and sol-
vent B was 0.1% acetic acid in 10% water / 90% ace-
tonitrile. Following isocratic solvent delivery for 5
minutes during the sample desalting step, a linear gradi-
ent was carried out for 120 min to 40% solvent B. 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. The informa-
tion-dependent acquisition (IDA) mode of operation was
employed in which a survey scan from m/z 400-1500 was
acquired followed by collision-induced dissociation
(CID) of the two most intense ions. Survey and MS/MS
spectra for each IDA cycle were accumulated for 1 and 3
sec, respectively.

Prior to MALDI-qTOFMS analysis, the digested samples
were bound to a C18 ZipTip microcolumn, washed sev-
eral times with 0.1% TFA, and eluted onto a MALDI target
with 1 iL matrix solution. The matrix solution was pre-
pared by dissolving 5 mg of a-cyano-4-hydroxycinnamic
acid (Sigma-Aldrich, St. Louis, MO, USA) in 1 mL of 50%
acetonitrile/0.1% TFA. Full scan mass spectra were
acquired for 1 minute using a N2 laser operated at 20 Hz.
For CID experiments in which MALDI was the source of
ion production, collision energies were maintained
between 75-110 eV using nitrogen as the collision gas.

Fragment ion data generated by the IDA and conventional
MS/MS modes of acquisition via the QSTAR were
searched against the NCBI nr sequence database using the
Mascot (Matrix Science, Boston, MA) database search
engine. Probability-based MOWSE scores above the
default significant value were considered for peptide
sequence identification in addition to validation by man-
ual interpretation of the tandem MS data. Manual inter-
pretation was also necessary for low-abundance and/or
poorly-ionized phosphopeptides that did not demon-
strate adequate MS/MS spectral quality for Mascot
processing.




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In silico analysis
The full-length BPV El sequence was extracted from the
Swiss-Prot sequence database, [Swiss-Prot:P03116] in
FASTA format. The sequence was submitted online to
NetPhos2.0 (http://www.cbs.dtu dk/services/NetPhos/,
[28]) for analysis of potential phosphate sites, and to Net-
PhosK (http://www.cbs.dtu.dk/services/NetPhosK/ [29])
for identification of potential kinases that may interact
with El.


Competing interests
The authors) declare that they have no competing inter-
ests.


Authors' contributions
MRL conceived of the study, directed the project, carried
out the in silico analysis, and drafted the manuscript. SMS
carried out all of the mass spectrometry and MS data anal-
ysis, and contributed to the draft of the manuscript. NE
and JR generated the recombinant baculovirus, and
expressed, purified, and analyzed protein samples. All
authors read and approved the final manuscript.

Acknowledgements
This work was supported by NIH AREA Grant R15 CA087051 to MRL.

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matchmaker E2. Genes Dev 2004, 18(16):1981-1996. scientist can read your work free of charge
52. Enemark EJ, Chen G, Vaughn DE, Stenlund A, Joshua-Tor L: Crystal scientist can read your work free of charge
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tiation protein El from papillomavirus. Mol Cell 2000, disseminating the results of biomedical research in our lifetime."
6(I):149-158.
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intermediates in the assembly of the papillomavirus replica- Your research papers will be:
tion initiation complex. Embo] 2002, 21(6): 1487-1496.
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