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Regulation of gene expression by adeno-associated virus

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Regulation of gene expression by adeno-associated virus
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Labow, Mark Aron, 1960-
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162 leaves : ill. ; 29 cm.

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Dependovirus ( jstor )
DNA ( jstor )
DNA replication ( jstor )
Gene expression ( jstor )
Genetic mutation ( jstor )
Genomes ( jstor )
Open reading frames ( jstor )
Plasmids ( jstor )
RNA ( jstor )
Transfection ( jstor )
Dependovirus ( mesh )
Dissertations, Academic -- Immunology and Medical Microbiology -- UF ( mesh )
Gene Expression Regulation ( mesh )
Immunology and Medical Microbiology thesis Ph.D ( mesh )
Transcription, Genetic ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 1987.
Bibliography:
Bibliography: leaves 147-161.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Mark Aron Labow.

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REGULATION OF GENE EXPRESSION BY
ADENO-ASSOCIATED VIRUS














BY

MARK ARON LABOW


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1987




REGULATION OF GENE EXPRESSION BY
ADENO-ASSOCIATED VIRUS
BY
MARK ARON LABOW
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1987


This dissertation is dedicated to my mother Audrey whose
support has made this possible and whose love has made this
worthwhile.


ACKNOWLEDGMENTS
I would like to thank Dr. Kenneth I. Berns for his
guidance, infinite patience, and love for biology (and
countless jokes and stories) which helped make several years
pass by very quickly.
I would like to thank the faculty of the University of
Florida who have given me a priceless education. In
particular I would like to thank William B. Gurley and
Nicholas Muzyczka for their advice and inspiration over the
years.
I wish to thank my friends Randy Horowitz and Paul L.
Hermanot for their wonderful company, help and advice.
I wish to thank Patricia Burfeind for her excellent
technical assistance and most of all, for her friendship.
Finally, I wish to thank Gwen Tse Wong for her faith,
advice and love.
in


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES V
ABBREVIATIONS USED ix
ABSTRACT X
CHAPTER I INTRODUCTION
The Parvoviruses 1
The Natural Occurrence of AAV 2
Latent Infection by AAV 4
The AAV Helper Functions 5
The Physical and Genetic Organization
of the AAV Genome 10
Molecular Genetics of AAV 16
Perspective 19
CHAPTER II METHODOLOGY
Cells and Culture Conditions 21
Viral Stocks 21
Preparation of Plasmid DNAs 21
Enzymes and Linkers 22
Construction of Mutant AAV Genomes ... 22
Radiolabeling of DNA 23
Diethylaminoethyl (DEAE)-Dextran DNA
Transfections 24
Calcium Phosphate DNA Transfections . 25
Genetic Selection of Mammalian Cells . 26
Chloramphenicol Acetyltranserase
(cat) Assays 26
RNA Extraction and Analysis 27
DNA Isolation 29
Southern Blotting and Hybridization
of Filters 30
Nuclear Runoff Transcription (NRT)
Analysis 30
Immunizations and Antibody Analysis . 31
Metabolic Labeling and
Immunoprecipitation Analysis .... 33
IV


CHAPTER III
AUTOREGULATION OF AAV GENE EXPRESSION
X Introduction 36
Results 37
Discussion 75
CHAPTER IV INHIBITION OF GENETIC TRANSFORMATION AND
EXPRESSION OF HETEROLOGOUS GENES BY THE
AAV REP GENE
Introduction 89
Results 91
Discussion 105
CHAPTER V IDENTIFICATION OF AAV REP GENE PRODUCTS
Introduction 112
Results 114
Discussion 123
CHAPTER VI CONSTRUCTION AND CHARACTERIZATION OF
HYBRID AAV/SV40 GENOMES
Introduction 126
Results 127
Discussion 137
CHAPTER VII CONCLUSION 146
LITERATURE CITED 147
BIOGRAPHICAL SKETCH
162


LIST OF TABLES
Table Page
3-1. CONSTRUCTION OF PLASMID GENOMES 38
4-1. INHIBITION OF GENETIC TRANSFORMATION BY
AAV GENOMES 100
4-2. INHIBITION OF TRANSFORMATION OF pTK BY
d!52-91/neo 101
4-3. DOSE DEPENDENCE OF AAV AND NEO DNA ON
TRANSFORMATION 103
4-4. EFFECT OF CLONED E4 SEQUENCES ON AAV MEDIATED
INHIBITION OF TRANSFORMATION 106
4-5. INHIBITION OF pSV2cat EXPRESSION BY
COTRANSFECTED AAV DNA 107
5-1. PRODUCTION OF ANTIPEPTIDE ANTISERA 115
vi


LIST OF FIGURES
Figure Page
1-1. Structure and genetic organization of the
AAV genome 15
3-1. Accumulation of RNAs by two types of rep
mutants 42
3-2. Accumulation of RNAs by mutant AAV genomes .... 43
3-3. Accumulation of RNAs by ori~ AAV genomes 48
3-4. Accumulation of RNAs by rep deletion mutant
genomes 49
3-5. Transcription of AAV genomes in isolated nuclei. 53
3-6. Complementation of expression of rep mutants ... 58
3-7. Complementation of expression by left side
deletion mutants 59
3-8. Accumulation of RNAs by left side deletion
mutants 63
3-9. Phenotypes of left side deletion mutants 65
3-10. Structures of AAV genomes with duplications
of p40 69
3-11. Expression of AAV genomes containing
duplications of p40 70
3-12. Stability of AAV RNAs 71
3-13. Transcription of AAV in isolated KB cell nuclei. 76
3-14. Transcription of AAV in isolated HeLa cell
nuclei 78
4-1. AAV genomes inhibit genetic transformation .... 95
4-2. Southern blot analysis of G-418r cell clones ... 96
vii


5-1. Immunoprecipitation analysis with antipeptide
antibody 119
5-2. Immunoprecipitation analysis of AAV proteins . 120
5-3. Identification of an AAV protein made early
during infection 121
5-4. Identification of phosphorylated capsid
proteins 122
6-1. Construction of AAV/SV40 hybrid genomes 133
6-2. Structure of hybrid AAV/SV40 genomes 135
6-3. Expression of AAV plasmids in human cells .... 136
6-4. Replication of AAV plasmids in various
cell lines 139
6-5. Replication of hybrid genomes in cos-7 cells . 141
6-6. Inhibition of DNA replication by the AAV
rep gene 142
viii


ABBREVIATIONS USED
AAV adeno-associated virus
AAV-2 AAV type-2
Ad adenovirus
BPV bovine papilloma virus
BSA bovine serum albumin
C- carboxyl
CT calf thymus
cat choloramphenicol acetyltransferase
cpm counts per minute
DEAE diethylaminoethyl
DMEM Dulbecco1s modified Eagles medium
DNA deoxyribonucleic acid
EDTA ethelene diaminetetraacetic acid
FCS fetal calf serum
G gravity
HAT Hypoxanthine-aminopterin-thymidine
HSV herpes simplex virus
kb kilobase
kd kilodaltons
KLH Keyhole limpet hemocyanin
md monomer duplex
ix


MEM
. minimal essential medium
mMT mouse metallothionein
moi multiplicity of infection
mRNA messenger RNA
mu map units
neo neomycin phosphotransferase
NRT nuclear runoff transcription
ORF open reading frame
ori origin
PBS phosphate buffered saline
RNA ribonucleic acid
RSB reticulocyte swelling buffer
SDS sodium dodecyl sulfate
SRBCs sheep red blood cells
ss single stranded
SV40 Simian Virus 40
T-antigen tumor-antigen
TLC thin layer chromatography
TRs Terminal Repeats
tk thymidine kinase
x


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
REGULATION OF GENE EXPRESSION BY
ADENO-ASSOCIATED VIRUS
By
Mark Aron Labow
May, 1987
Chairman: Kenneth I. Berns
Major Department: Immunology and Medical Microbiology
The ability of adeno-associated virus (AAV) to affect
gene expression from both its own genome and heterologous
genes has been examined using several different assays. The
expression of both wild type and mutant AAV genomes was
examined in human cells. The effects of the AAV genome on
heterologous genes were examined in stable transformation
and transient gene expression assays. The properties of
hybrid AAV/simian virus 40 (SV40) genomes were also
examined.
Mutations of the open reading frame within the AAV p5
and pl9 RNAs greatly inhibited both the accumulation of AAV
RNAs and AAV transcription in adenovirus infected human
cells. This defect in gene expression was complemented in
trans indicating that a gene product encoded within the p5
gene activates AAV transcription. Proteins encoded by the
xi


p5 and pl9 genes were also identified using antisera to
synthetic peptides
Deletion of certain sequences within the left side of
the AAV genome enhanced the accumulation of the p5 or pl9
RNAs. The effect of such deletions occurred only in cis.
Analysis of transcription from the wild type AAV genome
suggests that the cis-active negative regulation occurs
after transcription initiation.
The AAV genome was also shown to inhibit the activity
of heterologous genes. Cotransfection of AAV genomes
inhibited both the transformation by several different
dominant selectable genes and transient expression by
pSV2cat. Both of the inhibitory activities were shown to be
mediated by the AAV p5 and pl9 genes.
Finally, hybrid AAV genomes containing the SV40 early
promoter and origin of DNA replication were constructed.
The replication of the hybrid genomes, initiated by the SV40
origin, was inhibited by the p5 and pl9 genes in cells which
constitutively produce the SV40 tumor-antigen.
Xll


CHAPTER I
INTRODUCTION
The Parvoviruses
The parvoviruses are among the smallest of the
eukaryotic viruses. The family Parvoviridae consists of
three genera, the densoviruses, the autonomous parvoviruses
and the defective parvoviruses also known as the
dependoviruses (Siegl et al., 1985). The densoviruses
infect several species of insects. The autonomous
parvoviruses infect and cause disease in many vertebrate
species (Siegl, 1984). Although the dependoviruses also
infect many vertebrate species from birds to humans, they
are unique in that they require coinfection with a
genetically distinct helper virus in order to replicate
(Atchison et al., 1965; Melnick et al., 1965; Hoggan et al.,
1966; Blacklow et al., 1967a; Carter and Laughlin, 1984).
The dependoviruses were first discovered as contaminants of
purified Adenovirus (Ad) preparations and are thus referred
to as adeno-associated viruses (AAV). AAV type-2 (AAV-2) is
the most extensively characterized dependovirus and is the
focus of this dissertation.
The AAV virion is typical of all parvoviruses,
consisting of a naked icosohedral shell of approximately 20
1


2
nm in diameter. The capsid is composed of three virion
proteins, VP1, VP2, and VP3, with molecular weights of 87
kilodaltons (kd), 72 kd, and 63 kd, respectively (Salo and
Mayor, 1977; Johnson, 1984). The proteins are
immunologically cross reactive and contain extensive
overlapping amino-acid sequences (Johnson et al., 1971; Rose
et al., 1971; Johnson et al., 1975; Buller and Rose, 1978;
Lubeck et al. 1979). VP3 comprises approximately 90% of
total virion protein. Subspecies of VP1 and VP3 which differ
slightly in MW have also been reported (McPherson and Rose,
1983). The AAV virion is extremely stable, being resistant
to heat, ionic detergents, and certain proteases.
The capsid surrounds a linear single stranded (ss)
deoxyribonucleic acid (DNA) genome of approximately 5
kilobases (kb) (Mayor et al., 1969; Rose et al., 1969; Berns
and Rose, 1970; see Berns and Hauswirth, 1984, for review).
Although many autonomous parvoviruses preferentially package
the minus strand of DNA, AAV, as well as the autonomous
parvoviruses LuIII and B19, packages DNA molecules of both
polarities with equal frequency (Berns and Adler, 1972;
Majaniemi et al., 1981; Bates et al., 1984; Cotmore and
Tattersall, 1984).
The Natural Occurrence of AAV
The existence of AAV in nature is extremely widespread.
AAVs have been isolated from nearly all animal species which
have been specifically tested. Although natural hosts of


3
AAV appear to include humans, several species of monkeys,
cattle, horses, dogs, quails, and chickens, the broad
species specificity of many AAV prevents the actual
determination of species origin. While AAV-4 is only known
to replicate efficiently in vitro in primary African green
monkey cells, AAV-1, -2 and -3 appear to replicate in cells
from a wide range of species (Hoggan et al., 1966; Blacklow
et al., 1968b; see Cukor et al., 1984, for review). However
these AAV are somewhat species specific, in that they
efficiently replicate only in the presence of a helper virus
of the same species origin as that of the host cell. For
example human AAV-1, -2 and -3 will replicate in canine or
chicken cells as long as canine or chicken Ad are provided
as helper.
Infection by AAV-2 and 3 in humans is very common, as
is infection of captive monkeys by AAV-1, -2, and -3
(Blacklow et al., 1968a,b). A fifth primate AAV, AAV-5, has
been isolated from human genital tissue (Bantel-Shaal and
zur Hausen, 1984; Georg-Fries et al., 1984). An estimated
85% of the population of human adults are sero-positive for
AAV (Blacklow et al., 1967b, 1968a,b). Further, the mean
age of seroconversion for AAV appears to be between the ages
of 7 to 8 years of age. Although infection is very common,
no disease in humans or animals is associated experimentally
or epidemiologically with AAV. This benign nature of AAV


4
infection is consistent with the normal inability of AAV to
replicate in the absence of a helper virus.
Latent Infection by AAV
The defective nature of AAV replication raises the
question of how the virus retains its biological identity in
the absence of helper virus. In this regard, the AAV genome
appears to exist in a latent state as a silent provirus.
The latent state of AAV infection was originally discovered
during a program for screening for persistent viral
infections of primary cell culture lots to be used for
vaccine production. It was discovered that although no cell
lots were positive for AAV structural antigens, infection of
the cells with Ad resulted in the detection of AAV antigen
in 20% of the lots of African green monkey kidney cells and
1-2% of the lots of human embryonic kidney cells (Hoggan et
al., 1972) .
Latent infection with AAV can be established
experimentally. Several studies have shown that infection
of human tissue culture cells with AAV results in a carrier
state from which no AAV virion DNA or capsid antigen is
detected but upon superinfection with Ad, AAV is produced
(Hoggan et al., 1972; Handa et al., 1977; Laughlin et al.,
1986) The process of establishing latent AAV infection is
extremely efficient. Up to one third of the cell clones
derived after infection produce AAV after superinfection


5
with Ad. The ability to rescue AAV is stable for greater
than 100 cell passages and up to one third of the individual
cells produce AAV antigen after superinfection with a helper
virus. The AAV genome exists in the latently infected
cells as an integrated provirus. Examination of the
integration complexes indicates that the AAV genomes are
often present as head to tail concatamers and that
integration occurs at random sites within the host DNA
(Berns et al., 1975; Cheung et al., 1980; Hermonat, 1984;
Tratschin et al., 1985; Laughlin et al. 1986). Further,
the AAV genome is integrated colinearly with the terminal
repeats (TRs) at the junctions of integration, suggesting a
role for the TRs in the integration process.
The AAV Helper Functions
Many viruses provide the full helper effect for AAV.
As described above, AAV will replicate in cells of varied
species origins using Ad of that same species. The helper
functions are also provided by herpes simplex virus (HSV) I
and II (Buller et al., 1981). In addition, the replication
of at least one AAV, AAV-5, has been reported to be
supported by vaccinia virus (Schlehofer et al., 1986).
While the plethora of genetically distinct helper viruses
might indicate that only a single common feature of the
helper viruses is needed for AAV replication, studies with
Ad indicate an interplay of many helper virus functions.


6
Studies using mutant Ad indicate that all the helper
functions are provided by Ad early regions. The first
region of importance is the El region. This region contains
two transcriptional units, Ela and Elb. The two genes
comprise the transforming region of Ad (Graham et al.,
1974). The normal functions of the two genes appear
complex. Ela encoded products normally function to activate
transcription of the other Ad early regions (Berk et al.,
1979; Jones and Shenk, 1979). The normal functions of the
Elb gene products are less clear but appear to involve the
ability to selectively transport viral ribonucleic acid
(RNA) to the cytoplasm while blocking the same process for
cellular messages (Babiss and Ginsberg, 1984; Babiss et al.,
1985; Pilder et al. 1986). Several proteins are produced
from alternatively processed Ela and Elb RNAs. When
studied in human KB culture cells, Ad mutants defective in
the production of either Ela or Elb products were defective
helpers for AAV-2, but provided significant helper effect
when the Ad mutants were used at very high multiplicities
(Ostrove and Berns, 1980; Janick et al., 1981; Laughlin et
al., 1982). However, mutants that were defective for the
production of both Ela and Elb products were completely
defective for helper function at all multiplicities.
The role of the Ela gene products as helper functions
can be explained by the ability of Ela proteins to stimulate
transcription. Transcription of AAV is readily detected in


7
cells transformed by the El region of Ad (Ostrove et al.,
1981). Specific induction of expression from the AAV pl9
promoter by the Ela gene has also been reported (Tratschin
et al., 1984b). The role of the Elb gene is less clear. To
some extent the functions of Ela and Elb appear
interchangeable in that mutants in both El regions are more
defective for AAV helper functions than the individual
mutants. Also microinjection of helper virus genes into
AAV-2 infected Vero cells showed that in addition to the E2a
and E4 genes, helper effect, as measured by AAV capsid
protein synthesis, required both Ela and Elb genes when
injected in low copy number but either Ela or Elb alone when
injected in high copy number (Richardson and Westphal,
1984) .
The E2a gene encodes the 72 kd ssDNA binding protein
(see Kelly, 1984, for review). This protein is required for
Ad DNA replication
both in vivo
and
in vitro.
negative
regulation of E4
transcription
and
regulation
of
the
stability of Ad early messenger
RNA
(mRNA) (Babich
and
Nevins, 1981; Handa et al., 1983). The E2a gene product has
been reported to be involved in both the regulation of AAV
gene expression and AAV DNA replication. One group has
reported that although AAV DNA replication and production of
RNA were relatively normal, the accumulation of AAV
structural proteins and progeny ss-DNA were severely
inhibited when a temperature sensitive E2a mutant was used


8
as helper at the nonpermissive temperature (Myers et al.,
1980; Jay et al., 1981). However, another group reported
that the same mutant was as efficient a helper as wild type
Ad for AAV-2 replication but that the E2a gene product was
responsible for a species specific block in the translation
of AAV-2 capsid proteins, as well as certain late Ad
proteins, in monkey cells (Straus 1976a; McPherson et al.,
1982) .
In addition, one group has reported that efficient
expression of the E2a gene in cos-1 cells supported AAV DNA
replication in the absence of other viral early genes (Quinn
and Kitchingman, 1986). However this study was done only in
cos-1 cells which produce the Simian Virus 40 (SV40) tumor-
antigen (T-antigen) which may contribute to AAV DNA
replication (see below). The production of AAV capsid
proteins is also enhanced by the Ad VA-RNAs (Janick et al.,
1981; Janick et al., 1982).
Finally, the E4 gene appears to be required for AAV DNA
replication (Carter et al., 1983). Microinjection studies
have shown that AAV DNA replication required the El, E2a,
and E4 genes (Richardson and Westphal, 1981). However,
microinjection of E4 RNA alone was sufficient for AAV DNA
replication, indicating that the function of the other Ad
regions might be indirectly needed to induce expression of
the E4 gene. However, the role of E4 as a helper gene has
not been examined in detail. Also, E4 mRNA has been shown


9
to act as a complete helper only in one cell line (Vero
cells). This is an important point in that Ad replication
is affected differently by E4 mutations in different cell
lines (Halbert et al., 1985)
The ability of divergent viruses and genes to support
AAV replication suggests that the helper functions act
indirectly by modulating activities of cellular genes. For
example, the Ela gene has been suggested to stimulate
transcription indirectly by enhancing the activity of a
cellular factor which binds Ad promoters (Feldman et al.,
1982; Kovesdi et al., 1986). Also VAI RNAs enhance
translation in Ad infected cells indirectly by preventing
the cell from inactivating a translation initiation factor
(Thimmappaya et al., 1982; Kitajewski et al., 1986). If all
the helper functions act indirectly, then one prediction is
that there would be a certain cell type or physiologic state
which would support AAV growth in the absence of helper
virus.
Several groups have reported helper virus independent
replication of AAV. Schlehofer (1983, 1986) originally
reported that certain human and Chinese hamster cell lines
transformed with SV40 become permissive for AAV-5
replication upon addition of a potent carcinogen or
cyclohexamide. Similar results were obtained for AAV-2.
Yakobson et al. (in press) have shown that several cell
lines will replicate AAV, supporting the production of


10
infectious progeny, when pretreated with potent cell
synchronizing agents. Interestingly, the cells most
efficient at replicating AAV were Chinese hamster cell lines
transformed with SV40. The SV40 genome appeared to be a
contributing factor for AAV replication since monkey CV1
cells did not support AAV replication although cos-1 cells
(SV40-transformed CV-1 cells) supported AAV replication.
However a Chinese hamster cell line that did not contain the
SV40 genome also replicated AAV although at a much reduced
level.
It should be noted that under optimal conditions,
replication without helper virus was at least two logs lower
than that in the presence of helper virus. Further, only a
fraction of treated cells were able to replicate AAV in the
absence of helper virus. Thus AAV replication appears to
require a complex cell milieu which can be modulated by an
array of viral genes and physiologic states.
The Physical and Genetic Organization of the AAV Genome
The AAV genome has been extensively characterized and
completely sequenced (Lusby and Berns, 1982; Srivastava et
al., 1983). One outstanding feature of AAV DNA is the
presence of inverted TRs. The TRs are 14 5 bases long and
contain several internal palindromes (Gerry et al., 1973;
Kozcot et al., 1973; Fife et al., 1977; Lusby et al., 1980).
Thus AAV DNA can form several different secondary structures
including ss-circles held together with 145 base duplex


11
"panhandles" (Berns and Kelly 1974). The termini can also
fold upon themselves to form intrastrand T-shaped
structures. The TRs appear to act as the origin (ori) of
AAV DNA replication, apparently acting as primers for DNA
synthesis (Straus et al., 1976b; Hauswirth and Berns, 1977,
1979) Further the AAV TRs also contain a nicking site
allowing both the replication of 5' terminal sequences and
the resolution of replication intermediates. Thus AAV DNA
is replicated in a manner analogous to the model proposed by
Cavalier-Smith (1974) for the replication of linear
chromosomal DNA. The TRs may also be important in the
process of integration into and rescue out of foreign DNA by
the AAV genome.
Sequence analysis of the internal portion of the AAV
genome has revealed several large open reading frames (ORFs)
as illustrated in figure 1-1. The genome can be divided
into two domains, with long ORFs on either side. The two
largest ORFs are the major left and right side ORFs from 6-
47 map units (mu) (bases 320-2186) and from 60-92 mu (bases
2810-4321), respectively. The right side ORF predicts a
protein of size and composition corresponding to the
smallest capsid protein, VP3. Due to the overlapping nature
of the capsid proteins amino-acid sequences, the larger
virion proteins must also contain the body of this reading
frame.


12
All of the ORFs are present in different forms within
the AAV mRNAs. The known AAV RNAs have been characterized
from Ad-infected cells. The genome appears to encode three
sets of transcripts (see Carter et al., 1984, for review).
The RNAs are all overlapping, polyadenylated, and have
coterminal 3' ends at mp 96 (Laughlin et al., 1979b; Green
and Roeder, 1980a,b; Green et al., 1980; Lusby and Berns,
1982; Srivastava et al., 1983). However, three different
5'ends have been located at 5, 19, and 40 mu, giving rise to
unspliced RNAs of 4.2, 3.6, and 2.6 kb respectively. An
intron located between 40 and 48 mu has also been identified
and spliced RNAs of 3.9, 3.3, and 2.3 kb have been
detected.
The RNA start sites are each located downstream from
putative RNA polymerase II promoters, p5, pl9, and p4 0 as
indicated by nucleotide sequence. The promoters contain
elements typical of eukaryotic RNA polymerase II promoters
including TATA boxes located approximately 30 bases upstream
of the RNA cap sites as well as CAAT boxes located
approximately 80 bases upstream from the pl9 and p40
transcripts. The sequences including and surrounding the
TATA boxes of the p5 and pl9 promoters are highly related to
that surrounding the Ela gene of Ad-5 (Lusby and Berns,
1982). Transcription of the AAV promoters is also
sensitive to low concentrations of a-amanitin as expected


13
for RNA polymerase II genes (Bloom and Rose, 1978; Jay et
al., 1978).
The p4 0 RNAs are predominantly spliced and comprise
about 90% of the total AAV RNA accumulated in cells
coinfected with AAV and Ad. The larger p5 and pl9 RNAs are
accumulated in approximately equal amounts and exist as
predominantly unspliced molecules. All of the AAV RNAs are
found in both the nucleus and cytoplasm.
Several experiments show that the p40 messages encode
the AAV structural proteins. First, the capsid proteins are
still produced by mutant AAV genomes lacking the p5 and pl9
promoters (Janik et al., 1984; Hermonat et al. 1984;
Tratschin et al., 1984a). Second, all three of the
structural proteins are translated in vitro from isolated
2.3 kb size AAV RNA (Jay et al., 1981). This observation is
paradoxical in that the sequence and structure of the
spliced p40 message could only account for the production of
the smallest capsid protein VP3. VP2 appears to be
synthesized using additional reading frame encoded upstream
from and in the same reading frame as the major left side
ORF, initiating translation at an upstream ACG codon
(Becerra et al., 1985). The mechanism by which the largest
structural protein, VP1, is synthesized is unclear but has
been suggested to involve the use of alternatively spliced
mRNAs (Hermonat et al., 1984). Several additional ORFs of


Figure 1-1. Structure and genetic organization of the AAV genome. The
4.7 kb AAV genome is shown schematically divided into 100 mu. The 145-base
TRs are represented as small black boxes. The structure of the known AAV
mRNAs are shown and are aligned with the genome representation. The
locations of the three AAV transcription promoters, p5, pl9, and p40, are
indicated at the left (5'end) of the RNAs produced from them. The 3'
polyadenylation site for all AAV RNAs is indicated by wavy lines at the
right (3' end) of the RNAs. Spliced RNAs are drawn with gaps to indicate
the removed intron. ORFs present within the messages are depicted by black
rectangles within the RNA. The sizes of the transcripts (not including the
polyadenylation tail) are shown on the right side of the figure. The
boundaries of the known separable genetic regions of the AAV genome are
shown at the bottom of the figure.


15


16
unknown function overlap the carboxyl (C)-terminal regions
of both large ORFs.
Molecular Genetics of AAV
The defectiveness of AAV has suggested that all
functions required for AAV replication are provided by both
the host cell and the helper virus and that AAV simply
encodes its capsid proteins. However the existence of the
large left side ORF indicated that AAV might encode
nonstructural proteins which might be required for AAV
replication. This notion was supported by the observation
that AAV defective interfering particles, whose genomes
contain large heterogeneous internal deletions, required
coinfection with wild type AAV in order to replicate
(Laughlin et al., 1979a). However the identification of
specific AAV replication functions was hampered by the
requirement for a cytopathic helper virus; thus AAV could
not be plaqued nor could AAV mutants be selected.
This problem was circumvented by cloning the AAV genome
into bacterial plasmid vectors. The AAV genome was
originally cloned by GC-tailing of reannealed virion DNA and
insertion into pBR322 (Samulski et al., 1982). Clones
containing the intact AAV genome are biologically active
(Samulski et al., 1982; Laughlin et al. 1983). Upon
transfection into helper virus infected human tissue culture
cells, the cloned AAV genomes are rescued from the plasmid
sequences and AAV replication ensues. The process of rescue


17
and replication from the plasmid vectors is highly
efficient, approaching the yields of AAV DNA and virus
achieved by transfection of wild type virion DNA.
The cloning of the AAV genome in an infectious form
provides the ability to modify the genome in vitro and to
propagate large amounts of the mutant genomes in bacteria.
The effects of such modifications are then assayed by
transfection of the mutant genomes into Ad-infected culture
cells. This procedure was originally used to examine the
effects of deletions within the TRs on AAV DNA replication
(Samulski et al., 1983; Senapathy et al., 1984; Lefebvre et
al., 1984). Mutants which contained large deletions of both
TRs sequences failed to replicate their DNA. Replication of
these mutants could not be complemented in trans. confirming
the predicted role of the TRs as origins of AAV DNA
replication. Unexpectedly, mutants which contained only one
deleted TR were viable, producing AAV genomes with wild type
TR on both sides of the genome. Thus there appears to be
some mechanism for repairing deleted terminal DNA sequences.
A detailed genetic analysis of the internal AAV
sequences has been done in a similar manner. Deletion and
frameshift mutations have been created in cloned AAV
genomes. Deletion mutations were created by digestion of
cloned AAV genomes with various restriction endonucleases
(Hermonat et al., 1984; Tratschin et al., 1984a).
Frameshift insertion mutations have also been created by


18
insertion of synthetic nucleotide linkers at many places
throughout the genome. Analysis of the different mutants
revealed at least three distinct phenotypes and have again
divided the genome into two basic domains.
Mutations between 11 and 42 mu severely inhibit the
replication of AAV DNA. The defective replication of the
mutants, referred to as rep mutants, is complemented in
trans by cotransfected AAV genomes. Thus the ORFs contained
uniquely within the p5 and pl9 transcripts encode a
protein(s) (rep proteins) absolutely reguired for AAV DNA
replication. Mutants containing a frameshift at 11 mu are
also defective for replication, indicating that a product
encoded by the p5 ORF is essential for DNA replication. No
mutation which specifically affects the pl9 ORF has yet to
be created so the importance of this gene in AAV replication
has not yet been elucidated.
Mutations which affect the C-terminal region of the p5
and pl9 ORFs have variable phenotypes. A mutation which
specifically affects the C-terminus of the p5 and pl9 ORFs
contained within the spliced mRNAs has no apparent effect on
DNA replication, although a frameshift mutation at 42 mu
within the rep ORF contained within the AAV intron inhibits
AAV DNA replication by 100 fold. Thus potential rep
proteins encoded by the unspliced p5 and pl9 ORFs appear to
be critical for AAV DNA replication. Conversely, certain
mutants which contain deletions of the entire intron and C-


19
region of the rep proteins efficiently replicate their DNA
while other similar mutants do not (Tratschin et al.,
1984b). As such it is unclear what specific function(s) the
C-terminal coding regions of the rep ORFs have.
Although mutations within the right half of the genome
do not affect the accumulation of AAV replication
intermediates, these mutations do affect capsid protein
production and function. As expected deletion and
frameshift mutation within the body of the large right side
ORF (cap mutants) completely abolishes capsid production,
the seguestration of ss-DNA, and virus production. Also
mutations between 48 and 55 mu specifically affect the
syntheses of the larger structural proteins, VP1 and VP2 but
not VP3 (Janik et al., 1984). Thus VP3 appears to be a
primary translation product, rather than a proteolytic
cleavage product of the larger virion proteins.
Unexpectedly, mutants with lesions between 48-55 mu and
those with lesions in the body of the right side ORF have
different phenotypes. Mutants containing lesions between 48
and 55 mu (lip mutants) still produce sequestered progeny
ss-DNA but produce virions of low infectivity. Thus the
different virion proteins appear to have separable
functions.
Perspective
The biology of AAV in many senses is similar to that of
latent prokaryotic viruses. Like the lambda bacteriophage,


20
AAV can efficiently integrate into and be rescued from the
host chromosome. Further similarities exist between AAV and
the defective bacteriophage P2 (Six and Klug, 1973; Calender
et al., 1981). Both exist as latent proviruses which
reguire coinfection with a specific helper virus in order to
replicate. One hallmark of the bacterial viruses is an
ability to modulate the expression of their genes. Indeed
the study of the "genetic switch" (Ptashne, 1986) between
the lytic and lysogenic states of lambda infection has
proved to be one of the most informative models of the
control of gene expression. We began these studies in the
hope that AAV might similarly provide a useful model of
eukaryotic gene regulation.


CHAPTER II
METHODOLOGY
Cells and Culture Conditions
Human KB and HeLa monolayer and monkey cos-7 cell
cultures were maintained in Dulbecco's modified Eagles
medium (DMEM) containing 10% fetal calf serum (FCS), 2mM
glutamine, and 500 units/ml of penicillin and streptomycin.
Mouse B78H1 and Ltk- monolayer cultures were maintained in
either minimal essential media (MEM) or DMEM containing 5%
FCS, 5% calf serum, 2mM glutamine, 500 units/ml of
penicillin and streptomycin, 2.5 /g/ml Amphotericin B
(Fungizone, Gibco Laboratories), and 0.1 mg/ml Gentamycin
(Gibco Laboratories). In some experiments cells were
treated with 10 /g of actinomycin D (Sigma)/ml of media for
times indicated in the text.
Viral Stocks
Vj '
AAV was prepared from lysates of HeLa cells coinfected
with both Ad-2 and AAV-2. Ad-2 was inactivated by heating
of the lysates at 56 for 30 minutes. Ad-2 stocks were also
prepared from lysates of infected HeLa cells.
Preparation of Plasmid DNAs
All plasmid DNAs were propagated in Escherichia coli
HB101 cells. After inoculation of fresh cultures with 1/50
volume of an overnight culture, plasmid containing cells
21


22
were grown to a density of 80 Klett units (green filter,
Klett-Summerson colorimeter) in Luria broth (1% tryptone,
0.5% yeast extract, 0.5% NaCl, and 0.5% glucose).
Chloramphenicol was then added to a final concentration of
170 /g/ml and the cultures were grown for an additional 16-
20 hours. Plasmid DNAs were isolated as described by
Birnboim and Doly (1979). Form I plasmid DNA was further
purified by centrifugation in cesium-ethidium bromide
eguilibrium gradients as described by Maniatis et al.
(1982).
Enzymes and Linkers
Restriction enzymes, T4 DNA ligase, T4 DNA polymerase,
T4 polynucleotide kinase, Escherichia coli DNA polymerase I
and Klenow fragment were purchased from either New England
Biolabs, Bethesda Research Laboratories, or International
Biotechnologies Inc. and used as specified by the
manufacturer. Linkers were purchased from either Pharmacia
or New England Biolabs and were phosphorylated as specified
by the manufacturer.
Construction of Mutant AAV Genomes
Plasmid constructions are summarized in chapter III
(table 3-1). Form I pSM620 (Samulski et al., 1982) was
digested with various enzymes (Apal. Ncol, or Xhol) and
religated to yield various deletion mutants. Certain
deletion mutants were constructed by ligating previously
described plamsids, which had been digested with Bglll and


23
EcoRV. Mutants containing deletions of the TRs (ori~
mutants) were created by insertion of the large Smal or Ball
fragment of particular genomes into the EcoRV site of pBR322
(Bolivar et al., 1977), pAT153 (Twigg and Sherratt, 1980),
or pFB69 (Barany, 1985). The construction of pLB1701 and
derivatives is described in chapter VI.
All plasmid constructions were placed into and
propagated in HB101. Initially, small amounts of plasmid
DNAs were isolated for structural analysis by the method of
Holmes and Quigley (1981) or Birnboim and Doly (1979).
Plasmid constructs were analyzed by digestion with
appropriate restriction endonucleases and electrophoresis in
agarose and acrylamide gels.
Radiolabelinq of DNA
Radiolabeled DNAs to be used for probe were produced
either by nick translation (Rigby et al. 1977) or by the
random primer technique (Feinberg and Vogelstein, 1983) and
were used interchangeably. Unless stated otherwise in the
text, duplex AAV-virion DNA was labeled as probe. Nick
translation reactions contained 1 ig of DNA, 50 mM Tris-HCl
(pH 7.8), 5 mM MgCl, 10 mM 2-mercaptoethanol, 0.1 mM each of
dGTP, dATP, and TTP, 100 tCi of a32P-dCTP, 1 ng DNAase I
(Sigma), and 5 units of DNA polymerase I in a final reaction
volume of 50 ¡j.1 Unincorporated deoxyribonucleotides were
removed by chromatography on Sephadex G-75 (Pharmacia Fine
Chemicals) in 10 mM Tris-HCl (pH 8.0). The specific


24
activity of probe obtained by this procedure was between 2
to 5 x 107 counts per minute (cpm)/Vg DNA. Random primer
reactions consisted of 0.1 ^g of denatured DNA (boiled for 7
minutes), 0.2 M Hepes (pH 6.6), 0.15 optical density units
of random hexadeoxyribonucleotides (P-L No. 2166), 50 mM
Tris-HCl (pH 8.0), 50 mM MgCl, 10 mM 2-mercaptoethanol, 100
/iM each of dATP, dGTP, and TTP, 100 Ci of a32P-dCTP (ICN) ,
0.2 /g/ml bovine serum albumin (BSA) and 5 units Klenow
fragment. Reactions were incubated at room temperature for
2 hours and stopped by the addition of sodium dodecyl
sulfate (SDS) and ethelene diaminetetraacetic acid (EDTA) to
0.2% and 50 mM, respectively. At this point reactions had
incorporated between 90 to 99% of the label and probes were
used without further purification.
Diethvlaminoethvl (DEAE)-Dextran DNA Transfections
The DEAE-dextran transfection procedure was carried out
as dsecribed by McCutchan and Pagano (1968) with some
modifications. Either 100 mm or 150 mm dishes of
approximately 80% confluent KB cells were washed once with
phosphate buffered saline (PBS) and a DNA-dextran mixture
was added and left on the cells at room temperature for 30
minutes. DNA-dextran consisted of variable amounts of each
plasmid as indicated and 500 /g of DEAE-dextran (MW =1 x 103
kd, Pharmacia Fine Chemicals) per ml in MEM without serum.
Plates of cells, either 100 mm or 150 mm diameter, received
either 1 ml or 2.5 ml of the DNA-dextran mixture


25
respectively. The transfection mixture was then replaced
with fresh media containing 50 ¡M chloroquine diphosphate.
Experiments involving infection with Ad-2 contained Ad-2 at
a multiplicity of infection (moi) of 10 in the chloroquine
containing media. The cells were incubated for 3 hours at
37C and refed with fresh media.
Calcium Phosphate DNA Transfections
Calcium phosphate coprecipitation transfections were
carried out as described by Graham and van der Eb (1973) and
as modified by Wigler et al. (1979). For stable
transformation and genetic selection, plates were seeded the
night before transfection at densities of 1.25 x 105 cells
or 3 x 105 cells per dish of B78H1 or Ltk cells,
respectively, while 30-50% confluent plates were used for
transient expression assays. Precipitates were formed by
combining the following in the following order: DNA (plasmid
and carrier DNA for a total of 25 ¡xq of DNA) and H20 to
0.250 ml, 0.250 ml 0.5 M CaCl, and 0.5 ml of 2 x Hepes (42
mM Hepes, 0.27 M NaCl, 10 mM KC1, 1.4 mM Na2HP04 (2H20) pH
7.05). The precipitates were allowed to form at room
temperature for 20 minutes and then added directly to the
media of 100 mm plates of cells. For stable transformation,
the media and precipitate were replaced with fresh media the
following day and again with selective media (see below) the
next day. For transient expression assays, the precipitate
and media were removed at 4 to 6 hours after transfection


26
and replaced with 5 ml of 12% glycerol in PBS, incubated for
1 minute, and finally replaced with 10 ml of fresh media
containing 4 mM sodium butyrate as described by Gorman et
al. (1983).
Genetic Selection of Mammalian Cells
G-418 resistant (G-418r) cells were selected in media
containing 1 mg/ml G-418 Sulfate (Geneticin, Gibco) as
described by Southern and Berg (1982). Thymidine kinase
(tk) positive cells were selected in hypoxanthine-
aminopterin-thymidine (HAT) media as described by Wigler et
al. (1979) and consisted of 15 /g/ml hypoxanthine, 1 ng/ml
aminopterin, and 5 /g/ml thymidine in DMEM. Colonies were
grown for 10-18 days in selective media, after which the
plates were fixed with 10% formaldehyde and stained with
cresyl violet.
Chloramphenicol Acetvltransferase (cat) Assays
Assays for cat activity were carried out essentially as
described by Gorman et al. (1982). Plates of transfected
cells were washed three times with cold PBS, incubated under
1 ml of STE (40mM Tris-HCL (pH7.4), 0.15 M NaCl, 1 mM EDTA)
for 5 minute at room temperature. The cells were harvested
by scraping, pelleted by centrifugation, and resuspended in
110 /I of 0.25 M Tris-HCl (pH 7.8) and frozen and thawed
three times. Cell debris was removed by centrifugation and
the supernatant stored at -20C. Half of the supernatants
were used in the assays which consisted of 55 /j1 extract, 75


27
/il 1 M Tris-HCl (pH7.8), 25 n 1 Acetyl Coenzyme A (4 mg/ml,
made fresh each time), and 0.1-0.25 iC 14C-chloramphenicol
(New England Nuclear Inc.). The reactions were incubated at
37C for 1 hour and extracted with 1 ml of ethyl acetate.
The organic layer was isolated and dried in a Savant speed
vacuum centrifuge for 30 minutes. The desiccated samples
were resuspended in 25 ^1 of ethyl acetate and spotted on
thin layer chromatography (TLC) plates (Kodak silica or
Bakerflex silica plates), and eluted in a TLC chamber
equilibrated with chloroform and methanol (95:5). Cat
activity was determined by autoradiography of the TLC plates
followed by scintillation counting of spots cut from the
plates.
RNA Extraction and Analysis
At 33 to 36 hours post transfection, cells were
harvested by scraping, washed with cold PBS, and swelled on
ice in reticulocyte swelling buffer (RSB, 10 mM Tris-HCl (pH
7.4), 10 mM NaCl, 5 mM MgCl and 2 mM DTT) for 10 minutes.
The cells were lysed with 12 strokes of a Dounce homogenizer
and the nuclei removed by centrifugation. RNA was then
extracted from the cytoplasmic homogenate by a modification
of the procedure described by Lusby and Berns (1982).
Briefly the cytoplasmic extract was added to 4 ml of ETS (10
mM EDTA, 0.,2% SDS, and 10 mM Tris-HCl (pH 7.4), and 5 ml of
phenol heated to 7 0C, and placed in a 70C water bath for
15 minutes with occasional shaking. After quick chilling on


28
ice, the aqueous phase was isolated, reextracted with 4 ml
of phenol/chloroform (1:1) and the RNA precipitated with
ethanol.
For some experiments total cellular RNA was isolated
using the guanidinium/CsCl method as described by Maniatis
et al. (1982). Cell pellets were lysed by addition of 2.5
ml of a solution containing 4 M guanidinium isothiocyanate,
5 mM sodium citrate (pH7.0), 0.1 M /3-mercaptoethanol and
0.5% sarkosyl. One-gram of CsCl was then added and the
solution layered onto a 1.2 ml cushion of 5.7 M CsCl
containing 0.1 M EDTA (pH7.5) in Beckman SW 50.1 tubes
followed by centrifugation at 35,000 rpm for 12-16 hours at
20C. The supernatants were discarded and the pellets
resuspended in 400 1 of a solution containing 10 mM Tris-
HC1 (pH74), 5 mM EDTA, and 1% SDS. This solution was then
extracted twice with chloroform and 1-butanol (4:1) and,
after addition of 0.1 volume of 3 M sodium acetate (pH 4.8),
precipitated with ethanol. Concentrations were determined
by a2 60 an<^ by intensity of ribosomal RNA bands after
shadowing with UV light on cellulose TLC (Analtech, catalog
number 08011). RNA samples were prepared and fractionated
in 1.1% agarose gels containing 2.2 M formaldehyde as
described by Maniatis et al. (1982) using vertical slab gels
run for 5 to 8 hours at 100 volts. RNAs were transferred
onto nitrocellulose filters in 20 x SSC without further


29
treatment of the gels. The filters were then handled as
described below.
DNA Isolation
Low molecular weight DNAs were isolated by a
modification of the method of Hirt (1967). Cell monolayers
were washed in cold PBS and harvested by scraping in 1 ml of
Hirt buffer (0.6% SDS and 50 mM EDTA, pH7.4). Sodium
chloride was added to 1 M after incubation for 20 minutes at
room temperature and the samples incubated overnight at 4C.
The samples were then centrifuged for 45 minutes in an
Eppendorf centrifuge, the supernatants were collected,
extracted 2-3 times with an equal volume of
phenol:chloroform (1:1), and the nucleic acid precipitated
with ethanol. In some experiments this procedure was used
to isolate DNA directly from isolated nuclei.
Genomic DNAs were isolated by a modification of a
procedure reported by Cheung et al. (1980). Nuclei isolated
as described above were resuspended in 1 ml of 1 x SSC and
pronase and SDS were added to concentrations of 1 mg/ml and
0.1%, respectively, followed by incubation overnight at
37C. The solution was then gently extracted twice with an
equal volume of phenol:chloroform (1:1) and precipitated
with 2.5 volumes of ethanol. Nucleic acid was then gently
resuspended in 10 mM Tris-HCl (pH7.4) and incubated with 1
mg of RNase A (Sigma) for 60 minutes at 37C, followed by
one extraction with phenol:chloroform and ethanol
extraction


30
precipitation. DNAs were concentrations were determined by
A260
Southern Blotting and Hybridization of Filters
DNA gels (nondenaturing) were prepared and blotted to
nitrocellulose as described by Southern (1975). After
transfer, the filters were baked at 80C for two hours,
placed in a heat sealable bag with 15 ml of prehybridization
buffer (50% formamide, 5 x SSC, 0.5% SDS, 0.1% PVP, 0.1%
BSA, 0.1% Ficoll, 0.15 mg/ml sheared calf thymus (CT) DNA)
and incubated at 42C for 4 hours, and the buffer replaced
with fresh buffer containing denatured DNA probe and CT DNA,
and then incubated at 42C for 12 to 16 hours. The filters
were then washed twice for 15 minutes in 2 x SSC and 0.5%
SDS at 68C, twice for 60 minutes in 0.1 x SSC and 0.1% SDS
at 68C and once at room temperature for 20 minutes in 0.1 x
SSC and 0.5% SDS. Filters were dried and placed against
film for autoradiography.
Nuclear Runoff Transcription (NRT) Assays
Cells were harvested at 24 hours postinfection and
nuclei were obtained as described above except that DTT was
added to 2 mM to the RSB and KC1 added to 140 mM immediately
after homogenization. Nuclei were isolated by centrifugation
and prepared and analyzed as described (Falk-Pedersen et
al., 1985). Nuclei from 3 x 107 cells were resuspended 1 ml
NRT buffer (20 mM hepes (pH 7.9), 20% glycerol, 140 mM KCl,
10 mM MgCl, 2 mM DTT), repelleted at 2,000 times gravity (G)


31
for 4 minutes, and resuspended in 200 /I of transcription
cocktail (60 /I 32P-UTP (200 /iC) 1 /I of a solution
containing 1 mM each of GTP, ATP, and CTP, 2 mM DTT, 8 0 1
NRT buffer), incubated on ice for 3 minutes then incubated
at 30C for 15 minutes. Reactions were terminated by adding
1 ml of HSB (0.01M Tris-HCl (pH 7.4) 0.5 M NaCl, 0.05 M
MgCl, 2 mM CaCl) and 0.1 mg DNAase I and then incubated at
room temperature for 1 minute followed by the addition of 4
ml of ETS and 5 ml of 70C phenol. Labeled RNA was isolated
as described for cytoplasmic RNA. RNA was further purified
by precipitation in 2 M LiCl at 0C overnight. The amount
of incorporated label was determined by chromatography on
PEI cellulose TLC using 1 M HC1. Equal amounts of labeled
RNA for each experiment (approximately lxlO7 or 5xl07 acid
precipitable cpm for KB cells and HeLa cells, respectively)
were used as probes of southern blots containing restriction
digested AAV-2 and Ad-2 DNAs. Blots were hybridized and
washed as described above except that an additional wash was
carried out in 0.1 x SSC, 0.5% SDS, 10 mM NaP04, 1% Triton
X-100, and 1% Tween-20 for 1 hour at 68C.
Immunizations and Antibody Analysis
Synthetic peptides were prepared by Dr. Ben Dunn as
described by Dunn et al. (1983). Peptides contained 3H-
amino acids so all steps could be quantitated. Peptides
were coupled to either BSA or keyhole limpet hemocyanin
(KLH) with glutaraldehyde as described by Kagan and Glick


32
(1979). Glutaraldehyde (1 ml, 2 0 mM) was added dropwise to
a 2 ml solution containing 10-20 mg of BSA or KLH and a 30
molar excess of peptide in 0.1 M sodium phosphate (pH 7.5)
and incubated at room temperature with stirring for 2 hours.
Conjugated peptide was purified by chromatography on
Sephadex G-25 followed by dialysis against PBS for 12-16
hours with several changes of buffer. Conjugates contained
an approximate carrier to peptide molar ratio of 1:15 to
1:30.
Two-month-old New Zealand white rabbits were immunized
with conjugates containing a total of 300 g of peptide
emulsified in 2 ml of Freund's complete adjuvant by
intradermal injections at multiple sites across the back.
Animals were boosted with subcutaneous injections containing
200 nq of peptide/conjugate in 2 ml of Freund's incomplete
adjuvant at approximate 4 week intervals. Antisera were
drawn approximately 7 days after injections. Antisera were
further purified by chromatography on peptide columns
created by coupling of peptide to CnBr-activated Sepharose
4B prepared as specified by the manufacturer (Pharmacia Fine
Chemicals).
The presence of antipeptide antibody was determined by
hemagglutination assays using peptide coupled to sheep red
blood cells (SRBCs). SRBCs (0.2 ml, pelleted) were washed
three times with 2 ml of 0.1 M NaCl, pelleted by
centrifugation at 1000 x G for 5 minutes each time and then


33
resuspended in cold PBS (pH 8.2) containing 1%
glutaraldehyde. The cells were rotated gently at 4C for 30
minutes, washed 5 times in cold 0.15M NaCl, 5 times in cold
water, diluted to 2.5% in PBS (pH 7.2) and mixed with an
equal volume of a 1:60,000 solution of tannic acid in PBS
(pH 7.2) The cells were gently rotated at 4C for 20
minutes, washed twice with PBS (pH 7.2), resuspended as a
2.5% suspension in PBS (pH 6.4), incubated with 400 /ig of
peptide at 37C for 2 hours, washed twice with 1% normal
rabbit serum in PBS (pH 7.2), resuspended in the same
solution as a 2.5% suspension and frozen by emulsion in
liquid nitrogen in small aliquots and stored at -70C.
Hemagglutination assays were carried out as described.
All sera tested were preincubated at 37C to inactivate
complement. Reactions contained 25 1 of 0.5% coupled SRBCs
(diluted in 1% agammaglobulin horse sera) and 50 ul of
diluted rabbit antisera. Reactions were incubated at room
temperature for 4 hours in 96 well round bottom microtiter
plates.
Metabolic Labeling and Immunoprecipitation Analysis
KB cells (approximately 1 x 106 cells) were infected
with either Ad-2 (moi of 10) or coinfected with Ad-2 and
AAV-2 (moi of 20) washed twice with methionine-free or
phosphate free MEM, and then incubated for 1 hour in 0.5 ml
of the same media containing 2% dialyzed FCS and either 250
juC 35S-methionine or 500 fiCi 32P-phosphate. Labeled cells


34
were then washed twice in cold PBS on the plates and lysed
by scraping in 2.5 ml PLB (phospholysis buffer; lOmM sodium
phosphate (pH7.5) 100 mM NaCl, 1% Triton X-100, 0.5% sodium
desoxycholate, 0.1% SDS) containing 1 mM PMSF (Sigma). Cell
debris were removed by centrifugation at 1000 xG for 10
minutes and the lysates cleared by centrifugation at 100,000
x G for 1 hour at 4C. Samples were then diluted two fold
with PLB containing 10 mg/ml BSA.
Equal amounts of labeled proteins (approximately 5 x
107 cpm acid precipitable counts) were incubated overnight
on ice with 5 ^1 of antibody. Protein-A sepharose (Pharmacia
Fine Chemicals) was preincubated in PLB and BSA overnight at
4C with rocking, added (100 1 of a 10% mixture) and
reactions rocked gently at 4C for 45 minutes. Bound immune
complexes were then pelleted and washed 8 to 12 times in PLB
with BSA and twice in PLB, resuspended in SDS sample buffer
and boiled to elute antibody and antigen.
Samples were then fractionated on discontinuous
polyacrylamide gels as described by Laemmli (1970). Gels
consisted of a 4% stacking gel and 10% running gel and
samples were electrophoresed for 4 to 6 hours at 125 volts.
Samples were run next to prestained protein MW markers
(Bethesda Research Laboratories) Proteins were then fixed
in 50% methanol, 10% acetic acid and 1% commasie brilliant
blue for 20 minutes followed by destaining overnight in 10%
acetic acid. Gels were photographed, rinsed with water for


35
30 minutes and treated with 1 M sodium salicylate for 45
minutes (for 35S-labeled proteins only) before autoradiography.


CHAPTER III
AUTOREGULATION OF AAV GENE EXPRESSION
Introduction
As described in chapter I, all aspects of AAV
macromolecular synthesis are regulated by various helper
functions. However, these studies also show a great
plasticity in the requirement for specific helper virus
genes. Although AAV gene expression is positively regulated
by the Ela, Elb, E2a, and VA genes, none of these genes
appears absolutely essential for AAV gene expression. The
recent observation that AAV can replicate in certain cells
in the absence of helper functions supports the idea that no
helper virus genes are directly involved in or essential for
AAV gene expression. Thus the regulation of AAV gene
expression may largely depend on both cellular and AAV
encoded functions whose activities may be modulated by
helper virus gene products.
In this study, the regulation of the AAV genome has
been investigated. Mutant genomes created In vitro have
been studied in order to define the genetic elements
involved in the autoregulation of AAV gene expression. The
results show that autoregulation occurs on at least two
levels; first, a product encoded by the AAV rep gene is
essential for activation of AAV transcription, and second,
36


37
elements within the coding regions for the rep gene products
negatively regulate expression of the rep genes.
Results
Structure of mutant AAV genomes. The structures of
mutant AAV genomes created during this study are summarized
in table 3-1. Many of the cloned genomes were derived from
previously described mutants (Hermonat et al., 1984). The
name of the plasmid genome indicates the type of mutation.
Frameshift insertion mutations are indicated by ins followed
by the position of the insertion, indicated in mu. The
insertions consist of 8 base synthetic oligonucleotides
containing a Bglll restriction site. Deletion mutations are
indicated by dl followed by the location of the deletions.
Mutants containing nonrepairable deletions of both TRs are
designated as ori~. Certain mutants contain a tandem
duplication of AAV DNA and are indicated by dp and position
of the duplicated region. Other mutants contain an
insertion of the SV40 early promoter and are indicated by
/SV following the initial mutant description.
Accumulation of AAV RNAs by two types of rep mutants.
To determine whether an AAV nonstructural protein encoded by
the p5 or pl9 genes regulates AAV gene expression, the
accumulation of RNAs by two types of replication-defective
AAV genomes was compared. The first types contain 8 base
frameshift insertions at either 11 or 32 mu and thus are
defective for the production of the trans-acting p5 and pl9


38
TABLE 3-1
CONSTRUCTION OF PLASMID GENOMES
Plasmid
Description
dl03-ll (pLB1415o)
dlll-23 (pLB1415b)
dl23-37 (pLB14 06)
dl63-86 (pLBlOl)
dl63-80 (pLB102)
dl48-52 (pLB202)
d_115-86 (pLB306)
dl80-96 (pLB314)
inv!5-86 (pLB315)
pAAV/0-11 (pLB710)
pAAV/11-42 (pLB709)
pAAV/42-91 (pLB705)
pAAV/91-100 (pLB702)
620ori~
insllori" (pLB1207)
ins32ori~
ins32ori~b
ins42ori~ (pLB3411)
Ligation of BglII/EcoRVa digested
dl03-23 and insll
Ligation of Bglll/EcoRV digested
dl03-23 and insll
Ligation of Bglll/EcoRV digested
dl23-28 and dllO-37
ApaI deletion of pSM620
Apal deletion of pSM620
Xhol deletion of pSM620
Ncol deletion of pSM620
Ncol deletion of pSM620
Inversion of Ncol A of pSM620
pSM620 PstI C into PstI site of pBR322
pSM620 PstI B into PstI site of pBR322
pSM620 PstI A into PstI site of pBR322
pSM620 PstI D into PstI site of pBR322
pSM620 Smal A into EcoRV site ofpBR322
insll Smal A into EcoRV site of pBR322
ins32 Smal A into EcoRV site of pBR322
ins32 Ball A into EcoRV site of pBR322
ins42 Smal A into EcoRV site of pBR322


39
TABLE 3-1 (continued)
Plamid
Description
ins78ori~ (pLB2402)
ins78 Smal A into EcoRV site of pFB69
dl03-23ori (pLB1302)
dl03-23 Smal A into the EcoRV site of
pBR322
dll0-37ori~ (pLB1102)
dllO-37 Smal A into the EcoRV site of
pBR322
dl63-86ori~ (pLB801)
Apal deletion of 620ori
dl58-87ori (pLB2904)
dl58-87 Smal A into the EcoRV
site of pAT153
dp28-49 (pLBp40A)
Ligation of Bqlll/EcoRV digested
dl49-94 and d!23-28
dp37-58 (pLBp40B)
Ligation of Bglll/EcoRV digested
dl58-87 and dllO-37
dl03-23/SV (pLB605)
SV40 early promoter into Bglll
digested d!03-23
dl03-05/SV (pLB1701)
Insertion of Aval/BstEII of pSM620
into Bglll/BstEII digested dl03-23/SV
dl03-05/SVins32(pLB3901)
Ligation of SacI/BstEII digested ins32
and dl03-05/SV
dl03-05/SVori (pLB1801)
dl03-05/SV Smal A fragment into the
EcoRV digested pBR322
dl03-ll/SV (pLB4104)
Ligation of Bglll/BstEII digested
dl03-23/SV and insll
dl03-20/SV (pLB4004)
Ligation of Bglll/EcoRV digested
dl03-23/SV and BclI/EcoRV digested
pSM62 0


40
encoded rep gene products. The second type is represented
by a mutant containing deletions of both TRs, 620ori~. The
replication of 620ori~ is blocked by cis-active defects of
the origins of DNA replication. This plasmid was used as a
control rather than the parental pSM620 plasmid so that the
effect(s) of mutations within the rep genes can be directly
analyzed. Because mutations within the rep genes abolish
AAV DNA replication, the use of an ori~ control eliminates
possible differences due to increased copy number of
replicating genomes or by differing conformations of
replicating and nonreplicating transcription templates. The
RNAs accumulated by the AAV genomes were analyzed after
transfection into Ad2-infected KB cells. Total cytoplasmic
RNAs were isolated from the transfected cells at 30 hr post
transfection. The RNAs wer fractionated by formaldehyde gel
electrophoresis and analyzed by blotting and hybridization
to radiolabeled AAV DNA as shown in figure 3-1.
The 620ori~ mutant (lane 1) produced readily detectable
levels of all the AAV transcripts except for the spliced p5
RNA. This RNA is normally present in relatively low
abundance and is difficult to resolve from the unspliced pl9
RNA. The RNAs produced by 620ori~ were in the same
stoichiometry as that produced by wild type AAV (Green and
Roeder, 1980a; Laughlin et al., 1979b), indicating that DNA
replication was not required for qualitatively normal
expression of the AAV genes. The most abundant RNA


41
accumulated was the spliced 2.3 kb p40 transcript. The
larger p5 and pl9 RNAs were accumulated in significantly
lower amounts.
Although the nonreplicating 620ori~ genome produced
readily detectable levels of transcripts, the nonreplicating
ins mutants did not (lanes 2 and 3). Both insertion mutants
appeared equally defective for the production of RNAs. The
subtle nature of the insertion mutations suggests that a
product encoded within rep genes is required for efficient
expression of all the AAV genes.
Similar results were obtained when total cellular RNAs
were analyzed as shown in figure 3-2. The amounts of
transcripts accumulated by the rep mutants insll and ins32
were significantly lower than by 620ori~. again indicating a
critical role for the p5 gene product in gene expression.
The amount of p40 RNA detected was higher for insll than for
ins32 in this experiment, although this result was not
always reproducible. Low amounts of p5 RNA from both
insertion mutants were also detected.
The accumulation of RNAs by a deletion mutant, d!03-23.
was also analyzed in this experiment. D103-23 accumulated
an intermediate level of p40 RNA, higher than by either rep
insertion mutant but significantly lower than by 620ori~.
The overproduction of the p4 0 RNA by left side deletion
mutants was reproducible and is discussed below.


42
1 2 3
Figure 3-1. Accumulation of RNAs by two types of
rep mutants. KB cells were transfected with 20 nq of either
620ori~ (lane 1) ins32 (lane 2) or insll (lane 3) and
infected with Ad-2. At 30 hours postinfection, cytoplasmic
RNAs were isolated, and 2 0 ng of RNA from each experiment
were fractionated on a formaldehyde-agarose gel and
subsequently analyzed by blotting and hybridization to 32P-
labeled AAV virion DNA. The sizes of the RNAs are as shown
in figure 1 and indicated here are the 4.2 kb unspliced p5
RNA, the 3.6 kb unspliced (top of caret) and 3.3 kb spliced
(bottom of caret) pl9 RNAs, and the 2.3-kb spliced p40 RNA.
The 2.6-kb unspliced p40 RNA is seen directly above the 2.3
kb RNA.


43
4.2 -
3.6-
*
12 3 4
Figure 3-2. Accumulation of RNAs by mutant AAV
genomes. KB cells were transfected with either insll (lane
1) ins32 (lane 2) 620ori~ (lane 3) or dl03-23 (lane 4)
and infected with Ad-2 as described for figure 3-1. 20 g
of total cellular RNA from each experiment were analyzed as
described for figure 3-1. The sizes of the unspliced p5
(4.2 kb), pl9 (3.6 kb), and spliced p40 RNAs (2.3 kb) are
indicated on the right.


Accumulation of AAV RNAs by rep- ori- double mutants.
The AAV genomes used in the experiments described above
contained either of two types of mutations, both of which
blocked DNA replication. The rep mutants (ins and dl)
contained trans-complementable defects in the AAV rep gene,
while the defective replication of 620ori~ was
noncomplementable due to deletion mutations within the TRs
(which act as origins for AAV DNA replication) Thus the
difference in the accumulation of transcripts by the mutants
might be explained either by the loss of a positive
activator of expression in the rep mutants or by the removal
of a cis-active repressor of expression within the TRs of
620ori~.
To distinguish between these possibilities, we analyzed
the accumulation of transcripts by mutant AAV genomes
containing similar deletions of the TRs. The RNAs produced
by the genomes upon transfection into Ad2-infected cells are
shown in figure 3-3. Both rep insertion mutants (insllori-
and ins32ori ) produced
greatly
reduced
amounts
of
transcripts compared
with
620ori-
(lane
1)
Thus
the
defective expression
of the ins mutants
was
due to
the
lesions in the rep gene.
An ori- capsid deletion mutant, dl63-86ori~ accumulated
normal amounts and proportions of its deleted transcripts
(lane 4) This indicates that AAV capsid proteins have no
role in the accumulation of AAV RNAs.


45
Although most mutations within the rep gene abolish AAV
DNA replication, a mutant containing an insertion at 42 mu
is partially defective, replicating at about the 0.1% level
of wild type genomes (Hermonat et al., 1984). To determine
if this mutant is also defective in the accumulation of
RNAs, an ori~ version of ins42 (lane 6) was constructed and
analyzed. This mutant genome produced intermediate levels
of RNAs compared to ins32ori~ (lane 7) and 620ori~ (lane 5).
While the mutation at 42 mu reduces DNA replication by 100-
1000 fold (Hermonat et al. 1984) the accumulation of RNAs
was only inhibited by approximately 10 fold. This may
suggest that different regions of the AAV rep proteins are
required for DNA replication and regulation of gene
expression.
The predominant RNA accumulated by ins42ori~ was the
spliced p40 message, demonstrating that the mutation did not
severely affect the splicing efficiency of AAV messages.
Thus the defects caused by mutation at 42 mu do not appear
to be caused by defects in RNA splicing but by lesions
within the coding region contained in the AAV intron,
supporting the suggestion that products encoded by the
unspliced p5 and pl9 transcripts are essential for AAV
replication (Hermonat et al., 1984).
The results presented above indicate that a product
encoded within the AAV p5 and pl9 genes is required for the
efficient accumulation of AAV RNAs in helper virus infected


46
cells. In order to determine if this positive autoregulation
by the rep gene product occurred in the absence of helper
virus, the RNAs accumulated by cloned genomes upon
transfection into uninfected KB cells were analyzed. As
shown in figure 3-3 equally low levels of the 2.3 kb p40
transcript were accumulated by 620ori~ (lane 8) and
ins32ori~(lane 9) in the absence of Ad while only 620ori~
produced high levels of transcripts in the presence of Ad.
Thus efficient activation of expression only appeared to
occur in Ad infected cells.
We also compared the accumulation of RNAs by ori~
genomes which have deletions on the left side of the AAV
genome. A blot comparing the RNAs produced by dl03-23ori~.
dll0-37ori~. 620ori~. and ins32ori~ is shown in figure 3-4.
As expected, genomes containing mutant rep genes accumulated
much lower amounts of transcripts than 620ori~. However,
dl03-23ori~ produced higher levels of the 2.3 kb p40 message
than by the other rep mutants. Thus both ori~ and ori+
versions of dl03-23 show enhanced accumulation of the p40
RNA suggesting that upstream sequences negatively regulate
transcription from p40. D110-37ori~ did not overproduce the
p40 RNA suggesting that deletion of sequences between 03-10
mu was required for the enhanced accumulation of the p4 0
RNA.


Figure 3-3. Accumulation of RNAs by ori~ AAV genomes. Ad-2-infected
KB cells were transfected with 20 g of either 620ori~ (lanes 1 and 5),
insllori- (lane 2) ins32ori~ (lanes 3 and 7) d!63-86ori~ (lane 4) or
ins42ori~ (lane 6) and uninfected KB cells were transfected with either
620ori~ (lane 8) or ins32ori~ (lane 9) A 20 g amount of RNA from each
transfection was analyzed as described in the legend to figure 3-1. The
position of RNAs indicated are of wild type size and are the same as shown
in figure 3-1. The sizes of the transcripts of d!63-86ori~ are
approximately 1.1 kb smaller than the wild type transcripts.


P5-
p19<
p40~
P5-
p19<
p40"
5 6 7 8 9
CO


49
12 3 4
41
i* 4

p40l
Figure 3-4. Accumulation of RNAs by rep deletion
mutant genomes. Ad-2 infected KB cells were transfected
with 20 /g of 620ori~ (lane 1) ins32ori~ (lane 2) dllO-
37ori~ (lane 3), or dl03-23ori~ (lane 4). A 20 g amount of
RNA from each transfection was analyzed as described in the
legend to figure 3-1. Only the position of the spliced 2.3
kb p40 RNA is indicated.


50
Transcription of AAV genomes in isolated nuclei. The
data presented above showed that mutants with defective p5
and pl9 ORFs were defective in the accumulation of AAV
transcripts. In order to determine if the defect was at the
transcriptional or post-transcriptional level, the rates of
transcription from transfected AAV genomes were measured in
isolated nuclei (figure 3-5). Nuclei from Ad2-infected HeLa
cells transfected with either 620ori~ (panel A) ins32ori~
(panel B) or mock transfected (panel C) were isolated and
incubated to allow runoff elongation of preinitiated
transcription complexes.
Radiolabeled transcripts were isolated and equal
amounts hybridized to Southern blots containing 620ori~ DNA
which had been digested with PstI and Hindlll (lane 1) .
This digestion produces four fragments. Fragment a (3.8 kb)
contains pBR322 sequences in addition to 0.4 kb of AAV DNA
(91-98 mu). Fragments b and c contain only AAV DNA from 42-
91 mu and 11-40 mu, respectively. A fourth fragment, d,
contains AAV DNA from 2-11 mu as well as a small amount of
pBR322 DNA. Each blot also contained PstI digests of cloned
Ad5 DNA, either pXHOC (Ela and Elb sequences, lane 2) or
pXBAC (E4 sequences, lane 3) to confirm that equivalent
amounts of labeled nuclear RNA were used in each experiment.
Although equal amounts of labeled RNAs were used in
these experiments, the level of Ad transcription as
indicated by hybridization signal in panel B (ins32ori)


51
appears to be approximately two-fold higher than in panel A
(620ori). That this represents a consequence of 620ori~
expression seems unlikely since only a fraction of cells can
be expected to receive AAV DNA after transfection while
virtually all cells are expected to be infected with Ad.
However, an inhibition of Ad transcription by AAV is
documented below.
Although the level of Ad transcription detected was
higher upon transfection with ins32ori~. hybridization
signal to the AAV-specific fragments was observed only when
labeled nuclear RNA from 620ori~ transfected cells was used.
No AAV-specific transcription from ins32ori~ was detected in
isolated nuclei in this experiment. Thus, expression of the
rep insertion mutant was defective at the level of
transcription initiation.
Complementation of rep mutant gene expression. The
experiments described above suggest that an AAV gene
product(s) encoded in the p5 gene is required for activation
of AAV transcription. If so, the defect should be
complementable in trans. To test this, cotransfection
experiments were carried out using two rep- ori~ double
mutants, ins32ori~ and dll0-37ori~. Ori~ plasmids were used
so that any induction of expression would be assayed for in
the absence of an increase in potential template number due
to DNA replication. The plasmids were transfected into Ad-2
infected KB cells or cotransfected with a capsid deletion


Figure 3-5. Transcription of AAV genomes in isolated nuclei. HeLa
cells were transfected with 620ori~ (panel A), ins32ori~ (panel B), or only
carrier DNA (panel C) as described in chapter II using the calcium phosphate
precipitation method and subsequently infected with Ad-2. Nuclei were
isolated at 24 hours postinfection and transcription reactions were carried
out. Egual amounts of labeled nuclear RNAs were then hybridized to Southern
blots of AAV and adenovirus DNAs. Lane 1 contains PstI- and Hindlll-
digested 620ori~ DNA, which consists of four fragments: a 3.6 kb fragment
which consists of pBR322 DNA and AAV DNA from 91-98 mu (a), a 2.4 kb
fragment which consists of AAV DNA from 42-91 mu (b) a 1.35 kb fragment
which consists of AAV DNA from 11-40 mu (c), and a 0.6 kb fragment (d) which
consists of AAV DNA from 2-11 mu and 0.2 kb of pBR322. Lanes 2 and 3
contain Pstl-digested Ad-5 Ela and Elb DNA from pXHOC and E4 DNA from pXBAC,
respectively.


53
" |
U 04 W 9


54
mutant, dl49-94. Total cytoplasmic RNAs were analyzed by
Northern blotting and hybridization to a labeled fragment of
AAV DNA (52-91 mu) so that the complementing genomes
transcripts were not visualized. As shown in figure 3-6, no
transcripts were detected upon transfection of either
ins32ori~ (lane 2), dll0-37ori~ (lane 5) or, as expected due
to the specificity of the probe, upon transfection of the
capsid deletion mutant (lane 6) Cotransfection of the
capsid deletion mutant with ins32ori~ resulted in the
production of detectable levels of a 2.3 kb RNA, predicted
for the
p4 0
message
of the
rep
insertion
mutant. As
expected,
the
larger
RNAs of
the
insertion
mutant were
present in much lower amounts than the p40 message.
A 2.3 kb transcript was also detected in a
cotransfection experiment using dll0-37ori~ (lane 4) In
this case, a second transcript slightly slower in migration
was also detected in approximately equal amounts to that of
the 2.3 kb RNA. This extra band appeared to represent a p5
transcript since dll0-37ori~ contains a p5 promoter which
would produce a highly truncated spliced transcript of
approximately 2.6 kb. The p5 transcript is normally
accumulated at levels of 5-10% of the p40 transcript. Thus,
the p5 RNA of dll0-37ori~ appeared to be overproduced.
One explanation for these data was that expression from
the p5 promoter is artificially high due to transcription
off a nonreplicating template. Alternatively, expression


55
may have been enhanced by the fortuitous construction of a
positive regulatory element (e.g. an enhancer) during the
cloning procedures. The identification of the 2.6 kb
species as a p5 transcript in the above experiment is also
difficult due to the low amount of transcripts produced by
complementation of the nonreplicating genomes. Therefore
several experiments were done using various ori+ rep
deletion mutants.
In the first of these experiments the production of
transcripts by left side deletion mutants upon
complementation with capsid deletion mutants was examined.
Capsid deletion mutants dl58-87 and dl63-86 (figure 3-7,
lanes 1 and 2) produced readily detectable amounts of their
deleted transcripts while the rep deletion mutants did not
(lanes 3 and 6) Cotransfection of dl03-23 with d!63-86
resulted in the accumulation of large amounts of 2.3 kb p40
transcripts (lane 4) which must have been produced from
dl03-23 since the capsid mutant does not produce a 2.3 kb
message. Further, the lack of wild type size p5 and pl9
messages indicated that the 2.3 kb message did not arise
from a wild type genome generated by recombination.
13110-37 produced high levels of 2.3 kb p40 RNA when
complemented by dl58-87, as well as an equally abundant 2.6
kb RNA. Because dllO-37 contains only a p5 promoter in
addition to the p40 promoter contained within dl03-23, this
2.6 kb RNA is most likely a p5 transcript. The 2.6 kb RNA


56
also hybridizes to a p5 specific probe (see below). Thus
the p5 RNA was overproduced by replicating and
nonreplicating genomes.
One explanation for the increased accumulation of the
deleted p5 transcript is the juxtaposition of p5 to
regulatory elements of p40. In order to determine if
overproduction of p5 RNA required sequences within p40, a
deletion mutant containing only the p5 promoter, d!15-86.
was constructed and analyzed. RNAs accumulated by this
genome upon transfection with and without pSM620 (to supply
p5 and pl9 rep gene products) are also shown in Figure 3-7.
A high level of the deleted p5 transcript was detected upon
complementation. The structure of the deleted RNA also
eliminates the possibility that it was produced by a
recombinant genome. This RNA was produced in approximately
equal amounts with the pSM620 p40 transcript and in
approximately 16 fold excess with the pSM620 p5 transcript
(as determined by counting the radioactive bands cut out
from the nitrocellulose blot). Thus, overproduction of the
p5 message was independent of regulatory sequences within
the p40 promoter. Further, overproduction of the p5 message
is not unique to a single mutation, indicating that
expression was not enhanced by the inadvertent construction
of a positive regulatory element.
These complementation experiments clearly demonstrate
positive trans-activation by rep gene products, although we


57
cannot rule out the possibility that the high level of
transcript accumulation in these experiments may in part be
a dosage effect due to DNA replication and concomitant
increases in potential transcription templates.
The overproduction of p5 RNA by genomes containing
deletions between 15 and 37 mu suggests the existence of a
negative regulatory element. Since the presence of this
putative negative regulatory sequence in wild type genomes
does not appear to eliminate expression from p5, but rather
reduces its accumulation the putative regulatory element
will be referred to as a depressor (dep) of expression and
the mutant phenotype as a dep- phenotype. Because
complementation of dep- mutants did not affect the ratio of
transcripts produced by the complementing genome, the dep
mutations must be cis-active.
In order to more precisely map the putative dep
sequence, additional deletion mutants were constructed and
analyzed as shown in figure 3-8. In this experiment several
left side deletion mutants were cotransfected with a capsid
deletion mutant, d_163-86. No AAV RNA was detected after
transfection of the left side mutants without
complementation (data not shown). Complementation of d!23-
28 resulted in the production of the expected rep mutant
RNAs. The p5 and pl9 RNAs of dl23-28 were produced at
normally low levels compared with the 2.3 kb p40 RNA. Thus
dep does not appear to reside between 23-28 mu. Similarly


58
Figure 3-6. Complementation of expression of rep
mutants. KB cells were transfected with 20 /xg of either
620ori~ (lane 1), ins32ori~ (lane 2), ins32ori~ and dl49-94
(lane 3) dll0-37ori~ and dl49-94 (lane 4) dll0-37ori~
(lane 5), or dl49-94 (lane 6) and infected with Ad-2. RNA
was prepared and analyzed as described in figure 3-1 except
that the blot was probed with a 32P-labeled fragment of AAV
DNA from 52-91 mu. The position of RNAs indicated on the
left are the wild type spliced p40 RNA (2.3 kb) and on the
right the unique p5 transcript of dll0-37ori~ seen in lane 4
(2.6 kb).


59
\
Figure 3-7. Complementation of expression by left side
deletion mutants. KB cells were transfected with 20 g of
either d!58-87 (lane 1), d!63-86 (lane 2), dl03-23 (lane 3),
dl03-23 and dl63-86 (lane 4), dllO-37 and dl58-87 (lane 5),
dllO-37 (lane 6), pSM620 (lane 7), dll5-86 and pSM620 (lane
8), or dll5-86 (lane 9) and subsequently infected with Ad-2.
The locations of specific RNAs originating from capsid
deletion mutants are indicated by a superscript c. Only the
spliced p40 and unspliced pl9 and p5 RNAs are indicated.
The bottom of the caret points to the dl58-87 RNAs, and the
top of the caret points to the dl63-86 RNAs. The truncated
RNAs of the rep deletion mutants are indicated with the
superscript r.


60
complementation of either dl03-ll and dlll-23 also resulted
in production of rep mutant RNAs with wild-type
stoichiometry.
Although both dllO-37 and dlll-23 are missing the pl9
promoter, only dllO-37 overproduced a p5 RNA (lane 5). Thus
the dep phenotype is not simply a reflection of reduced
competition due to the deleted promoter, as seen for the
SV40 early promoter (Kadesch and Berg, 1986). The 2.6 kb
RNA produced upon complementation of dllO-37 was shown
clearly to be a p5 transcript by its hybridization to a p5
specific probe (figure 3-8B, lane 5).
Because neither dll0-23 nor dl23-28 overproduced any
RNAs, the dep seguence can be predicted to reside between
28-37 mu. Indeed, dl23-37 exhibited a unique phenotype upon
complementation. The truncated p5 transcript of dl23-37 was
produced at low levels compared to the 2.3 kb p40 transcript
and was predominantly unspliced (based on the size of the
RNA) However, complementation of the mutant resulted in
the accumulation of an RNA equal in abundance and slightly
larger in size than 2.3 kb. This RNA was a size predicted
for the spliced pl9 message of dl23-37. Thus d!23-37
appears to accumulate excess amount of the pl9 RNA but
normal amounts of the p5 RNA.
The results of the complementation experiments are
summarized in figure 3-9. Deletion of sequences between 23-
37 mu was sufficient for overproduction of pl9 RNA but


61
insufficient for overproduction of p5 RNA. Thus a
functional dep sequence for pl9 appears to reside between
23-37 mu. Further because deletion from 23-28 mu had no
effect on RNA levels, one dep region must reside between 28-
37 mu. However the functional dep sequence for p5 appears
to also require an element between 10-23 mu. These
conclusions are discussed below.
Another point worth noting is that although the p5 and
pl9 messages are normally accumulated in predominantly
unspliced form, the p5 RNA or pl9 RNAs overproduced by dllO-
37 or dl23-37, respectively, were a size predicted for
spliced messages. Thus the dep mutations appeared to
simultaneously increase both the inherent splicing
efficiency of an RNA as well as the abundance of that RNA.
Negative regulation of p40 expression by the dep
region. The expression of deletion mutants suggests that
the p5 and pl9 genes are controlled by negative regulatory
elements, one of which appears to exist between 28-37 mu of
the AAV genome. To determine if dep is a separable genetic
element, hybrid AAV genomes were created which placed a dep
region downstream from a p4 0 promoter. This was done
because expression from p40 appeared unaffected by the dep
mutations. Genomes which contain a tandem duplication of
sequences including p40 and which either include (dp28-49)
or do not include (dp>37-58) the putative dep region were
created and analyzed. The structures of the genomes are


Figure 3-8. Accumulation of RNAs by left side deletion mutants. A)
Ad-2 infected KB cells were transfected with 20 g of either dl63-86 alone
(lane 1), or cotransfected with dl23-28 (lane 2), dl03-ll (lane 3), dlll-23
(lane 4), dllO-37 (lane 5), or dl23-37 (lane 6). RNA from each transfection
was analyzed as in figure 3-1. Lane M contains PstI digested pSM620 DNA as
markers. The spliced p40 RNA of the capsid deletion mutant is indicated on
the left side of the figure with a superscript c. The wild-type-size
spliced p40 message produced by all left side deletion mutants is indicated
on the right side of the figure (p40r) The p5 and pl9 RNAs of left side
deletion mutants are indicated by filled boxes or open circles,
respectively. B) Same as in A except that the blot was probed with a 32P-
PvuII-Hindlll fragment from pLB709 containing AAV DNA from 0-11 mu. The
size of most abundant RNA (lane 5) is indicated on the right.


A
M 1 2 3 4 5 6
B
1 23456
.
c 0
< MWH

" p40r
*
*
-2.6
-p40c
uM.


Figure 3-9. Phenotypes of left side deletion mutants. The AAV genome
is drawn schematically, with the position of the AAV promoters indicated by
the upside down L. The slash marks represent a large portion of the capsid
gene left out for simplicity. The structures of left side deletion mutants
are drawn below and next to the mutant designation. The relative levels of
transcripts observed for each mutant are shown on the right as high (H), low
(L) or none (N) The ratings are based on comparison to that of the
complementer; levels approximately equal to that of the spliced p40 message
or the p5/pl9 messages are considered high or low, respectively.


p5
JL
11 -23.
23-28
10-37.
23-37.
15-86
pl9
-'w
Wr
-ov-
/UV
-Aa/-
p5 pl9 p40
L N H
L L H
H N H
L H H
H N N
o\
ui


66
diagramed in figure 3-10. Each genome contains an upstream
and downstream p40 promoter (p40u and p40c*, respectively) .
The structure of the p40^ transcription unit is identical
for both genomes thus the levels of this transcript (wild
type 2.3 kb in size) can be used for comparison with that of
the upstream p40. However, the upstream p40 promoters
differ, being placed upstream from different but overlapping
duplicated portions of the genome. Only in the case of
dp>28-49 does the duplicated region contain the dep
sequences.
As shown in figure 3-11, both genomes produced
approximately equal amounts of the downstream p40 message.
However dp28-49 accumulated significantly lower amounts of
the p40u transcript compared with its own p40d transcript or
with that of dp37-58. The ratio of the p40u to the p40^
transcripts of dp28-49 was qualitatively similar to the
p5/pl9 to p40 ratio of transcripts accumulated by wild type
AAV indicating that the dep element inhibited the
accumulation of the p40 transcript when placed within the
transcriptional unit.
As noted in the figure 3-10, several different sized
messages could be produced from the p40u promoter of either
of the two genomes depending on the number of splicing
events in a particular message. Interestingly, the
predominant p40u message produced by dp28-49 was a 3.5 kb
unspliced message while the predominant message produced by


67
dp37-58 appeared to be the 2.9 kb doubly spliced message.
Thus the inclusion of the dep sequence in dp28-49 also
appeared to inhibit splicing of the message. These data
again support the notion that dep functions both to affect
the steady state levels and splicing efficiency of
particular messages.
Stability of AAV mRNAs. Several mechanisms could explain
the action of the dep region. One possibility includes the
specific destabilization of the RNA. If true this would
predict that the wild type p5/pl9 and p4 0 messages would
have very different half lives. In order to examine the
stability of the AAV RNAs, AAV infected cells were treated
with actinomycin D (to block transcription) for various
times, and the RNAs analyzed by northern blotting.
As shown in figure 3-12, most of the AAV RNAs remained
constant in level up to 4 hours after addition of
actinomycin D. Although the 2.6 kb p40 message decreased
rapidly in abundance, the levels of the p5 and pl9 RNAs
remained relatively constant. These data suggest that only
the 2.6 kb message is unstable. Alternatively, the
disappearance of the 2.6 kb RNA may reflect a continued
processing of the message to the spliced 2.3 kb species
after the addition of actinomycin D, although this seems
unlikely since the levels of unspliced p5 and 19 RNAs
remained constant.


Figure 3-10. Structures of AAV genomes with duplications of p40. The
organization of the wild type genome (WT) dp28-49 and dp37-58 are drawn
schematically. The position of promoters, introns (carets), and the TRs
(black boxes) are indicated. The p40 promoters of the dp mutant genomes are
indicated with a superscripts u (upstream) or d (downstream). The
duplicated portion of the dp genomes are indicated by open rectangles below
the genome representation, separated by lines indicating the 5' boundary of
the duplicated region. The stippled area indicates the position of the dep
region present in the duplicated portions of dp28-49. The structure and
sizes of the p40 RNAs predicted for the genomes are also drawn below each
genome.


69
\ \ 'S
n
*1 o
u
o

CL
I
*> 10 m m
I I
"O
O.


70
1-23
Figure 3-11. Expression of AAV genomes containing
duplications of p40. Ad-2 infected KB cells were infected
with AAV (lane 1), mock transfected (-), or transfected with
dp28-49 (lane 2) or dp37-58 (lane 3) The wild type size
messages are indicated on the right and RNAs originating
from the upstream p4 0 promoters (2.9 and 3.5 kb) and the
downstream p40 message (2.3 kb), are indicated on the left.


71
Figure 3-12. Stability of AAV RNAs. KB cells were
coinfected with Ad-2 and AAV-2. Actinomycin-D was added to
separate plates at 24 hours postinfection. Cytoplasmic RNA
was then isolated at 0 (lane 1) 30 (lane 2) 60 (lane 3) ,
or 240 (lane 4) minutes after addition of the drug. The
positions of specific RNAs are indicated on the left side of
the figure.


72
Transcription of AAV in isolated nuclei. Another
mechanism by which the dep region might act would be through
negative regulation of transcription initiation. This
predicts that the p40 promoter and downstream sequences
would be preferentially transcribed. Transcription rates of
different regions of the AAV genomes were measured by NRT
analysis using nuclei from Ad-2 or Ad-2 and AAV-2 infected
cells, followed by hybridization to restriction digested AAV
DNA blotted to nitrocellulose. The cloned AAV DNAs used are
identical to that described for figure 3-5 and thus
recognize either p5 specific transcription (fragment d), p5
and pl9 transcription (fragment c) or transcription from
all AAV transcription units (fragments a and b).
In the first experiment infected KB cells were used
(figure 3-13). No AAV transcription was detected from Ad-2
infected cells while a large amount of hybridization signal
to AAV DNA was detected using RNA transcribed in nuclei from
coinfected cells (panel B, lane 3). Fragment b contains AAV
DNA from 42-91 mu and should hybridize to all complete AAV
transcripts. Fragment c contains AAV DNA from 11-40 mu and
should be specific for the p5 and pl9 messages.
Although the steady state levels of p40 RNA (which consists
of mostly fragment b sequences) are 10-20 fold higher than
that of the p5 and pl9 RNAs, more transcriptional activity
was detected with the p5 and pl9 specific probe (fragment c)
than that detected with probe complementary to all AAV RNAs.


73
Thus the rate of initiation from the p5 and or pl9 promoters
appears to be at least as high as that from the p40
promoter.
A high level of transcription from the p5 promoter is
specifically indicated by comparison of hybridization signal
to fragments a and d. Both fragments contain approximately
0.2 kb of complementarity to AAV RNAs either at the 3' end
of all AAV transcripts in fragment a or to the 5* end of the
p5 transcript in fragment d. An equal amount of signal was
detected with either fragment indicating that the rate of
transcription for the p5 promoter is very high and is not
proportional to the amount of p5 RNA relative to p40 RNA.
Similar results were obtained using nuclei from
infected HeLa cells (figure 3-14). Transcription rates of
different regions of the AAV genomes were measured by NRT
analysis using nuclei from Ad-2 or Ad-2 and AAV-2 infected
cells followed by hybridization to restriction digested AAV
DNA blotted to nitrocellulose. The cloned AAV DNAs used are
the same as in the previous figure. Transcription was
measured at both 12 (A and B) and 24 hours (C and D)
postinfection. Although the signal detected with fragment b
was somewhat higher than for fragment c in this experiment,
the transcription rate per nucleotide of probe was
approximately equal. In this particular experiment the
fragment d was run off the gel.


74
Thus, in experiments using either KB or HeLa cells,
transcription
from
the
p5
and
pl9
sequences appears
approximately
equal
to
that
of
p4 0
specific sequences.
These data indicate
that the
overall
ratios of AAV RNA
accumulated must be determined in part by mechanisms after
transcription initiation. Further because the rates of
transcription of the left half of AAV (11-40 mu) appeared at
least as highe as of the right half of the genome (42-91
mu), it is suggested that the p5 and pl9 messages are being
prematurely terminated.
Although transcription rates have not yet been measured
for dep mutants, the accumulation of RNAs by these mutants
is proportional to the observed transcription rates of the
wild type genome. It is proposed that the dep sequences act
posttranscriptionally and are responsible for the observed
difference between transcription rates and the steady state
levels of RNA p5 and pl9 RNAs.
Another observation made in the last two experiments is
that the amount of Ad transcription detected was
significantly lower in cells coinfected with AAV. Ad
transcription detected using probes for either the El, E4,
or late regions was significantly reduced, although the
inhibition of transcription appeared greater in KB cells
(approximately 10 fold) than in HeLa cells (approximately 3
fold). The inhibition observed in HeLa cells was similar at
both early (12 hours) and late (24 hours) times


75
postinfection. Thus, although the AAV rep gene products
activate AAV transcription, AAV gene expression can be
correlated with an inhibition of Ad transcription.
AAV coinfection is known to inhibit Ad replication.
Interestingly, AAV appears to inhibit the production of
progeny Ad to a much greater extent than Ad DNA replication.
Thus the inhibition of Ad transcription may be a major
effector of the inhibition of the helper virus replication.
Whether the inhibition of Ad transcription is a consequence
of a specific AAV gene product or due to competition for
limited transcriptional machinery is not known.
Discussion
In this study cloned AAV genomes have been analyzed in
order to delineate genetic elements involved in the
autoregulation of AAV gene expression. These data
illustrates that autoregulation occurs both by trans-active
AAV proteins and by cis active elements.
Positive regulation in trans by an AAV rep gene
product. Several experiments show that at least one AAV
protein encoded within the p5 gene is essential for trans
activation of AAV transcription. First, cloned genomes
containing small lesions within the ORF unique to the p5 and
pl9 transcripts accumulated extremely low levels of all the
AAV RNAs compared to genomes containing wild type p5 and pl9
genes. Virtually all left side mutations had a similar
effect. The subtle nature of certain mutations (8 base


76
A B
12 3 12 3
Figure 3-13. Transcription of AAV in isolated KB cell
nuclei. KB cells were infected with Ad-2 (panel A) or
coinfected with Ad-2 and AAV-2 (panel B) Nuclei were
isolated at 24 hours postinfection and transcription
reactions were carried out for 10 minutes. Equal amounts of
RNA were then hybridized to southern blots containing Pstl-
digested Ad-5 Ela and Elb DNA from pXHOC (lane 1) E4 DNA
from pXBAC (lane 2) or PstI- and HindiII-digested 620ori~
DNA (lane 3). The AAV fragments indicated are as described
for figure 3-5.


Figure 3-14. Transcription of AAV in isolated HeLa cell nuclei. HeLa
cells were infected with Ad-2 at an moi of 10 (panels A and C) or coinfected
with both Ad-2 and AAV-2 at an moi of 20 (panels B and D) Nuclei were
isolated at either 12 hours (panels A and B) or 24 hours (panels C and D)
posttransfection and labeled nuclear RNAs were used as probe for Southern
blots of restriction enzyme digested Ad-5 and AAV-2 DNAs. Lane 3 contains
PstI- and Hindlll digested 620ori~ DNA as described in the legend to figure
3-5. Lanes 1 and 2 contain PstI-digested Ad-5 DNAs corresponding to the E4
region (pXBAC) or late region, respectively.


8 L


79
frameshift
insertions)
indicates
that the
defects
in
expression
were due to
mutational
effects
on
the
rep
proteins.
Supporting this notion is the
observation
that
the
defect in the accumulation of RNA was complementable in
trans. However the amount of transcripts accumulated by the
rep~/ori~ mutants upon complementation by an ori+ genome was
significantly lower than that of 620ori~. This may reflect
a competition by the ori+ complementing AAV genome for
limited transcriptional machinery as noted by other workers
(see below). Complementation of expression of ori+ rep
mutants was very efficient.
Activation of AAV expression did not require DNA
replication. This was illustrated both by the efficient
expression of 620ori~ and by the ability to complement
expression of ori~ rep mutants. The stoichiometry of the
different messages was also identical to that produced by
replicating molecules. Thus AAV gene expression is not
divisible into simple early and late stages separated by DNA
replication.
Evidence that activation by the rep proteins is at the
level of transcription initiation has also been presented.
When transcription rates of transfected genomes in isolated
cell nuclei were measured, transcription from a rep+ genome
(620pri) was detected, although transcription from a rep~


80
genome (ins32ori_) was not. Thus, the rep mutant was
defective at the level of transcription initiation.
One question that may be addressed is whether
transactivation by rep is specific for AAV genes or is
promiscuous in activity as observed for the Ad Ela (Green et
al., 1983: Treisman et al., 1983; Gaynor et al, 1984; Stein
and Ziff, 1984; Kao et al., 1985) and herpes ICPO and ICP4
proteins (Everett, 1984; O'Hare and Hayward, 1985a,b). The
observation that AAV infection inhibits transcription of the
Ad genome rather than activating it strongly suggests that
transactivation is specific for the AAV genome although this
point requires further investigation.
Because a mutation which specifically blocked
production of p5 gene products (insll) inhibited the
accumulation of AAV RNAs, a role for a p5 gene product(s) in
transactivation of expression is specifically indicated. It
is not yet possible to determine if pl9 encoded proteins are
also required for this process. Although transactivation
appears independent of DNA replication, a p5 gene product is
required for both AAV DNA and RNA synthesis. Whether the
two functions of the rep gene products represent one or
multiple activities is not known.
However the phenotype of ins42 suggests that different
functional domains of the rep gene products might be
involved in RNA and DNA synthesis. Although the mutation at
42 mu inhibits DNA synthesis by 100-1000 fold, only a 10


81
fold reduction in RNA synthesis is seen. Thus the mutation
at 42 mu appears to differentially affect DNA replication
and transcription.
The AAV p5 gene product (s) appears to be functionally
analogous to the SV4 0 T-antigen. Both gene products are
required for viral DNA replication and transactivation of
viral transcription (Hartzell et al., 1984). As discussed
above, the AAV p5 gene product may also be composed of
separate functional domains as is the SV40 T-antigen.
Several workers have recently noticed significant amino acid
homology between the predicted p5 and SV40 T-antigen
proteins in a region conserved between most DNA dependent
ATPases (N. Muzyzka, C. Astell personal communications).
This suggests that the different proteins have similar
enzymatic activities.
It should be noted that all eukaryotic DNA virus
families, including Herpes, Ad, papilloma, and polyoma
viruses, examined in detail appear to encode transcription
activator proteins (Jones and Shenk, 1979; Everett, 1984;
Spalholz et al., 1985). Thus the ability to activate
expression may be as essential a feature to these exogenous
replicons as are origins of replication. This feature is of
obvious importance when one thinks of the competition for
limited transcription machinery between viral genes and the
vast excess of cellular genes.


82
Evidence for trans-active regulation of AAV gene
expression by the rep proteins has also been recently
reported by another laboratory. Tratschin et al. (1986)
have reported that expression of the cat gene, inserted
within the AAV genome under the control of the p40 promoter,
is enhanced by the rep gene. While this study did not
determine at what level activation occurred, our results are
consistent, although the results presented here indicate
that transcription of all AAV promoters is activated by rep
In addition this group identified a negative regulatory
function of the rep gene products on expression of the p40
cat gene. This negative regulation occurred in human 293
cells, which constitutively produce Ad-5 Ela and Elb gene
products, but not in HeLa cells. Although the results
presented in this section do not address this phenomenon, a
negative regulatory activity of rep on heterologous genes is
described in chapter IV.
Rhode (1985) has also reported that expression of the
cat gene under the control of the p3 8 capsid gene promoter
of an autonomous parvovirus, HI, is enhanced by an HI p4
gene product. Again, no work at the RNA level was done by
Rhode. Both Rhode and Tratschin et al. (1986) reported that
expression of genomes was most efficient when the
transactivator genes were carried on the same plasmid
compared to cotransfection of separate plasmids. This was
suggested to represent competition for limited transcription


83
machinery, possibly the parvoviral transactivator proteins
themselves.
Cis active negative regulation of expression.
Although the p5 gene product is associated with positive
regulation of AAV transcription, several experiments
indicate that seguences within the p5 ORF itself act as
negative regulators of p5 and pl9 gene expression. Several
left side deletion mutants accumulated significantly greater
than normal amounts of the p5 or pl9 transcripts upon
complementation with rep+ genomes. Normally the p5
transcript is accumulated at 5-10% the level of the p40
transcripts at late times in Ad infected cells. However,
the dep mutants accumulated equal amounts of either the p5
or pl9 and p40 transcripts. The effect of the dep mutations
were clearly cis-active as the expression of the
complementing genomes were qualitatively unaffected.
The relative increases in p5 or pl9 transcripts
(compared to the levels of p40 transcripts) accumulated by
the dep mutants were not due to reduced expression from p40.
First, the amounts of p40 RNA accumulated by all left side
mutants upon complementation were similar. Second, ori+ dep
mutant genomes produced as much or greater amounts of p40
transcript upon complementation than the complementers
themselves. Thus the dep phenotype was due to enhanced
accumulation of the affected RNAs.


84
A genome lacking all promoters except for p5 (dll5-86),
accumulated a highly truncated p5 message in excess of the
complementor's p5 message. The high level of p5 expression
from this mutant demonstrates that the dep phenotype was not
a result of the close proximity of the p5 and p40 promoters
in the mutants. The unigue structure of the truncated p5
RNA also eliminates the possibility that it was produced
from a recombinant genome of unknown structure.
Various deletion mutants were used to define the region
of the genome responsible for cis-active negative
regulation. The first mutant discovered to overproduce the
p5 transcript was 110-37 whose deletion removed the pl9
promoter. However, deletion of the pl9 promoter was not
sufficient to reproduce the dep phenotype as dlll-23
accumulated normal amounts of p5 transcripts. Thus one
would predict that the negative regulatory sequence would be
contained between 23-37 mu.
One deletion mutant, 123-37, exhibited a unique
pattern of accumulated transcripts. In this case the
truncated p5 transcript was still produced at low levels but
an additional band was detected which was slightly slower in
mobility and approximately equal in amount to the 2.3 kb p40
message. It is assumed that this species is a spliced pl9
RNA based on the predicted size of that message. Thus
removal of 23-37 mu was required but not sufficient for
overproduction of the p5 transcript but sufficient for


85
overaccumulation of the pl9 transcript. In explanation for
these observations it is proposed that two dep elements
exist within the AAV genome, one between 15-2 3 mu and
another between 28-37 mu. However since the presence of the
upstream dep region (between 15-23 mu) in one mutant, d!23-
37, does not appear to inhibit the production the pl9 RNAs,
the negative regulatory element most likely resides upstream
from the promoter (upstream from 19 mu).
The regulatory effect of sequences between 28-37 mu was
not specific to the p5 and pl9 promoters. When the p40
promoter, whose expression was unaffected by the dep
mutations, was placed upstream from the 28-37 mu sequences
the accumulation of the upstream p40 transcript was strongly
inhibited
relative to
a second downstream
p4 0.
The
inhibition
was not due
to the competition
by
the
second
downstream
p40 since
removal of 28-37
mu
from
the
duplication
significantly enhanced the expression
of
the
upstream but not the downstream p40 transcript.
Although the studies presented here have not elucidated
the mechanism by which the putative dep sequences act,
several aspects are noteworthy. First the action of the dep
sequences appear directional, acting on upstream
transcription units. Second, the 28-37 mu sequences can act
on at least one heterologous promoter (p40).
Several mechanisms could explain the observed negative
regulation of p5 and pl9 expression. One explanation is


86
that the putative dep elements specifically destabilize
RNAs. Initial results would indicate that the AAV RNAs do
not differ significantly in cytoplasmic stability; however
the messages may differ in nuclear stabilities.
Alternatively the dep elements might act as binding sites
for repressors of transcription initiation. If so, one
would predict that transcription rates of the wild type p5
and pl9 genes would be much lower than that for the p40
transcript. However, when transcription rates were measured
using different DNA probes, an approximately equal amount of
transcription for all AAV genes was detected.
An additional mechanism might involve premature
termination of transcription. Transcription of SV40 and an
autonomous parvovirus, Minute Parvovirus of Mice, has been
suggested to be regulated by some method of transcription
attenuation (Ben-Asher and Aloni, 1984). This mechanism
appears to be the most consistent with the observations made
here.
One other aspect of regulation by dep appears to be an
inhibition of splicing of the affected message. This
conclusion is based on the observation that in all cases an
increased splicing efficiency cosegregates with the dep
phenotype. The dep sequences also appear to inhibit splicing
of p40 transcripts when placed within a normally
predominantly spliced p40 transcription unit. These
observation suggest that dep exerts its effects in cis. by


87
altering the structure of RNAs. If so, such structural
changes can easily be envisioned to affect either
transcription termination or RNA stability as discussed
above.
Finally we have not addressed the question of which
protein(s) may act to facilitate regulation by dep or why
such regulation even exists. Because dep does not appear to
affect expression of the p40 capsid gene, it is interesting
to speculate that dep is needed to modulate the amount of
rep protein expression at a time after DNA replication has
begun and when large amounts of capsid proteins are needed.
Conversely, expression of the p40 capsid gene appears to be
negatively regulated by rep in 293 cells (Tratschin et al.,
1986). 293 cells only express the Ela and Elb genes of Ad;
resulting in a physiologic state similar to that present
early during Ad infection. Thus, although AAV transcription
cannot yet be divided into early and late phases, such a
distinction may be made at the posttranscriptional level.
Such multiple levels of control are strikingly similar
to the regulation of the SV40 genome. Early in infection
the T-antigen gene is highly expressed (Benoist and Chambn,
1981; Buchman et al., 1984) while expression of the late
region is repressed (Omilli et al., 1986). T-antigen
synthesis leads to both DNA replication and transactivation
of SV40 transcription although the synthesis of early RNA is
kept relatively low compared to late RNAs by repression of


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UNIVERSITY OF FLORIDA
-... "II i ii mi ii mi am mi
3 1262 08554 3881


REGULATION OF GENE EXPRESSION BY
ADENO-ASSOCIATED VIRUS
BY
MARK ARON LABOW
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1987

This dissertation is dedicated to my mother Audrey whose
support has made this possible and whose love has made this
worthwhile.

ACKNOWLEDGMENTS
I would like to thank Dr. Kenneth I. Berns for his
guidance, infinite patience, and love for biology (and
countless jokes and stories) which helped make several years
pass by very quickly.
I would like to thank the faculty of the University of
Florida who have given me a priceless education. In
particular I would like to thank William B. Gurley and
Nicholas Muzyczka for their advice and inspiration over the
years.
I wish to thank my friends Randy Horowitz and Paul L.
Hermanot for their wonderful company, help and advice.
I wish to thank Patricia Burfeind for her excellent
technical assistance and most of all, for her friendship.
Finally, I wish to thank Gwen Tse Wong for her faith,
advice and love.
in

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES VÜ
ABBREVIATIONS USED ix
ABSTRACT XÍ
CHAPTER I INTRODUCTION
The Parvoviruses 1
The Natural Occurrence of AAV 2
Latent Infection by AAV 4
The AAV Helper Functions 5
The Physical and Genetic Organization
of the AAV Genome 10
Molecular Genetics of AAV 16
Perspective 19
CHAPTER II METHODOLOGY
Cells and Culture Conditions 21
Viral Stocks 21
Preparation of Plasmid DNAs 21
Enzymes and Linkers 22
Construction of Mutant AAV Genomes ... 22
Radiolabeling of DNA 23
Diethylaminoethyl (DEAE)-Dextran DNA
Transfections 24
Calcium Phosphate DNA Transfections . . 25
Genetic Selection of Mammalian Cells . . 26
Chloramphenicol Acetyltransíerase
(cat) Assays 26
RNA Extraction and Analysis 27
DNA Isolation 29
Southern Blotting and Hybridization
of Filters 30
Nuclear Runoff Transcription (NRT)
Analysis 30
Immunizations and Antibody Analysis . . 31
Metabolic Labeling and
Immunoprecipitation Analysis .... 33
IV

CHAPTER III
AUTOREGULATION OF AAV GENE EXPRESSION
X Introduction 36
Results 37
Discussion 75
CHAPTER IV INHIBITION OF GENETIC TRANSFORMATION AND
EXPRESSION OF HETEROLOGOUS GENES BY THE
AAV REP GENE
Introduction 89
Results 91
Discussion 105
CHAPTER V IDENTIFICATION OF AAV REP GENE PRODUCTS
Introduction 112
Results 114
Discussion 123
CHAPTER VI CONSTRUCTION AND CHARACTERIZATION OF
HYBRID AAV/SV40 GENOMES
Introduction 126
Results 127
Discussion 137
CHAPTER VII CONCLUSION 146
LITERATURE CITED 147
BIOGRAPHICAL SKETCH
162

LIST OF TABLES
Table Page
3-1. CONSTRUCTION OF PLASMID GENOMES 38
4-1. INHIBITION OF GENETIC TRANSFORMATION BY
AAV GENOMES 100
4-2. INHIBITION OF TRANSFORMATION OF pTK BY
d!52-91/neo 101
4-3. DOSE DEPENDENCE OF AAV AND NEO DNA ON
TRANSFORMATION 103
4-4. EFFECT OF CLONED E4 SEQUENCES ON AAV MEDIATED
INHIBITION OF TRANSFORMATION 106
4-5. INHIBITION OF pSV2cat EXPRESSION BY
COTRANSFECTED AAV DNA 107
5-1. PRODUCTION OF ANTIPEPTIDE ANTISERA 115
vi

LIST OF FIGURES
Figure Page
1-1. Structure and genetic organization of the
AAV genome 15
3-1. Accumulation of RNAs by two types of rep
mutants 42
3-2. Accumulation of RNAs by mutant AAV genomes .... 43
3-3. Accumulation of RNAs by ori~ AAV genomes 48
3-4. Accumulation of RNAs by rep deletion mutant
genomes 49
3-5. Transcription of AAV genomes in isolated nuclei. . 53
3-6. Complementation of expression of rep mutants ... 58
3-7. Complementation of expression by left side
deletion mutants 59
3-8. Accumulation of RNAs by left side deletion
mutants 63
3-9. Phenotypes of left side deletion mutants 65
3-10. Structures of AAV genomes with duplications
of p40 69
3-11. Expression of AAV genomes containing
duplications of p40 70
3-12. Stability of AAV RNAs 71
3-13. Transcription of AAV in isolated KB cell nuclei. . 76
3-14. Transcription of AAV in isolated HeLa cell
nuclei 78
4-1. AAV genomes inhibit genetic transformation .... 95
4-2. Southern blot analysis of G-418r cell clones ... 96
vii

5-1. Immunoprecipitation analysis with antipeptide
antibody 119
5-2. Immunoprecipitation analysis of AAV proteins . . . 120
5-3. Identification of an AAV protein made early
during infection 121
5-4. Identification of phosphorylated capsid
proteins 122
6-1. Construction of AAV/SV40 hybrid genomes 133
6-2. Structure of hybrid AAV/SV40 genomes 135
6-3. Expression of AAV plasmids in human cells .... 136
6-4. Replication of AAV plasmids in various
cell lines 139
6-5. Replication of hybrid genomes in cos-7 cells . . . 141
6-6. Inhibition of DNA replication by the AAV
rep gene 142
viii

ABBREVIATIONS USED
AAV adeno-associated virus
AAV-2 AAV type-2
Ad adenovirus
BPV bovine papilloma virus
BSA bovine serum albumin
C- carboxyl
CT calf thymus
cat choloramphenicol acetyltransferase
cpm counts per minute
DEAE diethylaminoethyl
DMEM Dulbecco1s modified Eagles medium
DNA deoxyribonucleic acid
EDTA ethelene diaminetetraacetic acid
FCS fetal calf serum
G gravity
HAT Hypoxanthine-aminopterin-thymidine
HSV herpes simplex virus
kb kilobase
kd kilodaltons
KLH Keyhole limpet hemocyanin
md monomer duplex
ix

MEM
. minimal essential medium
mMT mouse metallothionein
moi multiplicity of infection
mRNA messenger RNA
mu map units
neo neomycin phosphotransferase
NRT nuclear runoff transcription
ORF open reading frame
ori origin
PBS phosphate buffered saline
RNA ribonucleic acid
RSB reticulocyte swelling buffer
SDS sodium dodecyl sulfate
SRBCs sheep red blood cells
ss single stranded
SV40 Simian Virus 40
T-antigen tumor-antigen
TLC thin layer chromatography
TRs Terminal Repeats
tk thymidine kinase
x

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
REGULATION OF GENE EXPRESSION BY
ADENO-ASSOCIATED VIRUS
By
Mark Aron Labow
May, 1987
Chairman: Kenneth I. Berns
Major Department: Immunology and Medical Microbiology
The ability of adeno-associated virus (AAV) to affect
gene expression from both its own genome and heterologous
genes has been examined using several different assays. The
expression of both wild type and mutant AAV genomes was
examined in human cells. The effects of the AAV genome on
heterologous genes were examined in stable transformation
and transient gene expression assays. The properties of
hybrid AAV/simian virus 40 (SV40) genomes were also
examined.
Mutations of the open reading frame within the AAV p5
and pl9 RNAs greatly inhibited both the accumulation of AAV
RNAs and AAV transcription in adenovirus infected human
cells. This defect in gene expression was complemented in
trans indicating that a gene product encoded within the p5
gene activates AAV transcription. Proteins encoded by the
xi

p5 and pl9 genes were also identified using antisera to
synthetic peptides
Deletion of certain sequences within the left side of
the AAV genome enhanced the accumulation of the p5 or pl9
RNAs. The effect of such deletions occurred only in cis.
Analysis of transcription from the wild type AAV genome
suggests that the cis-active negative regulation occurs
after transcription initiation.
The AAV genome was also shown to inhibit the activity
of heterologous genes. Cotransfection of AAV genomes
inhibited both the transformation by several different
dominant selectable genes and transient expression by
pSV2cat. Both of the inhibitory activities were shown to be
mediated by the AAV p5 and pl9 genes.
Finally, hybrid AAV genomes containing the SV40 early
promoter and origin of DNA replication were constructed.
The replication of the hybrid genomes, initiated by the SV40
origin, was inhibited by the p5 and pl9 genes in cells which
constitutively produce the SV40 tumor-antigen.
Xll

CHAPTER I
INTRODUCTION
The Parvoviruses
The parvoviruses are among the smallest of the
eukaryotic viruses. The family Parvoviridae consists of
three genera, the densoviruses, the autonomous parvoviruses
and the defective parvoviruses also known as the
dependoviruses (Siegl et al., 1985). The densoviruses
infect several species of insects. The autonomous
parvoviruses infect and cause disease in many vertebrate
species (Siegl, 1984). Although the dependoviruses also
infect many vertebrate species from birds to humans, they
are unique in that they require coinfection with a
genetically distinct helper virus in order to replicate
(Atchison et al., 1965; Melnick et al., 1965; Hoggan et al.,
1966; Blacklow et al., 1967a; Carter and Laughlin, 1984).
The dependoviruses were first discovered as contaminants of
purified Adenovirus (Ad) preparations and are thus referred
to as adeno-associated viruses (AAV). AAV type-2 (AAV-2) is
the most extensively characterized dependovirus and is the
focus of this dissertation.
The AAV virion is typical of all parvoviruses,
consisting of a naked icosohedral shell of approximately 20
1

2
nm in diameter. The capsid is composed of three virion
proteins, VP1, VP2, and VP3, with molecular weights of 87
kilodaltons (kd), 72 kd, and 63 kd, respectively (Salo and
Mayor, 1977; Johnson, 1984). The proteins are
immunologically cross reactive and contain extensive
overlapping amino-acid sequences (Johnson et al., 1971; Rose
et al., 1971; Johnson et al., 1975; Buller and Rose, 1978;
Lubeck et al. , 1979). VP3 comprises approximately 90% of
total virion protein. Subspecies of VP1 and VP3 which differ
slightly in MW have also been reported (McPherson and Rose,
1983). The AAV virion is extremely stable, being resistant
to heat, ionic detergents, and certain proteases.
The capsid surrounds a linear single stranded (ss)
deoxyribonucleic acid (DNA) genome of approximately 5
kilobases (kb) (Mayor et al., 1969; Rose et al., 1969; Berns
and Rose, 1970; see Berns and Hauswirth, 1984, for review).
Although many autonomous parvoviruses preferentially package
the minus strand of DNA, AAV, as well as the autonomous
parvoviruses LuIII and B19, packages DNA molecules of both
polarities with equal frequency (Berns and Adler, 1972;
Majaniemi et al., 1981; Bates et al., 1984; Cotmore and
Tattersall, 1984).
The Natural Occurrence of AAV
The existence of AAV in nature is extremely widespread.
AAVs have been isolated from nearly all animal species which
have been specifically tested. Although natural hosts of

3
AAV appear to include humans, several species of monkeys,
cattle, horses, dogs, quails, and chickens, the broad
species specificity of many AAV prevents the actual
determination of species origin. While AAV-4 is only known
to replicate efficiently in vitro in primary African green
monkey cells, AAV-1, -2 and -3 appear to replicate in cells
from a wide range of species (Hoggan et al., 1966; Blacklow
et al., 1968b; see Cukor et al., 1984, for review). However
these AAV are somewhat species specific, in that they
efficiently replicate only in the presence of a helper virus
of the same species origin as that of the host cell. For
example human AAV-1, -2 and -3 will replicate in canine or
chicken cells as long as canine or chicken Ad are provided
as helper.
Infection by AAV-2 and 3 in humans is very common, as
is infection of captive monkeys by AAV-1, -2, and -3
(Blacklow et al., 1968a,b). A fifth primate AAV, AAV-5, has
been isolated from human genital tissue (Bantel-Shaal and
zur Hausen, 1984; Georg-Fries et al., 1984). An estimated
85% of the population of human adults are sero-positive for
AAV (Blacklow et al., 1967b, 1968a,b). Further, the mean
age of seroconversion for AAV appears to be between the ages
of 7 to 8 years of age. Although infection is very common,
no disease in humans or animals is associated experimentally
or epidemiologically with AAV. This benign nature of AAV

4
infection is consistent with the normal inability of AAV to
replicate in the absence of a helper virus.
Latent Infection by AAV
The defective nature of AAV replication raises the
question of how the virus retains its biological identity in
the absence of helper virus. In this regard, the AAV genome
appears to exist in a latent state as a silent provirus.
The latent state of AAV infection was originally discovered
during a program for screening for persistent viral
infections of primary cell culture lots to be used for
vaccine production. It was discovered that although no cell
lots were positive for AAV structural antigens, infection of
the cells with Ad resulted in the detection of AAV antigen
in 20% of the lots of African green monkey kidney cells and
1-2% of the lots of human embryonic kidney cells (Hoggan et
al., 1972) .
Latent infection with AAV can be established
experimentally. Several studies have shown that infection
of human tissue culture cells with AAV results in a carrier
state from which no AAV virion DNA or capsid antigen is
detected but upon superinfection with Ad, AAV is produced
(Hoggan et al., 1972; Handa et al., 1977; Laughlin et al.,
1986) . The process of establishing latent AAV infection is
extremely efficient. Up to one third of the cell clones
derived after infection produce AAV after superinfection

5
with Ad. The ability to rescue AAV is stable for greater
than 100 cell passages and up to one third of the individual
cells produce AAV antigen after superinfection with a helper
virus. The AAV genome exists in the latently infected
cells as an integrated provirus. Examination of the
integration complexes indicates that the AAV genomes are
often present as head to tail concatamers and that
integration occurs at random sites within the host DNA
(Berns et al., 1975; Cheung et al., 1980; Hermonat, 1984;
Tratschin et al., 1985; Laughlin et al. , 1986). Further,
the AAV genome is integrated colinearly with the terminal
repeats (TRs) at the junctions of integration, suggesting a
role for the TRs in the integration process.
The AAV Helper Functions
Many viruses provide the full helper effect for AAV.
As described above, AAV will replicate in cells of varied
species origins using Ad of that same species. The helper
functions are also provided by herpes simplex virus (HSV) I
and II (Buller et al., 1981). In addition, the replication
of at least one AAV, AAV-5, has been reported to be
supported by vaccinia virus (Schlehofer et al., 1986).
While the plethora of genetically distinct helper viruses
might indicate that only a single common feature of the
helper viruses is needed for AAV replication, studies with
Ad indicate an interplay of many helper virus functions.

6
Studies using mutant Ad indicate that all the helper
functions are provided by Ad early regions. The first
region of importance is the El region. This region contains
two transcriptional units, Ela and Elb. The two genes
comprise the transforming region of Ad (Graham et al.,
1974). The normal functions of the two genes appear
complex. Ela encoded products normally function to activate
transcription of the other Ad early regions (Berk et al.,
1979; Jones and Shenk, 1979). The normal functions of the
Elb gene products are less clear but appear to involve the
ability to selectively transport viral ribonucleic acid
(RNA) to the cytoplasm while blocking the same process for
cellular messages (Babiss and Ginsberg, 1984; Babiss et al.,
1985; Pilder et al. , 1986). Several proteins are produced
from alternatively processed Ela and Elb RNAs. When
studied in human KB culture cells, Ad mutants defective in
the production of either Ela or Elb products were defective
helpers for AAV-2, but provided significant helper effect
when the Ad mutants were used at very high multiplicities
(Ostrove and Berns, 1980; Janick et al., 1981; Laughlin et
al., 1982). However, mutants that were defective for the
production of both Ela and Elb products were completely
defective for helper function at all multiplicities.
The role of the Ela gene products as helper functions
can be explained by the ability of Ela proteins to stimulate
transcription. Transcription of AAV is readily detected in

7
cells transformed by the El region of Ad (Ostrove et al.,
1981). Specific induction of expression from the AAV pl9
promoter by the Ela gene has also been reported (Tratschin
et al., 1984b). The role of the Elb gene is less clear. To
some extent the functions of Ela and Elb appear
interchangeable in that mutants in both El regions are more
defective for AAV helper functions than the individual
mutants. Also microinjection of helper virus genes into
AAV-2 infected Vero cells showed that in addition to the E2a
and E4 genes, helper effect, as measured by AAV capsid
protein synthesis, required both Ela and Elb genes when
injected in low copy number but either Ela or Elb alone when
injected in high copy number (Richardson and Westphal,
1984) .
The E2a gene encodes the 72 kd ssDNA binding protein
(see Kelly, 1984, for review). This protein is required for
Ad DNA replication
both in vivo
and
in vitro.
negative
regulation of E4
transcription
and
regulation
of
the
stability of Ad early messenger
RNA
(mRNA) (Babich
and
Nevins, 1981; Handa et al., 1983). The E2a gene product has
been reported to be involved in both the regulation of AAV
gene expression and AAV DNA replication. One group has
reported that although AAV DNA replication and production of
RNA were relatively normal, the accumulation of AAV
structural proteins and progeny ss-DNA were severely
inhibited when a temperature sensitive E2a mutant was used

8
as helper at the nonpermissive temperature (Myers et al.,
1980; Jay et al., 1981). However, another group reported
that the same mutant was as efficient a helper as wild type
Ad for AAV-2 replication but that the E2a gene product was
responsible for a species specific block in the translation
of AAV-2 capsid proteins, as well as certain late Ad
proteins, in monkey cells (Straus 1976a; McPherson et al.,
1982) .
In addition, one group has reported that efficient
expression of the E2a gene in cos-1 cells supported AAV DNA
replication in the absence of other viral early genes (Quinn
and Kitchingman, 1986). However this study was done only in
cos-1 cells which produce the Simian Virus 40 (SV40) tumor-
antigen (T-antigen) which may contribute to AAV DNA
replication (see below). The production of AAV capsid
proteins is also enhanced by the Ad VA-RNAs (Janick et al.,
1981; Janick et al., 1982).
Finally, the E4 gene appears to be required for AAV DNA
replication (Carter et al., 1983). Microinjection studies
have shown that AAV DNA replication required the El, E2a,
and E4 genes (Richardson and Westphal, 1981). However,
microinjection of E4 RNA alone was sufficient for AAV DNA
replication, indicating that the function of the other Ad
regions might be indirectly needed to induce expression of
the E4 gene. However, the role of E4 as a helper gene has
not been examined in detail. Also, E4 mRNA has been shown

9
to act as a complete helper only in one cell line (Vero
cells). This is an important point in that Ad replication
is affected differently by E4 mutations in different cell
lines (Halbert et al., 1985)
The ability of divergent viruses and genes to support
AAV replication suggests that the helper functions act
indirectly by modulating activities of cellular genes. For
example, the Ela gene has been suggested to stimulate
transcription indirectly by enhancing the activity of a
cellular factor which binds Ad promoters (Feldman et al.,
1982; Kovesdi et al., 1986). Also VAI RNAs enhance
translation in Ad infected cells indirectly by preventing
the cell from inactivating a translation initiation factor
(Thimmappaya et al., 1982; Kitajewski et al., 1986). If all
the helper functions act indirectly, then one prediction is
that there would be a certain cell type or physiologic state
which would support AAV growth in the absence of helper
virus.
Several groups have reported helper virus independent
replication of AAV. Schlehofer (1983, 1986) originally
reported that certain human and Chinese hamster cell lines
transformed with SV40 become permissive for AAV-5
replication upon addition of a potent carcinogen or
cyclohexamide. Similar results were obtained for AAV-2.
Yakobson et al. (in press) have shown that several cell
lines will replicate AAV, supporting the production of

10
infectious progeny, when pretreated with potent cell
synchronizing agents. Interestingly, the cells most
efficient at replicating AAV were Chinese hamster cell lines
transformed with SV40. The SV40 genome appeared to be a
contributing factor for AAV replication since monkey CV1
cells did not support AAV replication although cos-1 cells
(SV40-transformed CV-1 cells) supported AAV replication.
However a Chinese hamster cell line that did not contain the
SV40 genome also replicated AAV although at a much reduced
level.
It should be noted that under optimal conditions,
replication without helper virus was at least two logs lower
than that in the presence of helper virus. Further, only a
fraction of treated cells were able to replicate AAV in the
absence of helper virus. Thus AAV replication appears to
require a complex cell milieu which can be modulated by an
array of viral genes and physiologic states.
The Physical and Genetic Organization of the AAV Genome
The AAV genome has been extensively characterized and
completely sequenced (Lusby and Berns, 1982; Srivastava et
al., 1983). One outstanding feature of AAV DNA is the
presence of inverted TRs. The TRs are 14 5 bases long and
contain several internal palindromes (Gerry et al., 1973;
Kozcot et al., 1973; Fife et al., 1977; Lusby et al., 1980).
Thus AAV DNA can form several different secondary structures
including ss-circles held together with 145 base duplex

11
"panhandles" (Berns and Kelly 1974). The termini can also
fold upon themselves to form intrastrand T-shaped
structures. The TRs appear to act as the origin (ori) of
AAV DNA replication, apparently acting as primers for DNA
synthesis (Straus et al., 1976b; Hauswirth and Berns, 1977,
1979) . Further the AAV TRs also contain a nicking site
allowing both the replication of 5' terminal sequences and
the resolution of replication intermediates. Thus AAV DNA
is replicated in a manner analogous to the model proposed by
Cavalier-Smith (1974) for the replication of linear
chromosomal DNA. The TRs may also be important in the
process of integration into and rescue out of foreign DNA by
the AAV genome.
Sequence analysis of the internal portion of the AAV
genome has revealed several large open reading frames (ORFs)
as illustrated in figure 1-1. The genome can be divided
into two domains, with long ORFs on either side. The two
largest ORFs are the major left and right side ORFs from 6-
47 map units (mu) (bases 320-2186) and from 60-92 mu (bases
2810-4321), respectively. The right side ORF predicts a
protein of size and composition corresponding to the
smallest capsid protein, VP3. Due to the overlapping nature
of the capsid proteins amino-acid sequences, the larger
virion proteins must also contain the body of this reading
frame.

12
All of the ORFs are present in different forms within
the AAV mRNAs. The known AAV RNAs have been characterized
from Ad-infected cells. The genome appears to encode three
sets of transcripts (see Carter et al., 1984, for review).
The RNAs are all overlapping, polyadenylated, and have
coterminal 3' ends at mp 96 (Laughlin et al., 1979b; Green
and Roeder, 1980a,b; Green et al., 1980; Lusby and Berns,
1982; Srivastava et al., 1983). However, three different
51 ends have been located at 5, 19, and 40 mu, giving rise to
unspliced RNAs of 4.2, 3.6, and 2.6 kb respectively. An
intron located between 40 and 48 mu has also been identified
and spliced RNAs of 3.9, 3.3, and 2.3 kb have been
detected.
The RNA start sites are each located downstream from
putative RNA polymerase II promoters, p5, pl9, and p4 0 as
indicated by nucleotide sequence. The promoters contain
elements typical of eukaryotic RNA polymerase II promoters
including TATA boxes located approximately 30 bases upstream
of the RNA cap sites as well as CAAT boxes located
approximately 80 bases upstream from the pl9 and p40
transcripts. The sequences including and surrounding the
TATA boxes of the p5 and pl9 promoters are highly related to
that surrounding the Ela gene of Ad-5 (Lusby and Berns,
1982). Transcription of the AAV promoters is also
sensitive to low concentrations of a-amanitin as expected

13
for RNA polymerase II genes (Bloom and Rose, 1978; Jay et
al., 1978).
The p4 0 RNAs are predominantly spliced and comprise
about 90% of the total AAV RNA accumulated in cells
coinfected with AAV and Ad. The larger p5 and pl9 RNAs are
accumulated in approximately egual amounts and exist as
predominantly unspliced molecules. All of the AAV RNAs are
found in both the nucleus and cytoplasm.
Several experiments show that the p40 messages encode
the AAV structural proteins. First, the capsid proteins are
still produced by mutant AAV genomes lacking the p5 and pl9
promoters (Janik et al., 1984; Hermonat et al. , 1984;
Tratschin et al., 1984a). Second, all three of the
structural proteins are translated in vitro from isolated
2.3 kb size AAV RNA (Jay et al., 1981). This observation is
paradoxical in that the sequence and structure of the
spliced p40 message could only account for the production of
the smallest capsid protein VP3. VP2 appears to be
synthesized using additional reading frame encoded upstream
from and in the same reading frame as the major left side
ORF, initiating translation at an upstream ACG codon
(Becerra et al., 1985). The mechanism by which the largest
structural protein, VP1, is synthesized is unclear but has
been suggested to involve the use of alternatively spliced
mRNAs (Hermonat et al., 1984). Several additional ORFs of

Figure 1-1. Structure and genetic organization of the AAV genome. The
4.7 kb AAV genome is shown schematically divided into 100 mu. The 145-base
TRs are represented as small black boxes. The structure of the known AAV
mRNAs are shown and are aligned with the genome representation. The
locations of the three AAV transcription promoters, p5, pl9, and p40, are
indicated at the left (5'end) of the RNAs produced from them. The 3'
polyadenylation site for all AAV RNAs is indicated by wavy lines at the
right (31 end) of the RNAs. Spliced RNAs are drawn with gaps to indicate
the removed intron. ORFs present within the messages are depicted by black
rectangles within the RNA. The sizes of the transcripts (not including the
polyadenylation tail) are shown on the right side of the figure. The
boundaries of the known separable genetic regions of the AAV genome are
shown at the bottom of the figure.

15

16
unknown function overlap the carboxyl (C)-terminal regions
of both large ORFs.
Molecular Genetics of AAV
The defectiveness of AAV has suggested that all
functions required for AAV replication are provided by both
the host cell and the helper virus and that AAV simply
encodes its capsid proteins. However the existence of the
large left side ORF indicated that AAV might encode
nonstructural proteins which might be required for AAV
replication. This notion was supported by the observation
that AAV defective interfering particles, whose genomes
contain large heterogeneous internal deletions, required
coinfection with wild type AAV in order to replicate
(Laughlin et al., 1979a). However the identification of
specific AAV replication functions was hampered by the
requirement for a cytopathic helper virus; thus AAV could
not be plaqued nor could AAV mutants be selected.
This problem was circumvented by cloning the AAV genome
into bacterial plasmid vectors. The AAV genome was
originally cloned by GC-tailing of reannealed virion DNA and
insertion into pBR322 (Samulski et al., 1982). Clones
containing the intact AAV genome are biologically active
(Samulski et al., 1982; Laughlin et al. , 1983). Upon
transfection into helper virus infected human tissue culture
cells, the cloned AAV genomes are rescued from the plasmid
sequences and AAV replication ensues. The process of rescue

17
and replication from the plasmid vectors is highly
efficient, approaching the yields of AAV DNA and virus
achieved by transfection of wild type virion DNA.
The cloning of the AAV genome in an infectious form
provides the ability to modify the genome in vitro and to
propagate large amounts of the mutant genomes in bacteria.
The effects of such modifications are then assayed by
transfection of the mutant genomes into Ad-infected culture
cells. This procedure was originally used to examine the
effects of deletions within the TRs on AAV DNA replication
(Samulski et al., 1983; Senapathy et al., 1984; Lefebvre et
al., 1984). Mutants which contained large deletions of both
TRs sequences failed to replicate their DNA. Replication of
these mutants could not be complemented in trans. confirming
the predicted role of the TRs as origins of AAV DNA
replication. Unexpectedly, mutants which contained only one
deleted TR were viable, producing AAV genomes with wild type
TR on both sides of the genome. Thus there appears to be
some mechanism for repairing deleted terminal DNA sequences.
A detailed genetic analysis of the internal AAV
sequences has been done in a similar manner. Deletion and
frameshift mutations have been created in cloned AAV
genomes. Deletion mutations were created by digestion of
cloned AAV genomes with various restriction endonucleases
(Hermonat et al., 1984; Tratschin et al., 1984a).
Frameshift insertion mutations have also been created by

18
insertion of synthetic nucleotide linkers at many places
throughout the genome. Analysis of the different mutants
revealed at least three distinct phenotypes and have again
divided the genome into two basic domains.
Mutations between 11 and 42 mu severely inhibit the
replication of AAV DNA. The defective replication of the
mutants, referred to as rep mutants, is complemented in
trans by cotransfected AAV genomes. Thus the ORFs contained
uniquely within the p5 and pl9 transcripts encode a
protein(s) (rep proteins) absolutely required for AAV DNA
replication. Mutants containing a frameshift at 11 mu are
also defective for replication, indicating that a product
encoded by the p5 ORF is essential for DNA replication. No
mutation which specifically affects the pl9 ORF has yet to
be created so the importance of this gene in AAV replication
has not yet been elucidated.
Mutations which affect the C-terminal region of the p5
and pl9 ORFs have variable phenotypes. A mutation which
specifically affects the C-terminus of the p5 and pl9 ORFs
contained within the spliced mRNAs has no apparent effect on
DNA replication, although a frameshift mutation at 42 mu
within the rep ORF contained within the AAV intron inhibits
AAV DNA replication by 100 fold. Thus potential rep
proteins encoded by the unspliced p5 and pl9 ORFs appear to
be critical for AAV DNA replication. Conversely, certain
mutants which contain deletions of the entire intron and C-

19
region of the rep proteins efficiently replicate their DNA
while other similar mutants do not (Tratschin et al.,
1984b). As such it is unclear what specific function(s) the
C-terminal coding regions of the rep ORFs have.
Although mutations within the right half of the genome
do not affect the accumulation of AAV replication
intermediates, these mutations do affect capsid protein
production and function. As expected deletion and
frameshift mutation within the body of the large right side
ORF (cap mutants) completely abolishes capsid production,
the seguestration of ss-DNA, and virus production. Also
mutations between 48 and 55 mu specifically affect the
syntheses of the larger structural proteins, VP1 and VP2 but
not VP3 (Janik et al., 1984). Thus VP3 appears to be a
primary translation product, rather than a proteolytic
cleavage product of the larger virion proteins.
Unexpectedly, mutants with lesions between 48-55 mu and
those with lesions in the body of the right side ORF have
different phenotypes. Mutants containing lesions between 48
and 55 mu (lip mutants) still produce sequestered progeny
ss-DNA but produce virions of low infectivity. Thus the
different virion proteins appear to have separable
functions.
Perspective
The biology of AAV in many senses is similar to that of
latent prokaryotic viruses. Like the lambda bacteriophage,

20
AAV can efficiently integrate into and be rescued from the
host chromosome. Further similarities exist between AAV and
the defective bacteriophage P2 (Six and Klug, 1973; Calender
et al., 1981). Both exist as latent proviruses which
reguire coinfection with a specific helper virus in order to
replicate. One hallmark of the bacterial viruses is an
ability to modulate the expression of their genes. Indeed
the study of the "genetic switch" (Ptashne, 1986) between
the lytic and lysogenic states of lambda infection has
proved to be one of the most informative models of the
control of gene expression. We began these studies in the
hope that AAV might similarly provide a useful model of
eukaryotic gene regulation.

CHAPTER II
METHODOLOGY
Cells and Culture Conditions
Human KB and HeLa monolayer and monkey cos-7 cell
cultures were maintained in Dulbecco's modified Eagles
medium (DMEM) containing 10% fetal calf serum (FCS), 2mM
glutamine, and 500 units/ml of penicillin and streptomycin.
Mouse B78H1 and Ltk- monolayer cultures were maintained in
either minimal essential media (MEM) or DMEM containing 5%
FCS, 5% calf serum, 2mM glutamine, 500 units/ml of
penicillin and streptomycin, 2.5 /¿g/ml Amphotericin B
(Fungizone, Gibco Laboratories), and 0.1 mg/ml Gentamycin
(Gibco Laboratories). In some experiments cells were
treated with 10 /¿g of actinomycin D (Sigma)/ml of media for
times indicated in the text.
Viral Stocks
V' '
AAV was prepared from lysates of HeLa cells coinfected
with both Ad-2 and AAV-2. Ad-2 was inactivated by heating
of the lysates at 56° for 30 minutes. Ad-2 stocks were also
prepared from lysates of infected HeLa cells.
Preparation of Plasmid DNAs
All plasmid DNAs were propagated in Escherichia coli
HB101 cells. After inoculation of fresh cultures with 1/50
volume of an overnight culture, plasmid containing cells
?U
21

22
were grown to a density of 80 Klett units (green filter,
Klett-Summerson colorimeter) in Luria broth (1% tryptone,
0.5% yeast extract, 0.5% NaCl, and 0.5% glucose).
Chloramphenicol was then added to a final concentration of
170 /¿g/ml and the cultures were grown for an additional 16-
20 hours. Plasmid DNAs were isolated as described by
Birnboim and Doly (1979). Form I plasmid DNA was further
purified by centrifugation in cesium-ethidium bromide
eguilibrium gradients as described by Maniatis et al.
(1982).
Enzymes and Linkers
Restriction enzymes, T4 DNA ligase, T4 DNA polymerase,
T4 polynucleotide kinase, Escherichia coli DNA polymerase I
and Klenow fragment were purchased from either New England
Biolabs, Bethesda Research Laboratories, or International
Biotechnologies Inc. and used as specified by the
manufacturer. Linkers were purchased from either Pharmacia
or New England Biolabs and were phosphorylated as specified
by the manufacturer.
Construction of Mutant AAV Genomes
Plasmid constructions are summarized in chapter III
(table 3-1). Form I pSM620 (Samulski et al., 1982) was
digested with various enzymes (Apal. Ncol, or Xhol) and
religated to yield various deletion mutants. Certain
deletion mutants were constructed by ligating previously
described plamsids, which had been digested with Bglll and

23
EcoRV. Mutants containing deletions of the TRs (ori~
mutants) were created by insertion of the large Smal or Ball
fragment of particular genomes into the EcoRV site of pBR322
(Bolivar et al., 1977), pAT153 (Twigg and Sherratt, 1980),
or pFB69 (Barany, 1985). The construction of pLB1701 and
derivatives is described in chapter VI.
All plasmid constructions were placed into and
propagated in HB101. Initially, small amounts of plasmid
DNAs were isolated for structural analysis by the method of
Holmes and Quigley (1981) or Birnboim and Doly (1979).
Plasmid constructs were analyzed by digestion with
appropriate restriction endonucleases and electrophoresis in
agarose and acrylamide gels.
Radiolabeling of DNA
Radiolabeled DNAs to be used for probe were produced
either by nick translation (Rigby et al. , 1977) or by the
random primer technigue (Feinberg and Vogelstein, 1983) and
were used interchangeably. Unless stated otherwise in the
text, duplex AAV-virion DNA was labeled as probe. Nick
translation reactions contained 1 ¿ig of DNA, 50 mM Tris-HCl
(pH 7.8), 5 mM MgCl, 10 mM 2-mercaptoethanol, 0.1 mM each of
dGTP, dATP, and TTP, 100 ¿tCi of a32P-dCTP, 1 ng DNAase I
(Sigma), and 5 units of DNA polymerase I in a final reaction
volume of 50 ¿ul. Unincorporated deoxyribonucleotides were
removed by chromatography on Sephadex G-75 (Pharmacia Fine
Chemicals) in 10 mM Tris-HCl (pH 8.0). The specific

24
activity of probe obtained by this procedure was between 2
to 5 x 107 counts per minute (cpm)//xg DNA. Random primer
reactions consisted of 0.1 ¿xg of denatured DNA (boiled for 7
minutes), 0.2 M Hepes (pH 6.6), 0.15 optical density units
of random hexadeoxyribonucleotides (P-L No. 2166), 50 mM
Tris-HCl (pH 8.0), 50 mM MgCl, 10 mM 2-mercaptoethanol, 100
/xM each of dATP, dGTP, and TTP, 100 /xCi of a32P-dCTP (ICN) ,
0.2 /xg/ml bovine serum albumin (BSA) , and 5 units Klenow
fragment. Reactions were incubated at room temperature for
2 hours and stopped by the addition of sodium dodecyl
sulfate (SDS) and ethelene diaminetetraacetic acid (EDTA) to
0.2% and 50 mM, respectively. At this point reactions had
incorporated between 90 to 99% of the label and probes were
used without further purification.
Diethvlaminoethvl (DEAE)-Dextran DNA Transfections
The DEAE-dextran transfection procedure was carried out
as dsecribed by McCutchan and Pagano (1968) with some
modifications. Either 100 mm or 150 mm dishes of
approximately 80% confluent KB cells were washed once with
phosphate buffered saline (PBS) and a DNA-dextran mixture
was added and left on the cells at room temperature for 30
minutes. DNA-dextran consisted of variable amounts of each
plasmid as indicated and 500 /xg of DEAE-dextran (MW =1 x 103
kd, Pharmacia Fine Chemicals) per ml in MEM without serum.
Plates of cells, either 100 mm or 150 mm diameter, received
either 1 ml or 2.5 ml of the DNA-dextran mixture

25
respectively. The transfection mixture was then replaced
with fresh media containing 50 ¡M chloroquine diphosphate.
Experiments involving infection with Ad-2 contained Ad-2 at
a multiplicity of infection (moi) of 10 in the chloroquine
containing media. The cells were incubated for 3 hours at
37°C and refed with fresh media.
Calcium Phosphate DNA Transfections
Calcium phosphate coprecipitation transfections were
carried out as described by Graham and van der Eb (1973) and
as modified by Wigler et al. (1979). For stable
transformation and genetic selection, plates were seeded the
night before transfection at densities of 1.25 x 105 cells
or 3 x 105 cells per dish of B78H1 or Ltk“ cells,
respectively, while 30-50% confluent plates were used for
transient expression assays. Precipitates were formed by
combining the following in the following order: DNA (plasmid
and carrier DNA for a total of 25 /¿g of DNA) and H20 to
0.250 ml, 0.250 ml 0.5 M CaCl, and 0.5 ml of 2 x Hepes (42
mM Hepes, 0.27 M NaCl, 10 mM KC1, 1.4 mM Na2HP04 (2H20) , pH
7.05). The precipitates were allowed to form at room
temperature for 20 minutes and then added directly to the
media of 100 mm plates of cells. For stable transformation,
the media and precipitate were replaced with fresh media the
following day and again with selective media (see below) the
next day. For transient expression assays, the precipitate
and media were removed at 4 to 6 hours after transfection

26
and replaced with 5 ml of 12% glycerol in PBS, incubated for
1 minute, and finally replaced with 10 ml of fresh media
containing 4 mM sodium butyrate as described by Gorman et
al. (1983).
Genetic Selection of Mammalian Cells
G-418 resistant (G-418r) cells were selected in media
containing 1 mg/ml G-418 Sulfate (Geneticin, Gibco) as
described by Southern and Berg (1982). Thymidine kinase
(tk) positive cells were selected in hypoxanthine-
aminopterin-thymidine (HAT) media as described by Wigler et
al. (1979) and consisted of 15 /¿g/ml hypoxanthine, 1 ng/ml
aminopterin, and 5 /¿g/ml thymidine in DMEM. Colonies were
grown for 10-18 days in selective media, after which the
plates were fixed with 10% formaldehyde and stained with
cresyl violet.
Chloramphenicol Acetvltransferase (cat) Assays
Assays for cat activity were carried out essentially as
described by Gorman et al. (1982). Plates of transfected
cells were washed three times with cold PBS, incubated under
1 ml of STE (40mM Tris-HCL (pH7.4), 0.15 M NaCl, 1 mM EDTA)
for 5 minute at room temperature. The cells were harvested
by scraping, pelleted by centrifugation, and resuspended in
110 /¿I of 0.25 M Tris-HCl (pH 7.8) and frozen and thawed
three times. Cell debris was removed by centrifugation and
the supernatant stored at -20°C. Half of the supernatants
were used in the assays which consisted of 55 /¿I extract, 75

27
lil 1 M Tris-HCl (pH7.8), 25 n 1 Acetyl Coenzyme A (4 mg/ml,
made fresh each time), and 0.1-0.25 ^C 14C-chloramphenicol
(New England Nuclear Inc.). The reactions were incubated at
37°C for 1 hour and extracted with 1 ml of ethyl acetate.
The organic layer was isolated and dried in a Savant speed
vacuum centrifuge for 30 minutes. The desiccated samples
were resuspended in 25 ^1 of ethyl acetate and spotted on
thin layer chromatography (TLC) plates (Kodak silica or
Bakerflex silica plates), and eluted in a TLC chamber
equilibrated with chloroform and methanol (95:5). Cat
activity was determined by autoradiography of the TLC plates
followed by scintillation counting of spots cut from the
plates.
RNA Extraction and Analysis
At 33 to 36 hours post transfection, cells were
harvested by scraping, washed with cold PBS, and swelled on
ice in reticulocyte swelling buffer (RSB, 10 mM Tris-HCl (pH
7.4), 10 mM NaCl, 5 mM MgCl and 2 mM DTT) for 10 minutes.
The cells were lysed with 12 strokes of a Dounce homogenizer
and the nuclei removed by centrifugation. RNA was then
extracted from the cytoplasmic homogenate by a modification
of the procedure described by Lusby and Berns (1982).
Briefly the cytoplasmic extract was added to 4 ml of ETS (10
mM EDTA, 0.,2% SDS, and 10 mM Tris-HCl (pH 7.4), and 5 ml of
phenol heated to 7 0°C, and placed in a 70°C water bath for
15 minutes with occasional shaking. After quick chilling on

28
ice, the aqueous phase was isolated, reextracted with 4 ml
of phenol/chloroform (1:1) and the RNA precipitated with
ethanol.
For some experiments total cellular RNA was isolated
using the guanidinium/CsCl method as described by Maniatis
et al. (1982). Cell pellets were lysed by addition of 2.5
ml of a solution containing 4 M guanidinium isothiocyanate,
5 mM sodium citrate (pH7.0), 0.1 M /3-mercaptoethanol and
0.5% sarkosyl. One-gram of CsCl was then added and the
solution layered onto a 1.2 ml cushion of 5.7 M CsCl
containing 0.1 M EDTA (pH7.5) in Beckman SW 50.1 tubes
followed by centrifugation at 35,000 rpm for 12-16 hours at
20°C. The supernatants were discarded and the pellets
resuspended in 400 ¿¿1 of a solution containing 10 mM Tris-
HC1 (pH7•4) , 5 mM EDTA, and 1% SDS. This solution was then
extracted twice with chloroform and 1-butanol (4:1) and,
after addition of 0.1 volume of 3 M sodium acetate (pH 4.8),
precipitated with ethanol. Concentrations were determined
by a2 60 an<3 by intensity of ribosomal RNA bands after
shadowing with UV light on cellulose TLC (Analtech, catalog
number 08011). RNA samples were prepared and fractionated
in 1.1% agarose gels containing 2.2 M formaldehyde as
described by Maniatis et al. (1982) using vertical slab gels
run for 5 to 8 hours at 100 volts. RNAs were transferred
onto nitrocellulose filters in 20 x SSC without further

29
treatment of the gels. The filters were then handled as
described below.
DNA Isolation
Low molecular weight DNAs were isolated by a
modification of the method of Hirt (1967). Cell monolayers
were washed in cold PBS and harvested by scraping in 1 ml of
Hirt buffer (0.6% SDS and 50 mM EDTA, pH7.4). Sodium
chloride was added to 1 M after incubation for 20 minutes at
room temperature and the samples incubated overnight at 4°C.
The samples were then centrifuged for 45 minutes in an
Eppendorf centrifuge, the supernatants were collected,
extracted 2-3 times with an equal volume of
phenol:chloroform (1:1), and the nucleic acid precipitated
with ethanol. In some experiments this procedure was used
to isolate DNA directly from isolated nuclei.
Genomic DNAs were isolated by a modification of a
procedure reported by Cheung et al. (1980). Nuclei isolated
as described above were resuspended in 1 ml of 1 x SSC and
pronase and SDS were added to concentrations of 1 mg/ml and
0.1%, respectively, followed by incubation overnight at
37°C. The solution was then gently extracted twice with an
equal volume of phenol:chloroform (1:1) and precipitated
with 2.5 volumes of ethanol. Nucleic acid was then gently
resuspended in 10 mM Tris-HCl (pH7.4) and incubated with 1
mg of RNase A (Sigma) for 60 minutes at 37°C, followed by
one extraction with phenol:chloroform and ethanol
extraction

30
precipitation. DNAs were concentrations were determined by
A260 •
Southern Blotting and Hybridization of Filters
DNA gels (nondenaturing) were prepared and blotted to
nitrocellulose as described by Southern (1975) . After
transfer, the filters were baked at 80°C for two hours,
placed in a heat sealable bag with 15 ml of prehybridization
buffer (50% formamide, 5 x SSC, 0.5% SDS, 0.1% PVP, 0.1%
BSA, 0.1% Ficoll, 0.15 mg/ml sheared calf thymus (CT) DNA)
and incubated at 42°C for 4 hours, and the buffer replaced
with fresh buffer containing denatured DNA probe and CT DNA,
and then incubated at 42°C for 12 to 16 hours. The filters
were then washed twice for 15 minutes in 2 x SSC and 0.5%
SDS at 68°C, twice for 60 minutes in 0.1 x SSC and 0.1% SDS
at 68°C and once at room temperature for 20 minutes in 0.1 x
SSC and 0.5% SDS. Filters were dried and placed against
film for autoradiography.
Nuclear Runoff Transcription ÍNRT) Assays
Cells were harvested at 24 hours postinfection and
nuclei were obtained as described above except that DTT was
added to 2 mM to the RSB and KCl added to 140 mM immediately
after homogenization. Nuclei were isolated by centrifugation
and prepared and analyzed as described (Falk-Pedersen et
al., 1985). Nuclei from 3 x 107 cells were resuspended 1 ml
NRT buffer (20 mM hepes (pH 7.9), 20% glycerol, 140 mM KCl,
10 mM MgCl, 2 mM DTT), repelleted at 2,000 times gravity (G)

31
for 4 minutes, and resuspended in 200 /¿I of transcription
cocktail (60 /¿I 32P-UTP (200 /iC) , 1 /¿I of a solution
containing 1 mM each of GTP, ATP, and CTP, 2 mM DTT, 8 0 ¿¿1
NRT buffer), incubated on ice for 3 minutes then incubated
at 30°C for 15 minutes. Reactions were terminated by adding
1 ml of HSB (0.01M Tris-HCl (pH 7.4) 0.5 M NaCl, 0.05 M
MgCl, 2 mM CaCl) and 0.1 mg DNAase I and then incubated at
room temperature for 1 minute followed by the addition of 4
ml of ETS and 5 ml of 70°C phenol. Labeled RNA was isolated
as described for cytoplasmic RNA. RNA was further purified
by precipitation in 2 M LiCl at 0°C overnight. The amount
of incorporated label was determined by chromatography on
PEI cellulose TLC using 1 M HC1. Equal amounts of labeled
RNA for each experiment (approximately lxlO7 or 5xl07 acid
precipitable cpm for KB cells and HeLa cells, respectively)
were used as probes of southern blots containing restriction
digested AAV-2 and Ad-2 DNAs. Blots were hybridized and
washed as described above except that an additional wash was
carried out in 0.1 x SSC, 0.5% SDS, 10 mM NaP04, 1% Triton
X-100, and 1% Tween-20 for 1 hour at 68°C.
Immunizations and Antibody Analysis
Synthetic peptides were prepared by Dr. Ben Dunn as
described by Dunn et al. (1983). Peptides contained 3H-
amino acids so all steps could be quantitated. Peptides
were coupled to either BSA or keyhole limpet hemocyanin
(KLH) with glutaraldehyde as described by Kagan and Glick

32
(1979). Glutaraldehyde (1 ml, 2 0 mM) was added dropwise to
a 2 ml solution containing 10-20 mg of BSA or KLH and a 30
molar excess of peptide in 0.1 M sodium phosphate (pH 7.5)
and incubated at room temperature with stirring for 2 hours.
Conjugated peptide was purified by chromatography on
Sephadex G-25 followed by dialysis against PBS for 12-16
hours with several changes of buffer. Conjugates contained
an approximate carrier to peptide molar ratio of 1:15 to
1:30.
Two-month-old New Zealand white rabbits were immunized
with conjugates containing a total of 300 ¿¿g of peptide
emulsified in 2 ml of Freund's complete adjuvant by
intradermal injections at multiple sites across the back.
Animals were boosted with subcutaneous injections containing
200 nq of peptide/conjugate in 2 ml of Freund's incomplete
adjuvant at approximate 4 week intervals. Antisera were
drawn approximately 7 days after injections. Antisera were
further purified by chromatography on peptide columns
created by coupling of peptide to CnBr-activated Sepharose
4B prepared as specified by the manufacturer (Pharmacia Fine
Chemicals).
The presence of antipeptide antibody was determined by
hemagglutination assays using peptide coupled to sheep red
blood cells (SRBCs). SRBCs (0.2 ml, pelleted) were washed
three times with 2 ml of 0.1 M NaCl, pelleted by
centrifugation at 1000 x G for 5 minutes each time and then

33
resuspended in cold PBS (pH 8.2) containing 1%
glutaraldehyde. The cells were rotated gently at 4°C for 30
minutes, washed 5 times in cold 0.15M NaCl, 5 times in cold
water, diluted to 2.5% in PBS (pH 7.2) and mixed with an
equal volume of a 1:60,000 solution of tannic acid in PBS
(pH 7.2) . The cells were gently rotated at 4°C for 20
minutes, washed twice with PBS (pH 7.2), resuspended as a
2.5% suspension in PBS (pH 6.4), incubated with 400 /ig of
peptide at 37°C for 2 hours, washed twice with 1% normal
rabbit serum in PBS (pH 7.2), resuspended in the same
solution as a 2.5% suspension and frozen by emulsion in
liquid nitrogen in small aliquots and stored at -70°C.
Hemagglutination assays were carried out as described.
All sera tested were preincubated at 37°C to inactivate
complement. Reactions contained 25 /xl of 0.5% coupled SRBCs
(diluted in 1% agammaglobulin horse sera) and 50 ¿ul of
diluted rabbit antisera. Reactions were incubated at room
temperature for 4 hours in 96 well round bottom microtiter
plates.
Metabolic Labeling and Immunoprecipitation Analysis
KB cells (approximately 1 x 106 cells) were infected
with either Ad-2 (moi of 10) or coinfected with Ad-2 and
AAV-2 (moi of 20) , washed twice with methionine-free or
phosphate free MEM, and then incubated for 1 hour in 0.5 ml
of the same media containing 2% dialyzed FCS and either 250
nC 35S-methionine or 500 /xCi 32P-phosphate. Labeled cells

34
were then washed twice in cold PBS on the plates and lysed
by scraping in 2.5 ml PLB (phospholysis buffer; lOmM sodium
phosphate (pH7.5) 100 mM NaCl, 1% Triton X-100, 0.5% sodium
desoxycholate, 0.1% SDS) containing 1 mM PMSF (Sigma). Cell
debris were removed by centrifugation at 1000 xG for 10
minutes and the lysates cleared by centrifugation at 100,000
x G for 1 hour at 4°C. Samples were then diluted two fold
with PLB containing 10 mg/ml BSA.
Equal amounts of labeled proteins (approximately 5 x
107 cpm acid precipitable counts) were incubated overnight
on ice with 5 ^1 of antibody. Protein-A sepharose (Pharmacia
Fine Chemicals) was preincubated in PLB and BSA overnight at
4°C with rocking, added (100 ¿¿1 of a 10% mixture) and
reactions rocked gently at 4°C for 45 minutes. Bound immune
complexes were then pelleted and washed 8 to 12 times in PLB
with BSA and twice in PLB, resuspended in SDS sample buffer
and boiled to elute antibody and antigen.
Samples were then fractionated on discontinuous
polyacrylamide gels as described by Laemmli (1970). Gels
consisted of a 4% stacking gel and 10% running gel and
samples were electrophoresed for 4 to 6 hours at 125 volts.
Samples were run next to prestained protein MW markers
(Bethesda Research Laboratories) . Proteins were then fixed
in 50% methanol, 10% acetic acid and 1% commasie brilliant
blue for 20 minutes followed by destaining overnight in 10%
acetic acid. Gels were photographed, rinsed with water for

35
30 minutes and treated with 1 M sodium salicylate for 45
minutes (for 35S-labeled proteins only) before autoradiography.

CHAPTER III
AUTOREGULATION OF AAV GENE EXPRESSION
Introduction
As described in chapter I, all aspects of AAV
macromolecular synthesis are regulated by various helper
functions. However, these studies also show a great
plasticity in the requirement for specific helper virus
genes. Although AAV gene expression is positively regulated
by the Ela, Elb, E2a, and VA genes, none of these genes
appears absolutely essential for AAV gene expression. The
recent observation that AAV can replicate in certain cells
in the absence of helper functions supports the idea that no
helper virus genes are directly involved in or essential for
AAV gene expression. Thus the regulation of AAV gene
expression may largely depend on both cellular and AAV
encoded functions whose activities may be modulated by
helper virus gene products.
In this study, the regulation of the AAV genome has
been investigated. Mutant genomes created In vitro have
been studied in order to define the genetic elements
involved in the autoregulation of AAV gene expression. The
results show that autoregulation occurs on at least two
levels; first, a product encoded by the AAV rep gene is
essential for activation of AAV transcription, and second,
36

37
elements within the coding regions for the rep gene products
negatively regulate expression of the rep genes.
Results
Structure of mutant AAV genomes. The structures of
mutant AAV genomes created during this study are summarized
in table 3-1. Many of the cloned genomes were derived from
previously described mutants (Hermonat et al., 1984). The
name of the plasmid genome indicates the type of mutation.
Frameshift insertion mutations are indicated by ins followed
by the position of the insertion, indicated in mu. The
insertions consist of 8 base synthetic oligonucleotides
containing a Bglll restriction site. Deletion mutations are
indicated by dl followed by the location of the deletions.
Mutants containing nonrepairable deletions of both TRs are
designated as ori~. Certain mutants contain a tandem
duplication of AAV DNA and are indicated by dp and position
of the duplicated region. Other mutants contain an
insertion of the SV40 early promoter and are indicated by
/SV following the initial mutant description.
Accumulation of AAV RNAs by two types of rep mutants.
To determine whether an AAV nonstructural protein encoded by
the p5 or pl9 genes regulates AAV gene expression, the
accumulation of RNAs by two types of replication-defective
AAV genomes was compared. The first types contain 8 base
frameshift insertions at either 11 or 32 mu and thus are
defective for the production of the trans-acting p5 and pl9

38
TABLE 3-1
CONSTRUCTION OF PLASMID GENOMES
Plasmid
Description
dl03-ll (pLB1415o)
dlll-23 (pLB1415b)
dl23-37 (pLB14 06)
dl63-86 (pLBlOl)
dl63-80 (pLB102)
dl48-52 (pLB202)
dll5-86 (pLB306)
dl80-96 (pLB314)
inv!5-86 (pLB315)
pAAV/0-11 (pLB710)
pAAV/11-42 (pLB709)
pAAV/42-91 (pLB705)
pAAV/91-100 (pLB702)
620ori~
insllori" (pLB1207)
ins32ori~
ins32ori~b
ins42ori~ (pLB3411)
Ligation of BglII/EcoRVa digested
dl03-23 and insll
Ligation of Bglll/EcoRV digested
dl03-23 and insll
Ligation of Bglll/EcoRV digested
dl23-28 and dllO-37
ApaI deletion of pSM620
Apal deletion of pSM620
Xhol deletion of pSM620
Ncol deletion of pSM620
Ncol deletion of pSM620
Inversion of Ncol A of pSM620
pSM620 PstI C into PstI site of pBR322
pSM620 PstI B into PstI site of pBR322
pSM620 PstI A into PstI site of pBR322
pSM620 PstI D into PstI site of pBR322
pSM620 Smal A into EcoRV site ofpBR322
insll Smal A into EcoRV site of pBR322
ins32 Smal A into EcoRV site of pBR322
ins32 Ball A into EcoRV site of pBR322
ins42 Smal A into EcoRV site of pBR322

39
TABLE 3-1 (continued)
Plamid
Description
ins78ori~ (pLB2402)
ins78 Smal A into EcoRV site of pFB69
dl03-23ori“ (pLB1302)
dl03-23 Smal A into the EcoRV site of
pBR322
dll0-37ori~ (pLB1102)
dllO-37 Smal A into the EcoRV site of
pBR322
dl63-86ori~ (pLB801)
Apal deletion of 620ori“
dl58-87ori“ (pLB2904)
dl58-87 Smal A into the EcoRV
site of pAT153
dp28-49 (pLBp40A)
Ligation of Bqlll/EcoRV digested
dl49-94 and d!23-28
dp37-58 (pLBp40B)
Ligation of Bglll/EcoRV digested
dl58-87 and dllO-37
dl03-23/SV (pLB605)
SV40 early promoter into Bglll
digested d!03-23
dl03-05/SV (pLB1701)
Insertion of Aval/BstEII of pSM620
into Bglll/BstEII digested dl03-23/SV
dl03-05/SVins32(pLB3901)
Ligation of SacI/BstEII digested ins32
and dl03-05/SV
dl03-05/SVori“ (pLB1801)
dl03-05/SV Smal A fragment into the
EcoRV digested pBR322
dl03-ll/SV (pLB4104)
Ligation of Bglll/BstEII digested
dl03-23/SV and insll
dl03-20/SV (pLB4004)
Ligation of Bglll/EcoRV digested
dl03-23/SV and BclI/EcoRV digested
pSM62 0

40
encoded rep gene products. The second type is represented
by a mutant containing deletions of both TRs, 620ori~. The
replication of 620ori~ is blocked by cis-active defects of
the origins of DNA replication. This plasmid was used as a
control rather than the parental pSM620 plasmid so that the
effect(s) of mutations within the rep genes can be directly
analyzed. Because mutations within the rep genes abolish
AAV DNA replication, the use of an ori~ control eliminates
possible differences due to increased copy number of
replicating genomes or by differing conformations of
replicating and nonreplicating transcription templates. The
RNAs accumulated by the AAV genomes were analyzed after
transfection into Ad2-infected KB cells. Total cytoplasmic
RNAs were isolated from the transfected cells at 30 hr post¬
transfection. The RNAs wer fractionated by formaldehyde gel
electrophoresis and analyzed by blotting and hybridization
to radiolabeled AAV DNA as shown in figure 3-1.
The 620ori~ mutant (lane 1) produced readily detectable
levels of all the AAV transcripts except for the spliced p5
RNA. This RNA is normally present in relatively low
abundance and is difficult to resolve from the unspliced pl9
RNA. The RNAs produced by 620ori~ were in the same
stoichiometry as that produced by wild type AAV (Green and
Roeder, 1980a; Laughlin et al., 1979b), indicating that DNA
replication was not required for qualitatively normal
expression of the AAV genes. The most abundant RNA

41
accumulated was the spliced 2.3 kb p40 transcript. The
larger p5 and pl9 RNAs were accumulated in significantly
lower amounts.
Although the nonreplicating 620ori~ genome produced
readily detectable levels of transcripts, the nonreplicating
ins mutants did not (lanes 2 and 3). Both insertion mutants
appeared equally defective for the production of RNAs. The
subtle nature of the insertion mutations suggests that a
product encoded within rep genes is required for efficient
expression of all the AAV genes.
Similar results were obtained when total cellular RNAs
were analyzed as shown in figure 3-2. The amounts of
transcripts accumulated by the rep mutants insll and ins32
were significantly lower than by 620ori~. again indicating a
critical role for the p5 gene product in gene expression.
The amount of p40 RNA detected was higher for insll than for
ins32 in this experiment, although this result was not
always reproducible. Low amounts of p5 RNA from both
insertion mutants were also detected.
The accumulation of RNAs by a deletion mutant, d!03-23.
was also analyzed in this experiment. D103-23 accumulated
an intermediate level of p40 RNA, higher than by either rep
insertion mutant but significantly lower than by 620ori~.
The overproduction of the p4 0 RNA by left side deletion
mutants was reproducible and is discussed below.

42
1 2 3
Figure 3-1. Accumulation of RNAs by two types of
rep mutants. KB cells were transfected with 20 nq of either
620ori~ (lane 1) , ins32 (lane 2) , or insll (lane 3) and
infected with Ad-2. At 30 hours postinfection, cytoplasmic
RNAs were isolated, and 2 0 nq of RNA from each experiment
were fractionated on a formaldehyde-agarose gel and
subsequently analyzed by blotting and hybridization to 32P-
labeled AAV virion DNA. The sizes of the RNAs are as shown
in figure 1 and indicated here are the 4.2 kb unspliced p5
RNA, the 3.6 kb unspliced (top of caret) and 3.3 kb spliced
(bottom of caret) pl9 RNAs, and the 2.3-kb spliced p40 RNA.
The 2.6-kb unspliced p40 RNA is seen directly above the 2.3
kb RNA.

43
4.2 -
3.6-
*♦
12 3 4
Figure 3-2. Accumulation of RNAs by mutant AAV
genomes. KB cells were transfected with either insll (lane
1) , ins32 (lane 2) , 620ori~ (lane 3) , or dl03-23 (lane 4)
and infected with Ad-2 as described for figure 3-1. 20 ^g
of total cellular RNA from each experiment were analyzed as
described for figure 3-1. The sizes of the unspliced p5
(4.2 kb), pl9 (3.6 kb), and spliced p40 RNAs (2.3 kb) are
indicated on the right.

Accumulation of AAV RNAs by rep- ori- double mutants.
The AAV genomes used in the experiments described above
contained either of two types of mutations, both of which
blocked DNA replication. The rep mutants (ins and dl)
contained trans-complementable defects in the AAV rep gene,
while the defective replication of 620ori~ was
noncomplementable due to deletion mutations within the TRs
(which act as origins for AAV DNA replication) . Thus the
difference in the accumulation of transcripts by the mutants
might be explained either by the loss of a positive
activator of expression in the rep mutants or by the removal
of a cis-active repressor of expression within the TRs of
620ori~.
To distinguish between these possibilities, we analyzed
the accumulation of transcripts by mutant AAV genomes
containing similar deletions of the TRs. The RNAs produced
by the genomes upon transfection into Ad2-infected cells are
shown in figure 3-3. Both rep insertion mutants (insllori-
and ins32ori ) produced
greatly
reduced
amounts
of
transcripts compared
with
620ori-
(lane
1) •
Thus
the
defective expression
of the ins mutants
was
due to
the
lesions in the rep gene.
An ori- capsid deletion mutant, dl63-86ori~ accumulated
normal amounts and proportions of its deleted transcripts
(lane 4) . This indicates that AAV capsid proteins have no
role in the accumulation of AAV RNAs.

45
Although most mutations within the rep gene abolish AAV
DNA replication, a mutant containing an insertion at 42 mu
is partially defective, replicating at about the 0.1% level
of wild type genomes (Hermonat et al., 1984). To determine
if this mutant is also defective in the accumulation of
RNAs, an ori~ version of ins42 (lane 6) was constructed and
analyzed. This mutant genome produced intermediate levels
of RNAs compared to ins32ori~ (lane 7) and 620ori~ (lane 5).
While the mutation at 42 mu reduces DNA replication by 100-
1000 fold (Hermonat et al. , 1984) the accumulation of RNAs
was only inhibited by approximately 10 fold. This may
suggest that different regions of the AAV rep proteins are
required for DNA replication and regulation of gene
expression.
The predominant RNA accumulated by ins42ori~ was the
spliced p40 message, demonstrating that the mutation did not
severely affect the splicing efficiency of AAV messages.
Thus the defects caused by mutation at 42 mu do not appear
to be caused by defects in RNA splicing but by lesions
within the coding region contained in the AAV intron,
supporting the suggestion that products encoded by the
unspliced p5 and pl9 transcripts are essential for AAV
replication (Hermonat et al., 1984).
The results presented above indicate that a product
encoded within the AAV p5 and pl9 genes is required for the
efficient accumulation of AAV RNAs in helper virus infected

46
cells. In order to determine if this positive autoregulation
by the rep gene product occurred in the absence of helper
virus, the RNAs accumulated by cloned genomes upon
transfection into uninfected KB cells were analyzed. As
shown in figure 3-3 equally low levels of the 2.3 kb p40
transcript were accumulated by 620ori~ (lane 8) and
ins32ori~(lane 9) in the absence of Ad while only 620ori~
produced high levels of transcripts in the presence of Ad.
Thus efficient activation of expression only appeared to
occur in Ad infected cells.
We also compared the accumulation of RNAs by ori~
genomes which have deletions on the left side of the AAV
genome. A blot comparing the RNAs produced by dl03-23ori~.
dll0-37ori~. 620ori~. and ins32ori~ is shown in figure 3-4.
As expected, genomes containing mutant rep genes accumulated
much lower amounts of transcripts than 620ori~. However,
dl03-23ori~ produced higher levels of the 2.3 kb p40 message
than by the other rep mutants. Thus both ori~ and ori+
versions of dl03-23 show enhanced accumulation of the p40
RNA suggesting that upstream sequences negatively regulate
transcription from p40. D110-37ori~ did not overproduce the
p40 RNA suggesting that deletion of sequences between 03-10
mu was required for the enhanced accumulation of the p4 0
RNA.

Figure 3-3. Accumulation of RNAs by ori~ AAV genomes. Ad-2-infected
KB cells were transfected with 20 g of either 620ori~ (lanes 1 and 5),
insllori- (lane 2) , ins32ori~ (lanes 3 and 7) , d!63-86ori~ (lane 4) , or
ins42ori~ (lane 6) and uninfected KB cells were transfected with either
620ori~ (lane 8) or ins32ori~ (lane 9) . A 20 g amount of RNA from each
transfection was analyzed as described in the legend to figure 3-1. The
position of RNAs indicated are of wild type size and are the same as shown
in figure 3-1. The sizes of the transcripts of d!63-86ori~ are
approximately 1.1 kb smaller than the wild type transcripts.

12 3 4
5 6 7 8 9
4^
CO

49
12 3 4
M
B
p4oi
Figure 3-4. Accumulation of RNAs by rep deletion
mutant genomes. Ad-2 infected KB cells were transfected
with 20 /¿g of 620ori~ (lane 1) , ins32ori~ (lane 2) , dllO-
37ori~ (lane 3), or dl03-23ori~ (lane 4). A 20 ¿¿g amount of
RNA from each transfection was analyzed as described in the
legend to figure 3-1. Only the position of the spliced 2.3
kb p40 RNA is indicated.

50
Transcription of AAV genomes in isolated nuclei. The
data presented above showed that mutants with defective p5
and pl9 ORFs were defective in the accumulation of AAV
transcripts. In order to determine if the defect was at the
transcriptional or post-transcriptional level, the rates of
transcription from transfected AAV genomes were measured in
isolated nuclei (figure 3-5). Nuclei from Ad2-infected HeLa
cells transfected with either 620ori~ (panel A) , ins32ori~
(panel B) , or mock transfected (panel C) were isolated and
incubated to allow runoff elongation of preinitiated
transcription complexes.
Radiolabeled transcripts were isolated and equal
amounts hybridized to Southern blots containing 620ori~ DNA
which had been digested with PstI and Hindlll (lane 1) .
This digestion produces four fragments. Fragment a (3.8 kb)
contains pBR322 sequences in addition to 0.4 kb of AAV DNA
(91-98 mu). Fragments b and c contain only AAV DNA from 42-
91 mu and 11-40 mu, respectively. A fourth fragment, d,
contains AAV DNA from 2-11 mu as well as a small amount of
pBR322 DNA. Each blot also contained PstI digests of cloned
Ad5 DNA, either pXHOC (Ela and Elb sequences, lane 2) or
pXBAC (E4 sequences, lane 3) to confirm that equivalent
amounts of labeled nuclear RNA were used in each experiment.
Although equal amounts of labeled RNAs were used in
these experiments, the level of Ad transcription as
indicated by hybridization signal in panel B (ins32ori“)

51
appears to be approximately two-fold higher than in panel A
(620ori“). That this represents a consequence of 620ori~
expression seems unlikely since only a fraction of cells can
be expected to receive AAV DNA after transfection while
virtually all cells are expected to be infected with Ad.
However, an inhibition of Ad transcription by AAV is
documented below.
Although the level of Ad transcription detected was
higher upon transfection with ins32ori~. hybridization
signal to the AAV-specific fragments was observed only when
labeled nuclear RNA from 620ori~ transfected cells was used.
No AAV-specific transcription from ins32ori~ was detected in
isolated nuclei in this experiment. Thus, expression of the
rep insertion mutant was defective at the level of
transcription initiation.
Complementation of rep mutant gene expression. The
experiments described above suggest that an AAV gene
product(s) encoded in the p5 gene is required for activation
of AAV transcription. If so, the defect should be
complementable in trans. To test this, cotransfection
experiments were carried out using two rep- ori~ double
mutants, ins32ori~ and dll0-37ori~. Ori~ plasmids were used
so that any induction of expression would be assayed for in
the absence of an increase in potential template number due
to DNA replication. The plasmids were transfected into Ad-2
infected KB cells or cotransfected with a capsid deletion

Figure 3-5. Transcription of AAV genomes in isolated nuclei. HeLa
cells were transfected with 620ori~ (panel A), ins32ori~ (panel B), or only
carrier DNA (panel C) as described in chapter II using the calcium phosphate
precipitation method and subsequently infected with Ad-2. Nuclei were
isolated at 24 hours postinfection and transcription reactions were carried
out. Egual amounts of labeled nuclear RNAs were then hybridized to Southern
blots of AAV and adenovirus DNAs. Lane 1 contains PstI- and Hindlll-
digested 620ori~ DNA, which consists of four fragments: a 3.6 kb fragment
which consists of pBR322 DNA and AAV DNA from 91-98 mu (a), a 2.4 kb
fragment which consists of AAV DNA from 42-91 mu (b) , a 1.35 kb fragment
which consists of AAV DNA from 11-40 mu (c), and a 0.6 kb fragment (d) which
consists of AAV DNA from 2-11 mu and 0.2 kb of pBR322. Lanes 2 and 3
contain Pstl-digested Ad-5 Ela and Elb DNA from pXHOC and E4 DNA from pXBAC,
respectively.

53
CO
U cs
CO
CO
CM
CO
CM
n
• i
O-Q
I • •
$ • •
I • •
¿ -Ó

54
mutant, dl49-94. Total cytoplasmic RNAs were analyzed by
Northern blotting and hybridization to a labeled fragment of
AAV DNA (52-91 mu) so that the complementing genomes
transcripts were not visualized. As shown in figure 3-6, no
transcripts were detected upon transfection of either
ins32ori~ (lane 2), dll0-37ori~ (lane 5) or, as expected due
to the specificity of the probe, upon transfection of the
capsid deletion mutant (lane 6) . Cotransfection of the
capsid deletion mutant with ins32ori~ resulted in the
production of detectable levels of a 2.3 kb RNA, predicted
for the
p4 0
message
of the
rep
insertion
mutant. As
expected,
the
larger
RNAs of
the
insertion
mutant were
present in much lower amounts than the p40 message.
A 2.3 kb transcript was also detected in a
cotransfection experiment using dll0-37ori~ (lane 4) . In
this case, a second transcript slightly slower in migration
was also detected in approximately equal amounts to that of
the 2.3 kb RNA. This extra band appeared to represent a p5
transcript since dll0-37ori~ contains a p5 promoter which
would produce a highly truncated spliced transcript of
approximately 2.6 kb. The p5 transcript is normally
accumulated at levels of 5-10% of the p40 transcript. Thus,
the p5 RNA of dll0-37ori~ appeared to be overproduced.
One explanation for these data was that expression from
the p5 promoter is artificially high due to transcription
off a nonreplicating template. Alternatively, expression

55
may have been enhanced by the fortuitous construction of a
positive regulatory element (e.g. an enhancer) during the
cloning procedures. The identification of the 2.6 kb
species as a p5 transcript in the above experiment is also
difficult due to the low amount of transcripts produced by
complementation of the nonreplicating genomes. Therefore
several experiments were done using various ori+ rep
deletion mutants.
In the first of these experiments the production of
transcripts by left side deletion mutants upon
complementation with capsid deletion mutants was examined.
Capsid deletion mutants dl58-87 and dl63-86 (figure 3-7,
lanes 1 and 2) produced readily detectable amounts of their
deleted transcripts while the rep deletion mutants did not
(lanes 3 and 6) . Cotransfection of dl03-23 with d!63-86
resulted in the accumulation of large amounts of 2.3 kb p40
transcripts (lane 4) which must have been produced from
dl03-23 since the capsid mutant does not produce a 2.3 kb
message. Further, the lack of wild type size p5 and pl9
messages indicated that the 2.3 kb message did not arise
from a wild type genome generated by recombination.
D110-37 produced high levels of 2.3 kb p40 RNA when
complemented by dl58-87, as well as an equally abundant 2.6
kb RNA. Because dllO-37 contains only a p5 promoter in
addition to the p40 promoter contained within dl03-23, this
2.6 kb RNA is most likely a p5 transcript. The 2.6 kb RNA

56
also hybridizes to a p5 specific probe (see below). Thus
the p5 RNA was overproduced by replicating and
nonreplicating genomes.
One explanation for the increased accumulation of the
deleted p5 transcript is the juxtaposition of p5 to
regulatory elements of p40. In order to determine if
overproduction of p5 RNA required sequences within p40, a
deletion mutant containing only the p5 promoter, d!15-86.
was constructed and analyzed. RNAs accumulated by this
genome upon transfection with and without pSM620 (to supply
p5 and pl9 rep gene products) are also shown in Figure 3-7.
A high level of the deleted p5 transcript was detected upon
complementation. The structure of the deleted RNA also
eliminates the possibility that it was produced by a
recombinant genome. This RNA was produced in approximately
equal amounts with the pSM620 p40 transcript and in
approximately 16 fold excess with the pSM620 p5 transcript
(as determined by counting the radioactive bands cut out
from the nitrocellulose blot). Thus, overproduction of the
p5 message was independent of regulatory sequences within
the p40 promoter. Further, overproduction of the p5 message
is not unique to a single mutation, indicating that
expression was not enhanced by the inadvertent construction
of a positive regulatory element.
These complementation experiments clearly demonstrate
positive trans-activation by rep gene products, although we

57
cannot rule out the possibility that the high level of
transcript accumulation in these experiments may in part be
a dosage effect due to DNA replication and concomitant
increases in potential transcription templates.
The overproduction of p5 RNA by genomes containing
deletions between 15 and 37 mu suggests the existence of a
negative regulatory element. Since the presence of this
putative negative regulatory sequence in wild type genomes
does not appear to eliminate expression from p5, but rather
reduces its accumulation the putative regulatory element
will be referred to as a depressor (dep) of expression and
the mutant phenotype as a dep- phenotype. Because
complementation of dep- mutants did not affect the ratio of
transcripts produced by the complementing genome, the dep
mutations must be cis-active.
In order to more precisely map the putative dep
sequence, additional deletion mutants were constructed and
analyzed as shown in figure 3-8. In this experiment several
left side deletion mutants were cotransfected with a capsid
deletion mutant, dl63-86. No AAV RNA was detected after
transfection of the left side mutants without
complementation (data not shown). Complementation of d!23-
28 resulted in the production of the expected rep mutant
RNAs. The p5 and pl9 RNAs of dl23-28 were produced at
normally low levels compared with the 2.3 kb p40 RNA. Thus
dep does not appear to reside between 23-28 mu. Similarly

58
Figure 3-6. Complementation of expression of rep
mutants. KB cells were transfected with 20 /xg of either
620ori~ (lane 1), ins32ori~ (lane 2), ins32ori~ and dl49-94
(lane 3) , dll0-37ori~ and dl49-94 (lane 4) , dll0-37ori~
(lane 5), or dl49-94 (lane 6) and infected with Ad-2. RNA
was prepared and analyzed as described in figure 3-1 except
that the blot was probed with a 32P-labeled fragment of AAV
DNA from 52-91 mu. The position of RNAs indicated on the
left are the wild type spliced p40 RNA (2.3 kb) and on the
right the unique p5 transcript of dll0-37ori~ seen in lane 4
(2.6 kb).

59
\
Figure 3-7. Complementation of expression by left side
deletion mutants. KB cells were transfected with 20 ¿¿g of
either d!58-87 (lane 1), d!63-86 (lane 2), dl03-23 (lane 3),
<¿103-23 and dl63-86 (lane 4), dllO-37 and dl58-87 (lane 5),
dllO-37 (lane 6), pSM620 (lane 7), dll5-86 and pSM620 (lane
8), or dll5-86 (lane 9) and subsequently infected with Ad-2.
The locations of specific RNAs originating from capsid
deletion mutants are indicated by a superscript c. Only the
spliced p40 and unspliced pl9 and p5 RNAs are indicated.
The bottom of the caret points to the dl58-87 RNAs, and the
top of the caret points to the dl63-86 RNAs. The truncated
RNAs of the rep deletion mutants are indicated with the
superscript r.

60
complementation of either dl03-ll and dlll-23 also resulted
in production of rep mutant RNAs with wild-type
stoichiometry.
Although both dllO-37 and dlll-23 are missing the pl9
promoter, only dllO-37 overproduced a p5 RNA (lane 5). Thus
the dep phenotype is not simply a reflection of reduced
competition due to the deleted promoter, as seen for the
SV40 early promoter (Kadesch and Berg, 1986). The 2.6 kb
RNA produced upon complementation of dllO-37 was shown
clearly to be a p5 transcript by its hybridization to a p5
specific probe (figure 3-8B, lane 5).
Because neither dll0-23 nor dl23-28 overproduced any
RNAs, the dep seguence can be predicted to reside between
28-37 mu. Indeed, dl23-37 exhibited a unique phenotype upon
complementation. The truncated p5 transcript of dl23-37 was
produced at low levels compared to the 2.3 kb p40 transcript
and was predominantly unspliced (based on the size of the
RNA) . However, complementation of the mutant resulted in
the accumulation of an RNA equal in abundance and slightly
larger in size than 2.3 kb. This RNA was a size predicted
for the spliced pl9 message of dl23-37. Thus d!23-37
appears to accumulate excess amount of the pl9 RNA but
normal amounts of the p5 RNA.
The results of the complementation experiments are
summarized in figure 3-9. Deletion of sequences between 23-
37 mu was sufficient for overproduction of pl9 RNA but

61
insufficient for overproduction of p5 RNA. Thus a
functional dep sequence for pl9 appears to reside between
23-37 mu. Further because deletion from 23-28 mu had no
effect on RNA levels, one dep region must reside between 28-
37 mu. However the functional dep sequence for p5 appears
to also require an element between 10-23 mu. These
conclusions are discussed below.
Another point worth noting is that although the p5 and
pl9 messages are normally accumulated in predominantly
unspliced form, the p5 RNA or pl9 RNAs overproduced by dllO-
37 or dl23-37, respectively, were a size predicted for
spliced messages. Thus the dep mutations appeared to
simultaneously increase both the inherent splicing
efficiency of an RNA as well as the abundance of that RNA.
Negative regulation of p40 expression by the dep
region. The expression of deletion mutants suggests that
the p5 and pl9 genes are controlled by negative regulatory
elements, one of which appears to exist between 28-37 mu of
the AAV genome. To determine if dep is a separable genetic
element, hybrid AAV genomes were created which placed a dep
region downstream from a p4 0 promoter. This was done
because expression from p40 appeared unaffected by the dep
mutations. Genomes which contain a tandem duplication of
sequences including p40 and which either include (dp28-49)
or do not include (dp>37-58) the putative dep region were
created and analyzed. The structures of the genomes are

Figure 3-8. Accumulation of RNAs by left side deletion mutants. A)
Ad-2 infected KB cells were transfected with 20 g of either dl63-86 alone
(lane 1), or cotransfected with dl23-28 (lane 2), dl03-ll (lane 3), dlll-23
(lane 4), dllO-37 (lane 5), or dl23-37 (lane 6). RNA from each transfection
was analyzed as in figure 3-1. Lane M contains PstI digested pSM620 DNA as
markers. The spliced p40 RNA of the capsid deletion mutant is indicated on
the left side of the figure with a superscript c. The wild-type-size
spliced p40 message produced by all left side deletion mutants is indicated
on the right side of the figure (p40r) . The p5 and pl9 RNAs of left side
deletion mutants are indicated by filled boxes or open circles,
respectively. B) Same as in A except that the blot was probed with a 32P-
PvuII-Hindlll fragment from pLB709 containing AAV DNA from 0-11 mu. The
size of most abundant RNA (lane 5) is indicated on the right.

A
M 1 2 3 4 5 6
B
1 23456
o\
U>

Figure 3-9. Phenotypes of left side deletion mutants. The AAV genome
is drawn schematically, with the position of the AAV promoters indicated by
the upside down L. The slash marks represent a large portion of the capsid
gene left out for simplicity. The structures of left side deletion mutants
are drawn below and next to the mutant designation. The relative levels of
transcripts observed for each mutant are shown on the right as high (H) , low
(L) , or none (N) . The ratings are based on comparison to that of the
complementer; levels approximately equal to that of the spliced p40 message
or the p5/pl9 messages are considered high or low, respectively.

p5
JL
11 -23.
23-28
10-37.
23-37.
15-86
pl9
-'w—
â– Wr
-ov-
-/uv-
-4a/-
p5 pl9 p40
L N H
L L H
H N H
L H H
H N N

(Jl

66
diagramed in figure 3-10. Each genome contains an upstream
and downstream p40 promoter (p40u and p40c*, respectively) .
The structure of the p40^ transcription unit is identical
for both genomes thus the levels of this transcript (wild
type 2.3 kb in size) can be used for comparison with that of
the upstream p40. However, the upstream p40 promoters
differ, being placed upstream from different but overlapping
duplicated portions of the genome. Only in the case of
dp>28-49 does the duplicated region contain the dep
sequences.
As shown in figure 3-11, both genomes produced
approximately equal amounts of the downstream p40 message.
However dp28-49 accumulated significantly lower amounts of
the p40u transcript compared with its own p40d transcript or
with that of dp37-58. The ratio of the p40u to the p40^
transcripts of dp28-49 was qualitatively similar to the
p5/pl9 to p40 ratio of transcripts accumulated by wild type
AAV indicating that the dep element inhibited the
accumulation of the p40 transcript when placed within the
transcriptional unit.
As noted in the figure 3-10, several different sized
messages could be produced from the p40u promoter of either
of the two genomes depending on the number of splicing
events in a particular message. Interestingly, the
predominant p40u message produced by dp28-49 was a 3.5 kb
unspliced message while the predominant message produced by

67
dp37-58 appeared to be the 2.9 kb doubly spliced message.
Thus the inclusion of the dep sequence in dp28-49 also
appeared to inhibit splicing of the message. These data
again support the notion that dep functions both to affect
the steady state levels and splicing efficiency of
particular messages.
Stability of AAV mRNAs. Several mechanisms could explain
the action of the dep region. One possibility includes the
specific destabilization of the RNA. If true this would
predict that the wild type p5/pl9 and p4 0 messages would
have very different half lives. In order to examine the
stability of the AAV RNAs, AAV infected cells were treated
with actinomycin D (to block transcription) for various
times, and the RNAs analyzed by northern blotting.
As shown in figure 3-12, most of the AAV RNAs remained
constant in level up to 4 hours after addition of
actinomycin D. Although the 2.6 kb p40 message decreased
rapidly in abundance, the levels of the p5 and pl9 RNAs
remained relatively constant. These data suggest that only
the 2.6 kb message is unstable. Alternatively, the
disappearance of the 2.6 kb RNA may reflect a continued
processing of the message to the spliced 2.3 kb species
after the addition of actinomycin D, although this seems
unlikely since the levels of unspliced p5 and 19 RNAs
remained constant.

Figure 3-10. Structures of AAV genomes with duplications of p40. The
organization of the wild type genome (WT) , dp28-49 and dp37-58 are drawn
schematically. The position of promoters, introns (carets), and the TRs
(black boxes) are indicated. The p40 promoters of the dp mutant genomes are
indicated with a superscripts u (upstream) or d (downstream). The
duplicated portion of the dp genomes are indicated by open rectangles below
the genome representation, separated by lines indicating the 5' boundary of
the duplicated region. The stippled area indicates the position of the dep
region present in the duplicated portions of dp28-49. The structure and
sizes of the p40 RNAs predicted for the genomes are also drawn below each
genome.

69
\ \ 'S
« n
*1 oí
u
”o
♦
CL
â–  I
» «SI M ff>
•*> 10 m m
I I
=b
o.

70
1-23
Figure 3-11. Expression of AAV genomes containing
duplications of p40. Ad-2 infected KB cells were infected
with AAV (lane 1), mock transfected (-), or transfected with
dp28-49 (lane 2) or dp37-58 (lane 3) . The wild type size
messages are indicated on the right and RNAs originating
from the upstream p4 0 promoters (2.9 and 3.5 kb) and the
downstream p40 message (2.3 kb), are indicated on the left.

71
12 3 4
%
nf? —
Figure 3-12. Stability of AAV RNAs. KB cells were
coinfected with Ad-2 and AAV-2. Actinomycin-D was added to
separate plates at 24 hours postinfection. Cytoplasmic RNA
was then isolated at 0 (lane 1) , 30 (lane 2) , 60 (lane 3) ,
or 240 (lane 4) minutes after addition of the drug. The
positions of specific RNAs are indicated on the left side of
the figure.

72
Transcription of AAV in isolated nuclei. Another
mechanism by which the dep region might act would be through
negative regulation of transcription initiation. This
predicts that the p40 promoter and downstream sequences
would be preferentially transcribed. Transcription rates of
different regions of the AAV genomes were measured by NRT
analysis using nuclei from Ad-2 or Ad-2 and AAV-2 infected
cells, followed by hybridization to restriction digested AAV
DNA blotted to nitrocellulose. The cloned AAV DNAs used are
identical to that described for figure 3-5 and thus
recognize either p5 specific transcription (fragment d), p5
and pl9 transcription (fragment c) , or transcription from
all AAV transcription units (fragments a and b).
In the first experiment infected KB cells were used
(figure 3-13). No AAV transcription was detected from Ad-2
infected cells while a large amount of hybridization signal
to AAV DNA was detected using RNA transcribed in nuclei from
coinfected cells (panel B, lane 3). Fragment b contains AAV
DNA from 42-91 mu and should hybridize to all complete AAV
transcripts. Fragment c contains AAV DNA from 11-40 mu and
should be specific for the p5 and pl9 messages.
Although the steady state levels of p40 RNA (which consists
of mostly fragment b sequences) are 10-20 fold higher than
that of the p5 and pl9 RNAs, more transcriptional activity
was detected with the p5 and pl9 specific probe (fragment c)
than that detected with probe complementary to all AAV RNAs.

73
Thus the rate of initiation from the p5 and or pl9 promoters
appears to be at least as high as that from the p40
promoter.
A high level of transcription from the p5 promoter is
specifically indicated by comparison of hybridization signal
to fragments a and d. Both fragments contain approximately
0.2 kb of complementarity to AAV RNAs either at the 3' end
of all AAV transcripts in fragment a or to the 5* end of the
p5 transcript in fragment d. An equal amount of signal was
detected with either fragment indicating that the rate of
transcription for the p5 promoter is very high and is not
proportional to the amount of p5 RNA relative to p40 RNA.
Similar results were obtained using nuclei from
infected HeLa cells (figure 3-14). Transcription rates of
different regions of the AAV genomes were measured by NRT
analysis using nuclei from Ad-2 or Ad-2 and AAV-2 infected
cells followed by hybridization to restriction digested AAV
DNA blotted to nitrocellulose. The cloned AAV DNAs used are
the same as in the previous figure. Transcription was
measured at both 12 (A and B) and 24 hours (C and D)
postinfection. Although the signal detected with fragment b
was somewhat higher than for fragment c in this experiment,
the transcription rate per nucleotide of probe was
approximately equal. In this particular experiment the
fragment d was run off the gel.

74
Thus, in experiments using either KB or HeLa cells,
transcription
from
the
p5
and
pl9
sequences appears
approximately
equal
to
that
of
p4 0
specific sequences.
These data indicate
that the
overall
ratios of AAV RNA
accumulated must be determined in part by mechanisms after
transcription initiation. Further because the rates of
transcription of the left half of AAV (11-40 mu) appeared at
least as highe as of the right half of the genome (42-91
mu), it is suggested that the p5 and pl9 messages are being
prematurely terminated.
Although transcription rates have not yet been measured
for dep mutants, the accumulation of RNAs by these mutants
is proportional to the observed transcription rates of the
wild type genome. It is proposed that the dep sequences act
posttranscriptionally and are responsible for the observed
difference between transcription rates and the steady state
levels of RNA p5 and pl9 RNAs.
Another observation made in the last two experiments is
that the amount of Ad transcription detected was
significantly lower in cells coinfected with AAV. Ad
transcription detected using probes for either the El, E4,
or late regions was significantly reduced, although the
inhibition of transcription appeared greater in KB cells
(approximately 10 fold) than in HeLa cells (approximately 3
fold). The inhibition observed in HeLa cells was similar at
both early (12 hours) and late (24 hours) times

75
postinfection. Thus, although the AAV rep gene products
activate AAV transcription, AAV gene expression can be
correlated with an inhibition of Ad transcription.
AAV coinfection is known to inhibit Ad replication.
Interestingly, AAV appears to inhibit the production of
progeny Ad to a much greater extent than Ad DNA replication.
Thus the inhibition of Ad transcription may be a major
effector of the inhibition of the helper virus replication.
Whether the inhibition of Ad transcription is a consequence
of a specific AAV gene product or due to competition for
limited transcriptional machinery is not known.
Discussion
In this study cloned AAV genomes have been analyzed in
order to delineate genetic elements involved in the
autoregulation of AAV gene expression. These data
illustrates that autoregulation occurs both by trans-active
AAV proteins and by cis active elements.
Positive regulation in trans by an AAV rep gene
product. Several experiments show that at least one AAV
protein encoded within the p5 gene is essential for trans¬
activation of AAV transcription. First, cloned genomes
containing small lesions within the ORF unique to the p5 and
pl9 transcripts accumulated extremely low levels of all the
AAV RNAs compared to genomes containing wild type p5 and pl9
genes. Virtually all left side mutations had a similar
effect. The subtle nature of certain mutations (8 base

76
A B
12 3 12 3
Figure 3-13. Transcription of AAV in isolated KB cell
nuclei. KB cells were infected with Ad-2 (panel A) or
coinfected with Ad-2 and AAV-2 (panel B) . Nuclei were
isolated at 24 hours postinfection and transcription
reactions were carried out for 10 minutes. Equal amounts of
RNA were then hybridized to southern blots containing Pstl-
digested Ad-5 Ela and Elb DNA from pXHOC (lane 1) , E4 DNA
from pXBAC (lane 2) or PstI- and HindiII-digested 620ori~
DNA (lane 3). The AAV fragments indicated are as described
for figure 3-5.

Figure 3-14. Transcription of AAV in isolated HeLa cell nuclei. HeLa
cells were infected with Ad-2 at an moi of 10 (panels A and C) or coinfected
with both Ad-2 and AAV-2 at an moi of 20 (panels B and D) . Nuclei were
isolated at either 12 hours (panels A and B) or 24 hours (panels C and D)
posttransfection and labeled nuclear RNAs were used as probe for Southern
blots of restriction enzyme digested Ad-5 and AAV-2 DNAs. Lane 3 contains
PstI- and Hindlll digested 620ori~ DNA as described in the legend to figure
3-5. Lanes 1 and 2 contain PstI-digested Ad-5 DNAs corresponding to the E4
region (pXBAC) or late region, respectively.

N> >
CO
f
$' #
"0 W
9
ro oo
oo
I ft ft
NO O
CO
8 L

79
frameshift
insertions)
indicates
that the
defects
in
expression
were due to
mutational
effects
on
the
rep
proteins.
Supporting this notion is the
observation
that
the
defect in the accumulation of RNA was complementable in
trans. However the amount of transcripts accumulated by the
rep~/ori~ mutants upon complementation by an ori+ genome was
significantly lower than that of 620ori~. This may reflect
a competition by the ori+ complementing AAV genome for
limited transcriptional machinery as noted by other workers
(see below). Complementation of expression of ori+ rep
mutants was very efficient.
Activation of AAV expression did not require DNA
replication. This was illustrated both by the efficient
expression of 620ori~ and by the ability to complement
expression of ori~ rep mutants. The stoichiometry of the
different messages was also identical to that produced by
replicating molecules. Thus AAV gene expression is not
divisible into simple early and late stages separated by DNA
replication.
Evidence that activation by the rep proteins is at the
level of transcription initiation has also been presented.
When transcription rates of transfected genomes in isolated
cell nuclei were measured, transcription from a rep+ genome
(620pri“) was detected, although transcription from a rep~

80
genome (ins32ori-) was not. Thus, the rep mutant was
defective at the level of transcription initiation.
One question that may be addressed is whether
transactivation by rep is specific for AAV genes or is
promiscuous in activity as observed for the Ad Ela (Green et
al., 1983: Treisman et al., 1983; Gaynor et al, 1984; Stein
and Ziff, 1984; Kao et al., 1985) and herpes ICPO and ICP4
proteins (Everett, 1984; O'Hare and Hayward, 1985a,b). The
observation that AAV infection inhibits transcription of the
Ad genome rather than activating it strongly suggests that
transactivation is specific for the AAV genome although this
point requires further investigation.
Because a mutation which specifically blocked
production of p5 gene products (insll) inhibited the
accumulation of AAV RNAs, a role for a p5 gene product(s) in
transactivation of expression is specifically indicated. It
is not yet possible to determine if pl9 encoded proteins are
also required for this process. Although transactivation
appears independent of DNA replication, a p5 gene product is
required for both AAV DNA and RNA synthesis. Whether the
two functions of the rep gene products represent one or
multiple activities is not known.
However the phenotype of ins42 suggests that different
functional domains of the rep gene products might be
involved in RNA and DNA synthesis. Although the mutation at
42 mu inhibits DNA synthesis by 100-1000 fold, only a 10

81
fold reduction in RNA synthesis is seen. Thus the mutation
at 42 mu appears to differentially affect DNA replication
and transcription.
The AAV p5 gene product (s) appears to be functionally
analogous to the SV4 0 T-antigen. Both gene products are
required for viral DNA replication and transactivation of
viral transcription (Hartzell et al., 1984). As discussed
above, the AAV p5 gene product may also be composed of
separate functional domains as is the SV40 T-antigen.
Several workers have recently noticed significant amino acid
homology between the predicted p5 and SV40 T-antigen
proteins in a region conserved between most DNA dependent
ATPases (N. Muzyzka, C. Astell personal communications).
This suggests that the different proteins have similar
enzymatic activities.
It should be noted that all eukaryotic DNA virus
families, including Herpes, Ad, papilloma, and polyoma
viruses, examined in detail appear to encode transcription
activator proteins (Jones and Shenk, 1979; Everett, 1984;
Spalholz et al., 1985). Thus the ability to activate
expression may be as essential a feature to these exogenous
replicons as are origins of replication. This feature is of
obvious importance when one thinks of the competition for
limited transcription machinery between viral genes and the
vast excess of cellular genes.

82
Evidence for trans-active regulation of AAV gene
expression by the rep proteins has also been recently
reported by another laboratory. Tratschin et al. (1986)
have reported that expression of the cat gene, inserted
within the AAV genome under the control of the p40 promoter,
is enhanced by the rep gene. While this study did not
determine at what level activation occurred, our results are
consistent, although the results presented here indicate
that transcription of all AAV promoters is activated by rep♦
In addition this group identified a negative regulatory
function of the rep gene products on expression of the p40
cat gene. This negative regulation occurred in human 293
cells, which constitutively produce Ad-5 Ela and Elb gene
products, but not in HeLa cells. Although the results
presented in this section do not address this phenomenon, a
negative regulatory activity of rep on heterologous genes is
described in chapter IV.
Rhode (1985) has also reported that expression of the
cat gene under the control of the p3 8 capsid gene promoter
of an autonomous parvovirus, HI, is enhanced by an HI p4
gene product. Again, no work at the RNA level was done by
Rhode. Both Rhode and Tratschin et al. (1986) reported that
expression of genomes was most efficient when the
transactivator genes were carried on the same plasmid
compared to cotransfection of separate plasmids. This was
suggested to represent competition for limited transcription

83
machinery, possibly the parvoviral transactivator proteins
themselves.
Cis active negative regulation of expression.
Although the p5 gene product is associated with positive
regulation of AAV transcription, several experiments
indicate that seguences within the p5 ORF itself act as
negative regulators of p5 and pl9 gene expression. Several
left side deletion mutants accumulated significantly greater
than normal amounts of the p5 or pl9 transcripts upon
complementation with rep+ genomes. Normally the p5
transcript is accumulated at 5-10% the level of the p40
transcripts at late times in Ad infected cells. However,
the dep mutants accumulated equal amounts of either the p5
or pl9 and p40 transcripts. The effect of the dep mutations
were clearly cis-active as the expression of the
complementing genomes were qualitatively unaffected.
The relative increases in p5 or pl9 transcripts
(compared to the levels of p40 transcripts) accumulated by
the dep mutants were not due to reduced expression from p40.
First, the amounts of p40 RNA accumulated by all left side
mutants upon complementation were similar. Second, ori+ dep
mutant genomes produced as much or greater amounts of p40
transcript upon complementation than the complementers
themselves. Thus the dep phenotype was due to enhanced
accumulation of the affected RNAs.

84
A genome lacking all promoters except for p5 (¿115-86),
accumulated a highly truncated p5 message in excess of the
complementor's p5 message. The high level of p5 expression
from this mutant demonstrates that the dep phenotype was not
a result of the close proximity of the p5 and p40 promoters
in the mutants. The unigue structure of the truncated p5
RNA also eliminates the possibility that it was produced
from a recombinant genome of unknown structure.
Various deletion mutants were used to define the region
of the genome responsible for cis-active negative
regulation. The first mutant discovered to overproduce the
p5 transcript was ¿110-37 whose deletion removed the pl9
promoter. However, deletion of the pl9 promoter was not
sufficient to reproduce the dep phenotype as dlll-23
accumulated normal amounts of p5 transcripts. Thus one
would predict that the negative regulatory sequence would be
contained between 23-37 mu.
One deletion mutant, ¿123-37, exhibited a unique
pattern of accumulated transcripts. In this case the
truncated p5 transcript was still produced at low levels but
an additional band was detected which was slightly slower in
mobility and approximately equal in amount to the 2.3 kb p40
message. It is assumed that this species is a spliced pl9
RNA based on the predicted size of that message. Thus
removal of 23-37 mu was required but not sufficient for
overproduction of the p5 transcript but sufficient for

85
overaccumulation of the pl9 transcript. In explanation for
these observations it is proposed that two dep elements
exist within the AAV genome, one between 15-2 3 mu and
another between 28-37 mu. However since the presence of the
upstream dep region (between 15-23 mu) in one mutant, d!23-
37, does not appear to inhibit the production the pl9 RNAs,
the negative regulatory element most likely resides upstream
from the promoter (upstream from 19 mu).
The regulatory effect of seguences between 28-37 mu was
not specific to the p5 and pl9 promoters. When the p40
promoter, whose expression was unaffected by the dep
mutations, was placed upstream from the 28-37 mu sequences
the accumulation of the upstream p40 transcript was strongly
inhibited
relative to
a second downstream
p4 0.
The
inhibition
was not due
to the competition
by
the
second
downstream
p40 since
removal of 28-37
mu
from
the
duplication
significantly enhanced the expression
of
the
upstream but not the downstream p40 transcript.
Although the studies presented here have not elucidated
the mechanism by which the putative dep sequences act,
several aspects are noteworthy. First the action of the dep
sequences appear directional, acting on upstream
transcription units. Second, the 28-37 mu sequences can act
on at least one heterologous promoter (p40).
Several mechanisms could explain the observed negative
regulation of p5 and pl9 expression. One explanation is

86
that the putative dep elements specifically destabilize
RNAs. Initial results would indicate that the AAV RNAs do
not differ significantly in cytoplasmic stability; however
the messages may differ in nuclear stabilities.
Alternatively the dep elements might act as binding sites
for repressors of transcription initiation. If so, one
would predict that transcription rates of the wild type p5
and pl9 genes would be much lower than that for the p40
transcript. However, when transcription rates were measured
using different DNA probes, an approximately equal amount of
transcription for all AAV genes was detected.
An additional mechanism might involve premature
termination of transcription. Transcription of SV40 and an
autonomous parvovirus, Minute Parvovirus of Mice, has been
suggested to be regulated by some method of transcription
attenuation (Ben-Asher and Aloni, 1984) . This mechanism
appears to be the most consistent with the observations made
here.
One other aspect of regulation by dep appears to be an
inhibition of splicing of the affected message. This
conclusion is based on the observation that in all cases an
increased splicing efficiency cosegregates with the dep
phenotype. The dep sequences also appear to inhibit splicing
of p40 transcripts when placed within a normally
predominantly spliced p40 transcription unit. These
observation suggest that dep exerts its effects in cis. by

87
altering the structure of RNAs. If so, such structural
changes can easily be envisioned to affect either
transcription termination or RNA stability as discussed
above.
Finally we have not addressed the question of which
protein(s) may act to facilitate regulation by dep or why
such regulation even exists. Because dep does not appear to
affect expression of the p40 capsid gene, it is interesting
to speculate that dep is needed to modulate the amount of
rep protein expression at a time after DNA replication has
begun and when large amounts of capsid proteins are needed.
Conversely, expression of the p40 capsid gene appears to be
negatively regulated by rep in 293 cells (Tratschin et al.,
1986). 293 cells only express the Ela and Elb genes of Ad;
resulting in a physiologic state similar to that present
early during Ad infection. Thus, although AAV transcription
cannot yet be divided into early and late phases, such a
distinction may be made at the posttranscriptional level.
Such multiple levels of control are strikingly similar
to the regulation of the SV40 genome. Early in infection
the T-antigen gene is highly expressed (Benoist and Chambón,
1981; Buchman et al., 1984) while expression of the late
region is repressed (Omilli et al., 1986). T-antigen
synthesis leads to both DNA replication and transactivation
of SV40 transcription although the synthesis of early RNA is
kept relatively low compared to late RNAs by repression of

88
early region transcription by T-antigen itself (Tijan,
1982) . The ability to negatively autoregulate expression
has been shown to be a common feature of viral transcription
transactivators, including the Ad Ela, Herpes ICP4, and SV40
T-antigen gene products (Tijan et al., 1982; O'Hare and
Hayward, 1985a,b; Hearing and Shenk, 1985; Smith et al.,
1985). It will be interesting to determine if regulation by
dep is also mediated by a rep gene product.
In summary, despite the small size of the AAV genome,
AAV gene expression appears to be controlled by a variety of
mechanisms. Thus the small size of the genome becomes a
great advantage due to the simplicity with which it can be
manipulated and analyzed. Further, the AAV genome is
subject both to regulation by cellular and helper virus
functions. Thus, the study of AAV gene expression should be
of pivotal importance in the elucidation of the interplay
between complex viral regulatory elements and cellular
metabolism.

CHAPTER IV
INHIBITION OF GENETIC TRANSFORMATION AND GENE EXPRESSION
OF HETEROLOGOUS GENES BY THE AAV REP GENE
Introduction
The ability to inhibit the replication of the helper
appears to be a common feature of defective viruses.
Certainly most defective interfering particles which require
the wild type virus to replicate inhibit the replication of
the parental virus. In this case the inhibition of the
helper virus reflects a simple competition between the
independent and dependent genome for limited gene products.
However for the helper dependent bacterial virus p4, which
is genetically distinct from the helper, inhibition is an
active process with several of the defective virus gene
products acting directly to alter the expression and
replication of the helper virus (Calendar, 1986).
Although AAV appears to have no detectable pathologic
or cytopathic effects on the host cell, several inhibitory
effects by AAV on the replication and oncogenicity of its
helper viruses have been documented. First, AAV coinfection
inhibits the replication of Ad DNA as well as the yield of
infectious virus (Hoggan et al., 1966; Casto et al.,
1967a,b; Parks et al. , 1968; Carter et al. , 1979).
Previously it was thought that the ability of AAV to inhibit
89

90
the replication of Ad was due to competition for replication
factors. However, the fact that AAV is genetically
unrelated to Ad raises the possibility that AAV might
actively inhibit Ad replication.
The mechanisms by which AAV inhibits the activity of
the helper viruses are of particular interest in that AAV
also inhibits the oncogenic properties of the helper viruses
as well as the tumorigenic properties of helper virus
transformed cells. AAV inhibits the ability of Ad to
transform cells and to cause tumors when injected into
susceptible animals (Kirchstein et al. , 1968; Casto and
Goodheart, 1972; Mayor et al., 1973; de la Maza and Carter,
1981) . AAV infection of an Ad-5 transformed cell line,
H14b, dramatically inhibits the ability of the cells to form
tumors after inoculation into newborn hamsters (Ostrove et
al., 1981). Although no loss of viability occurred in the
transformed cells after AAV infection, the production of the
Ad-5 58 kd tumor antigen was reduced by approximately 80%.
AAV also inhibits the oncogenicity of HSV transformed
cells, although no effects on HSV gene expression are yet
known (Cukor et al., 1975; Blacklow et al. , 1978). AAV
infection of HSV transformed cells before inoculation into
hamsters has also been reported to completely eliminate
metastasis of the resulting tumors. Finally, a recent
report demonstrated that AAV infection reduces the
tumorigenic properties of ras transformed mouse cells (Katz

91
and Carter, 1986) . Because AAV virions were used in these
studies the assignment of inhibitory functions to specific
regions of the AAV genome was not possible.
The experiments presented here show that AAV genomes
greatly inhibit stable transformation by dominant selectable
marker genes upon cotranfection into mammalian cells. AAV
genomes are also shown to inhibit the expression of a
heterologous gene, pSV2cat, in transient gene expression
assays. Expression of intact rep genes was required for
both inhibitory activities. Both inhibitory activities
occurred in the absence of helper virus functions and were
partially blocked by addition of a cloned Ad-5 E4 gene in
the transfection assays.
Results
AAV mediated inhibition of transformation. The
following experiments were performed in an attempt to create
mammalian cell lines which constitutively expressed the
trans-active AAV p5 and pl9 gene products. In doing so, the
strategy was to use a cotransfection and coselection
technique using a dominant selectable marker. In the first
experiment pGCcos3neo (Graf et al., 1974), which contains
the bacterial neo gene under the control of the SV40 early
promoter and early processing signals was used as the marker
plasmid. The plasmid is derived from pSV2neo (Southern and
Berg, 1982) and differs mainly by the addition of a lambda
cos sequence outside of the neo gene. This construct

92
confers resistance to the drug G-418 in mammalian cells.
This construct was transfected alone (0.1 pg) or
cotransfected with an excess amount of various cloned AAV
genomes (5 /¿g) . An excess of AAV DNA was used in order to
ensure that every cell that received the neo gene also
received AAV DNA. Transfection experiments were initially
carried out in B78H1 cells because of their high rate of
transformation after DNA mediated gene transfer (Graf et
al., 1984). Resistant cells were selected in media
containing 1 mg/ml G-418 for 10-18 days and finally fixed
with formaldehyde and stained. Plates of cells containing
low numbers of colonies were grown for several additional
days to allow detection of any slow growing colonies. The
plates of such an experiment are shown in figure 4-1.
Transfection of the neo gene alone resulted in the
formation of approximately 200 G-418r colonies (A). However
cotransfection with pSM620, a plasmid containing the entire
AAV genome, reduced the number of colonies formed by
approximately 99% (B) . Thus the AAV genome was inhibitory
to transformation.
In order to determine what specific AAV functions or
sequences were involved in the inhibition, cloned mutant AAV
genomes were also cotransfected with pGCcps3neo.
Cotransfection with cloned AAV genomes containing a large
deletion of the capsid gene, dl63-86 (D) , or deletions of
both TRs, 620ori~ (C) also inhibited transformation.

93
However, cotransfection with a mutant containing a
frameshift insertion at 32 mu, ins32ori~ (F) , within the
unique p5 and pl9 ORFs had no effect on transformation.
Cotransfection of insll (E) also inhibited transformation
but to a lower extent, resulting in the production of an
intermediate number of colonies (approximately 20) . From
these data we concluded that expression of one or both of
the rep genes were required for the inhibitory effect.
Also, because both an ori~ and a capsid deletion mutant
inhibited transformation, neither the replication of AAV DNA
nor the production of AAV virus appeared required for the
inhibition of transformation.
Genomic DNA analysis of G-418 cell clones. Several of
the resistant colonies generated were cloned and analyzed.
Colonies arising from cotransfection of pGCcos3neo and
either 620ori~ or a rep deletion mutant, dl03-23 were
isolated using cloning cylinders and the cells were grown up
in large amounts for the isolation of genomic DNA. Genomic
DNA was digested with PstI. fractionated by agarose
electrophoresis, and analyzed by blotting and hybridization
to 32P-labeled AAV DNA. As shown in figure 4-2, no AAV DNA
was retained in the rare cell colonies derived from
cotransfection with 620ori~ but was retained in colonies
derived from cotransfection with the rep~ genome. This data
supports the notion that the AAV rep genes were strongly
inhibitory to transformation and genetic selection.

Figure 4-1. AAV genomes inhibit genetic transformation. In this
experiment B78H1 cells were transfected with 0.1 g of pGCcos3neo alone (A)
or cotransfected with 5 g of pSM620 (B) , 620ori~ (C) , dl63-86 (D) , insll
(E) , or ins32pri (F) . Colonies were selected for 14-21 days in media
containing 1 mg/ml of G-418 after which the cells were fixed and stained.

95
• •

96
6 7
1 2 3 4 5
«
-2.4
-1.4
Figure 4-2. Southern blot analysis of G-418r cell
clones. Several G-418r resistant colonies arising after
cotransfection of pGCcos3neo and either 620ori~ (lanes 1-4) or
<1103-23 (lanes 5-7) . A 10 ¡iq amount of DNA from each
experiment was digested with PstI and fractionated by agarose
gel electrophoresis and analyzed by blotting to nitrocellulose
and hybridization to 32P-labeled AAV DNA. The sizes of the
two predominant bands in lanes 5-7 are shown on the right side
of the figure.

97
Specificity of the AAV mediated inhibition of
transformation. Several experiments were carried out to
determine if the inhibitory effect of the AAV rep genes was
specific for either the cell line used, the neo gene or for
the SV40 early regulatory sequence of pGCcos3neo. Table 4-1
contains the results of several experiments carried out in
either B78H1 or murine Ltk- cell lines using various cloned
AAV genomes and various selectable markers. Cotransfection
with 620ori~ and dJL63-86 (only done in B78H1 cells)
inhibited transformation by pGCcos3neo in all experiments
while the rep mutant ins32ori~ did not. The AAV mutant
insll inhibited transformation in two experiments shown
(A,B) but not in two additional experiments. The reason for
this variability is not known, but it should be noted that
the latter experiments were done in Ltk” cells while
experiments A and B were done in B78H1. Further, the
inhibition by insll in experiments A and B was not as great
as by genomes containing wild type p5 and pl9 genes. The
mutant ins42 failed to inhibit transformation in any
experiment.
Transformation by pTK (Wigler et al., 1979), containing
the HSVI thymidine kinase gene regulated by its endogenous
promoter, and transformation by the neo gene under control
of the cellular mouse metallothionein promoter (mMT) was
inhibited by cotransfection of 620ori~. In this case the
mMTneo gene was contained in a plasmid vector alone (pMTneq)

98
or in a plasmid (pBPV/MTneo) which also contained the
transforming region of the bovine papilloma virus genome
(Law et al., 1983). Thus the inhibition of transformation
was not specific for a single selectable marker or promoter.
Several experiments indicated that transformation by
one vector was to some extent immune to rep dependent
inhibition. These experiments involved dl52-91/neo
(Hermonat and Muzyczka, 1984) which contains a neo gene
under the control of the SV40 early promoter inserted within
the deleted region of an AAV mutant, dl52-91. This vector
has been used to create recombinant virus stocks capable of
transforming mammalian cells to G-418r with high frequency.
All three of the AAV promoters as well as intact p5 and pl9
rep genes are retained in this construction. This genome
replicates with wild type efficiency in the presence of Ad,
indicating that the rep genes function nearly as efficiently
as in a wild type genome.
Transformation to G-418r with this vector would be
expected to be relatively inefficient since every neo gene
is physically associated with the AAV rep genes. However
transformation by d!52-91/neo was about as efficient as
pGCcos3neo (table 4-1). This high rate of transformation
was not due to poor expression of the p5 and pl9 genes of
d!52-91/neo as indicated by the following. First,
cotransfection of additional 620ori~ DNA with d!52-91/neo
resulted in 10-50 times greater numbers of colonies observed

99
for cotransfections with pGCcos3neo. Although cotransfection
of 620ori~ with dl52-91/neo did reduce the number of
colonies, some of this inhibitory effect may have been of a
competitive nature since ins32ori~ exhibited about half
the inhibitory effect. Second d!58-87/neo inhibited
transformation of pTK in trans upon cotransfection (table 4-
2) . Thus, transformation by dl52-91/neo appeared resistant
to inhibition by the rep genes.
The notion that transformation by one vector was
refractory to suppression by the rep gene products suggests
some specificity of regulation. This data also indicates
that inhibition of transformation does not reflect a lethal
effect of rep gene expression to the host cell.
Dose dependent inhibition of transformation. In the
above experiments, large excesses of AAV DNA compared to
selectable marker DNA were used. In order to further
characterize the inhibitory effect cotransfections using
various amounts of AAV or pGCcos3neo DNA were done (table 4-
3) . In these experiments both B78H1 and Ltk- cells were
used. In either case using a constant 0.1 nq of marker DNA,
increasing the amounts of AAV DNA (620ori“) decreased the
number of resistant colonies. Cotransfection with as little
as 0.2 nq of 620ori~ DNA (1/1 rep gene/neo ratio) resulted
in an 80-90% inhibition while higher concentrations of AAV
DNA increased the amount of inhibition up to 99.9% and 97%
for B78H1 and Ltk- cells, respectively. The number of

INHIBITION OF GENETIC TRANSFORMATION BY AAV GENOMES
Number of colonies3
AAV DNAb
Expt
Cell line
Promoter/Marker0
-
620ori~
insl1“
ins32ori~
ins42ori~
dl63-86
A.
B78H1
pGCcos3neo
268
i
21
312 .
1
B.
B78H1
pGCcos3neo
252
0
82
0
C.
B78H1
pGCcos3neo
800
17
220
800
D.
Ltk~
PGCcos3neo
220
6
162
113
E.
Ltk~
pGCcos3neo
252
1
104
225
124
F.
Ltk"
ptk
115
1
500
G.
Ltk"
ptk
104
1
138
H.
Ltk"
pBPV/MMTneo
88
3
108
I.
Ltk"
pMMTneo
224
4
230
J.
Ltk"
d!52-91/neo
220
51
132
K.
Ltk"
dl52-91/neo
360
55
114
aThe numbers shown are for individual plates and represent the total number of colonies on single dishes containing under
200 colonies and an approximate number for dishes containing over 200 colonies.
^Transfections containing AAV DNA recieved 5 ug of supercoiled AAV containing plasmids.
cTransfections with pGCcos3neo, ptk,pBPV/MMTneo or pMMTneo, or d!52-91/neo contained O.lug, 0.05pg, O.lug, or 0.2pg of each
marker gene, respectively.
100

101
TABLE 4-2. INHIBITION OF TRANSFORMATION
OF pTK BY d!52-91/neo
Number of Colonies3
Cotransfected DNA*3
Experiment
-
620ori“
dl52-91/neo
ins32ori'
A.
185
12
15
420
B.
206
5
8
148
aThe numbers of colonies per dish are reported as in
table 1.
^5 fig of AAV containing plasmids were transfected with
0.1 ng of pTK DNA except for (-) which contained only carrier
DNA. All transfections were adjusted to 20 ng of DNA with
carrier.

102
colonies resulting after transfection of a constant amount
of AAV DNA could be increased by the cotransfection of
increasing amounts of pGCcos3neo DNA. Interestingly similar
inhibitory effects were seen at 1/1 ratios of AAV to neo DNA
whether 0.1 or 5 y.q of neo DNA were used. While the sample
size of this experiment is small, these data suggest that
the amount of inhibition depends on the ratio of AAV to
marker DNA used.
The effect of cloned helper virus genes on
transformation. The experiments above describe an
inhibitory activity of the AAV rep genes on genetic
transformation in the absence of helper virus functions.
Various helper virus genes are known to regulate AAV gene
expression and replication, therefore it was of interest to
determine if helper virus genes modulated the rep mediated
inhibitory activity. The Ela gene of Ad appears to directly
activate expression of the AAV pl9 rep gene (Tratschin et
al., 1984b). However experiments attempting to examine the
effect of the Ela gene on AAV-mediated inhibition are
difficult to interpret due to the known inhibitory action of
the Ela gene products on transcription of heterologous genes
such as the SV40 early promoter (Valcich and Ziff, 1985).
Indeed, cotransfection of pXHOC, containing both the Ela and
Elb genes inhibited transformation by pGCcos3neo and three
part cotransfections with both pXHOC and 620ori~ DNA

103
TABLE 4-3. DOSE DEPENDENCE OF AAV AND NEO DNA
ON TRANFORMATION
Experiment
Cell Line
Number
of Colonies3
620ori“ DNA
(¿¿g/Transfection)
0
•
o
1.0
5.0
Ab
B78H1
1000
76
31
1
Bb
Ltk"
420
87
17
14
Cc
Ltkc
480
(0.1)
6
600
(1.0)
39
2000
(5.0)
240
aThe numbers shown are reproted as in table 4-1.
^In these two experiments 0.1 /¿g of pGCcos3neo was
transfected with the indicated amounts of 620ori~.
cIn this experiment variable amounts of pGCcos3neo. as
indicated in parentheses, were transfected with or without
620ori~ (5 fj.g) .

104
inhibited transformation by pGCcos3neo to greater extents
than by either gene alone (table 4-4).
The effect of the E4 gene on the inhibition of
transformation was also determined. The E4 gene of Ad is of
interest in that it may be the only helper virus gene
absolutely essential for AAV DNA replication (Richardson and
Westphal, 1981). The addition of the cloned E4 gene (pXBAC)
to 620ori~/pGCcos3neo cotransfections resulted in a
significant increase in the number of G-418r colonies
generated in the presence of 620ori~. Control experiments
did not reveal any increase in transformation upon addition
of a heterologous plasmid (pFB69, a derivative of pBR322) in
place of pXBAC. Also restriction enzyme digestion of pXBAC
before transfection prevented the increase in colony
formation indicating that the intact E4 was required for its
effect. Thus the E4 gene appears to modulate the inhibitory
activity of the rep gene.
Inhibition of transient gene expression. Several
mechanisms could account for the ability of the AAV rep gene
to inhibit transformation by the tested markers. One
explanation, in accord with the known role of the rep gene
products as regulators of gene expression would be an
ability to negatively regulate the expression of the marker
genes. To test if the AAV genome could inhibit the
expression of a heterologous gene the effect of AAV DNA on
the expression of pSV2cat (Gorman et al., 1982), containing

105
the cat gene under the control of the SV4 0 early promoter
was analyzed.
Transfections with pSV2cat alone or with either 620ori~
or ins32ori~ were carried out in Ltk~ cells. At 40-48 hours
post transfection, extracts from the cells were made and
tested for the amount of cat activity. The data obtained
from several experiments is summarized in table 4-5.
Cotransfection of pSV2cat with the rep insertion mutant had
no effect although cotransfection with 620ori~ inhibited the
amount of cat activity generated. Cotransfection of 620ori~
DNA resulted in a 3 to 10 fold reduction in transient
pSV2cat expression. Thus in transient assays, expression of
the rep gene products inhibited expression of at least one
heterologous gene. Further, the addition of pXBAC to the
transfections partly relieved the inhibitory effect.
Discussion
In this section we describe two new activities of the
AAV rep gene products. First, the expression of the rep
gene products strongly inhibits the genetic transformation
by several dominant selectable markers. Second, rep gene
expression also appears to inhibit the expression of at
least one heterologous gene, pSV2cat. Previously, the rep
proteins were shown to have an essential role in AAV DNA
replication and positive regulation of AAV transcription
(Hermonat et al., 1984; Tratschin et al., 1984a; Labow et
al., 1986).

106
Table 4-4. EFFECT OF CLONED E4 SEQUENCES ON AAV
MEDIATED INHIBITION OF TRANSFORMATION
Number of Colonies3
Cotransfected DNA*3
Experiment
-
620ori_
pXHOC 62 0ori“/pXBAC 620ori"/pFB69
A.
460
14
14 170
B.
200
6
112(18°)
C.
372
8
- 48 0
aThe numbers of colonies per dish are reported as in
table 4-1.
^In this experiment, 0.1 ¿*g of pGCcps3neo was
transfected alone (-) or with additional plasmids as
indicated. Three-^g of 620ori~ and 6 nq of pXBAC DNAs
(containing the E4 gene) were used in experiments A and B
and 1 lg of 620ori~ and 10 pg of pXBAc DNAs were used in
experiment C.
cThe number shown in parenthesis inidactes the number of
colonies obtained in a duplicate dish that received pXBAC DNA
which had been digested with PstI and Hindlll prior to
transfection.

107
TABLE 4-5. INHIBITION OF pSV2cat EXPRESSION BY
COTRANSFECTED AAV DNA
CAT activity3
Cotransfected DNAb
Experiment
ins32ori- 620ori- 620ori-/E4
A.
46
(1)
58
(1.2)
15
(0.32)
26
(0.56)
B.
41
(1)
38
(0.93)
4
(0.10)
14
(0.34)
C.
42
(1)
32
(0.76)
7
(0.17)
ND
aCat activity was assayed as described in chapter II.
The percentage of choloramphenicol converted to acetylated
forms is listed along with the fraction of activity compared
to transfections with pSV2cat alone, as shown in
parentheses.
^The plasmid SV2cat (0.2 pg) was transfected alone (-)
or with various plasmid DNAs (5/xg each) as described in
chapter II.

108
As is the case with AAV DNA replication and
transcription, the p5 gene product was required for optimal
inhibition of transformation since mutations specific for
this ORF (at 11 mu) prevented inhibition. However in the
case of inhibition of transformation, AAV genomes which
contained only a functional pl9 gene were able to inhibit
transformation, although less efficiently than wild type
genomes. Inhibition by the pl9 gene was observed only in
B78H1 cells and not in Ltk- cells. One possible explanation
for this observation may be that the pl9 gene is less
efficiently expressed in Ltk- cells than in B78H1 cells.
Although we have no data supporting this notion, expression
of pl9 has been reported to be inducible by Ela gene
products (Tratschin et al., 1984b). Because certain cell
lines have an endogenous Ela like activity (Imperiale, et.
al., 1984; Kao et al., 1985) it is likely that pl9
expression will vary in different cell lines.
The inhibitory effects of the rep genes were general in
that transformation by at least two different selectable
markers was inhibited {neo and tk). Further inhibition was
not promoter specific in that markers controlled by either
of three promoters were inhibited. However, some
specificity of inhibition was implied in that transformation
by d!52-91/neo was much less inhibited than that by the
other vectors described. It is unclear why this vector
transformed with high efficiency in the presence of the rep

109
genes, although it should be noted that it is not known if
expression of neo is driven by the SV40 early promoter or by
the p40 promoter retained in d!52-91/neo.
Selection against the rep gene during genetic selection
has been suggested by several previous studies. Upon
transformation using recombinant AAV containing a neo gene
under the control of either the SV4 0 early promoter or the
AAV p40 promoter, integrated AAV DNA in the G-418r cells
often contained mutations inactivating the rep genes
(Hermonat 1984; Tratschin et al., 1985). However we
observed less inhibition by the rep genes using d!52-91/neo
than with non-AAV vectors. It should be noted that in the
previous studies by Hermonat and Tratschin, human cells were
infected with transducing AAV virus-vectors while the
experiments presented here were done using murine cells and
DNA mediated transfer. The two different procedures may not
be comparable. Also we have not determined if the rep genes
of d!52-91/neo remained intact in the resulting G-418r
cells.
Several possible mechanisms could explain the
inhibitory action of the AAV rep genes on transformation.
First, expression of the p5 and or pl9 genes might be lethal
to cells. This possibility seems least likely for several
reasons. As described above, the efficient transformation
by d!52-92/neo argues against rep gene lethality. Also the
observation that the number of colonies arising after

110
transfection with a given amount of AAV DNA can be increased
by adding more of the selectable marker argues against a
lethal effect of a given amount of AAV DNA.
A second possibility would be an ability to block the
integration or stabilization of the transgenes. We have no
data concerning this mechanism. A third possibility would
be an inhibitory activity on the expression of the
selectable gene. In support of this notion, we have shown
that the expression of pSV2cat is inhibited by the AAV rep
genes. Previously Ostrove et al. (1981) reported that AAV
infection of Ad transformed H14B cells resulted in the
inhibition of the synthesis of the 55 kd Elb tumor antigen
in the absence of an apparent loss of cell viability. This
inhibition of tumor antigen synthesis was not accompanied by
a reduction in Elb RNA levels suggesting that the inhibition
of expression may occur at the translational level. Our
data suggests that this inhibition was due to rep gene
expression.
While this study was in progress Tratschin et al.
(1986) reported that the rep genes inhibited expression of a
p40 controlled cat gene. This inhibitory activity occurred
in 293 cells which contain the Ela and Elb Ad genes but did
not occur in HeLa cells, supporting a general repression
function for the AAV rep genes. The rep dependent
inhibition of p40 expression was not associated with a
decrease in p40 RNA (B. Carter, personal communication)

Ill
again suggesting that regulation occurs at the level of
translation.
We have also reported that the inhibitory effects on
transformation are ameliorated by the E4 gene of Ad. Part
of the inhibitory effect of pSV2cat expression was also
decreased by the E4 gene. Similarly, the inhibitory
activity of rep on p40 expression seen by Tratschin et al.
(1986) in 293 cells was prevented by infection with Ad,
although a role specifically for E4 was not looked for. Thus
we conclude that the activity of rep gene products is
modulated in some way by an E4 gene product (s) . These
results suggest a cascade of changing functions of the rep
gene products during viral infection and further demonstrate
the complex interplay of viral gene products on cellular
processes.

CHAPTER V
IDENTIFICATION OF AAV REP GENE PRODUCTS
Introduction
Nucleotide sequence analysis indicates that the left
side of the AAV genome contains a large ORF which is
uniquely contained within the p5 and pl9 transcripts. Thus
these RNAs could encode several nonstructural proteins.
Because the p5 and pl9 messages exist in both spliced and
unspliced forms, four proteins could be produced from the
left side ORF. The p5 unspliced and spliced transcripts
could encode proteins of approximately 70 kd and 60 kd,
respectively. The pl9 unspliced and spliced messages could
encode proteins of approximately 44 and 34 kd, respectively
(see Carter et al., 1984 for review).
The proteins encoded by the p5 and pl9 ORFs are
essential for virus growth. As described above, mutations
within the p5 and pl9 ORFs abolish AAV DNA replication
(Hermonat et al., 1984; Tratschin et al., 1984a) and inhibit
AAV transcription (Labow et al., 1986). The AAV rep gene
products also affect the activity and expression of
heterologous genes (chapter IV) . Thus the rep proteins
appear to be pleiotropic regulators of gene expression.
Proteins encoded within the left side of autonomous
parvovirus genomes have been identified. They are nuclear
112

113
phosphoproteins of approximately 85 and 25 kd (Cotmore et.
al., 1983; Rhode and Paradiso, 1983). These proteins were
identified using antisera from infected animals and by in
vitro translation of specific mRNAs.
The putative rep proteins have not been previously
detected in infected cells. Several aspects of AAV biology
have made difficult the identification of these proteins.
First, the requirement for a helper virus leads to a complex
production of proteins in productively infected cells.
Antisera raised against infected cells would be directed
against both helper virus and AAV proteins. Second, the
levels of the rep proteins are expected to be low. The p5
and pl9 messages are normally present at 10% of the level of
the p40 message. Early studies also failed to detect p5 or
pl9 messages on polysomes.
In order to identify the AAV rep proteins, antisera to
synthetic peptides corresponding to different regions of the
left side ORF were produced by immunization of New Zealand
white rabbits and the sera used in immunoprecipitation
experiments of productively infected cells. Using this
technique, two proteins present specifically in cells
productively infected with AAV have been identified. The
two proteins are approximately 70 kd and 44 kd and appear to
be encoded by the p5 and pl9 RNAs, respectively.

114
Results
Preparation of Anti-peptide antisera. Several rabbits
were immunized with either of three peptide-carrier protein
conjugates:
peptide A (pA): Lys-Arg-Pro-Ala-Pro-Ser-Asp-Ala-Asp-Ile
peptide B (pB): Ser-Leu-Thr-Lys-Thr-Ala-Pro-Asp-Tpr-Leu
peptide C (pC): Lys-Glu-Trp-Glu-Leu-Pro-Pro-Asp-Ser-
Asp-Met-Asp-Leu-Asn-Leu-Ile
The peptide sequences of pA, pB, and pC are encoded
between 1797-1826, 1140-1172, or 417-449 base pairs,
respectively, on the AAV genome. The presence of
antipeptide antibody was determined by hemagglutination
assays as described. The titers (reciprocal of the lowest
dilution giving a positive agglutination reaction) obtained
are shown in table 5-1. Only those sera having any positive
reaction are shown. The highest titer sera was obtained for
pB. Anti-peptide antibody was further purified by affinity
chromatography on peptide-conjugated sepharose. While
highly concentrated antisera was obtained for pB, only
somewhat concentrated pA sera was obtained. We were unable
to concentrate the pC sera.
Immunoorecipitation of AAV proteins with anti-peptide
antisera. In the first experiment 5 nl of each sera
(affinity purified pA and pB; unfractionated pC sera) were
used in an immunoprecipitation experiment using 35S-
methionine labeled protein extracts from either Ad-2 or Ad-2

115
TABLE 5-1
PRODUCTION OF ANTIPEPTIDE ANTISERAa
Titera
Rabbitb
Serac
pA
pB
pC
E
PE
4
-
-
E
I
32
-
-
E
AP
256
—
—
B
PI
—
8
—
B
I
-
512
-
B
AP
—
>5000
-
R
PI
—
—
16
R
I
-
-
128
R
AP
—
—
16
aTiters shown are the reciprocal of the highest
dilutions giving a positive hemagglutination reaction against
SRBCs which had been coated with the indicated synthetic
peptide (pA, pB, or pC).
^Titers shown are group for the individual rabbit used;
either E (Ethyl), B (Bernie), or R (Ricky).
cThe sera tested were either preimmune sera (PI)
collected two weeks prior to the initial immunizations,
immune sera (I) collected approximately 7 days post¬
immunization, or affinity purified sera (AP).

116
and AAV-2 infected cells. The immunoprecipitation reactions
were carried out and analyzed as described in chapter II.
As shown in figure 5-1 a high amount of background was seen
in all experiments. However an additional band of
approximately 70 kd was identified using pB sera at both
time points (lanes 3,4). This protein was not seen using
extracts from cells infected only with Ad (lane 10) or when
using preimmune sera (lanes 7, 8). A second unique species
of approximately 44 kd was detected using pB sera, although
poorly resolved from the background bands.
Although the 70 kd protein appeared to be an AAV
specific protein, its molecular weight was very similar to
the capsid protein VP2 (72 kd). In order to distinguish the
two proteins an immunoprecipitation experiment was done
along with both anticapsid and anti-pB sera and the
specificity of immunoprecipitation was tested by competition
with excess unlabeled peptides used for the immunizations
(figure 5-2), As shown the 70 kd protein was detected at
both time points examined (12 or 2 0 hours post infection) .
The precipitation of the 70 kd protein was completely
inhibited by competition with the peptide used for
immunizations but not by a heterologous peptide. Thus the
70 kd protein appeared to be an AAV specific protein encoded
by the left side ORF. At 12 hours post infection a 44 kd
protein, again not well resolved in this experiment, also
appeared to be specifically recognized in AAV infected cells

117
by pB sera but was not specifically detected at the later
time point.
Immunoprecipitation experiments using proteins extracts
labeled at very early times (6 hours postinfection) were
also carried out. As shown in figure 5-3, a unique 44 kd
protein was detected using anti-pB sera and extracts from
cells coinfected with both Ad-2 and AAV-2 but not from Ad-2
infected cells. Again, this protein was not detected with
preimmune-sera or with extracts from cells infected with Ad.
Thus the synthesis of the 44 kd protein appeared enhanced at
early times after infection.
Identification of phosphorvlated capsid proteins.
Immunoprecipitation experiments using extracts labeled with
32P-P04 were also carried out (figure 5-4). No AAV specific
proteins were detected using either preimmune or antipeptide
sera. However a control experiment using anticapsid sera
detected several proteins. Proteins of 63, 72, and 87 kd
were detected. These molecular weights correspond closely
in size to the AAV capsid proteins. A fourth protein of 75
kd was also detected by the anticapsid sera and may
represent a modified virion protein. Phosphorylation of the
AAV capsid proteins has not been previously reported and the
function(s) of this modification is not known.

Figure 5-1. Immunoprecipitation analysis with antipeptide antibody.
In this experiment equal amounts of 35S-labeled proteins from KB cells
infected with Ad-2 (lanes 9-11) or coinfected with Ad-2 and AAV-2 (lanes 1-
8) were immunoprecipitated with either pA (lanes 1, 2, and 10) pB (lanes 3,
4, and 11), pC (lanes 5, 6, and 9), or preimmune sera (lanes 7 and 8).
Proteins were labeled at 24 hours postinfection except for those used in
lanes 1, 3, and 5 which were labeled at 20 hours postinfection. The
position of the two bands seen only in lanes 3 and 4 are indicated on the
left side of the figure. The sizes of MW standards are indicated on the
right.

/
1 2 3_ 4 5 6
7 8 9 10 11
-68
-44

120
Figure 5-2. Iinmunoprecipitation analysis of AAV
proteins. The following experiments used labeled proteins
from KB cells infected with Ad-2 (lanes 1, 5, and 7) or
coinfected with Ad-2 and AAV-2 (lanes 2-4, 6, and 8-11.
Proteins were labeled at either 12 hours (lanes 2 and 4-6)
or 20 hours (lanes 1,3, and 7-11) postinfection. The
labeled proteins were then immunoprecipitated using either
anticapsid (lanes 1-3), preimmune (lanes 4 and 7) or anti-
pB sera (lanes 5, 6, and 9-11). The experiments in lanes 10
and 11 included a 10 ^g amount of either pA or . pB
respectively as a competitor. Lanes 1-3 were exposed to
film for two days while lanes 4-11 were exposed for 10 days.
The sizes of MW standards are shown on the right.

121
12 3 4 5
Figure 5-3. Identification of an AAV protein made
early during infection. In this experiment KB cells were
infected with Ad-2 (lane 4) or coinfected with Ad-2 and AAV-
2. Proteins were labeled at 6 hours postinfection,
harvested, and immunoprecipitated using preimmune (lanes 1
and 2) , anti-pB sera (lane 3 and 4) , or anti-pA sera (lane
5). An arrow indicates a 44 kd polypeptide observed only in
AAV infected cells. The size of MW standards are indicated
on the right.

122
Figure 5-4. Identification of phosphorylated capsid
proteins. KB cells were infected with Ad-2 (lanes 1 and 4)
or coinfected with both Ad-2 and AAV-2 (lanes 2, 3 and 5)
and labeled with 32P-P04 for 1 hour at 20 hours
postinfection. Labeled proteins were then extracted and
immunoprecipitated with either anticapsid sera (lanes 1 and
2) , preimmune (lane 3), or anti-pB sera (lanes 4-5). The
size of unlabeled molecular weight markers are shown on the
right and the size of the capsid proteins are shown on the
left.

123
Discussion
In this section, the identification of two
nonstructural AAV proteins has been reported. The proteins
have molecular weights of approximately 7 0 kd and 44 kd,
based on their mobility in acrylamide gels. The proteins
appear to be encoded by the AAV p5 and pl9 genes based on
the following. First, the proteins were only detected in
cells coinfected with both Ad-2 and AAV-2. Second, they
were detected with antisera directed against an amino-acid
sequence shared specifically by the ORFs of the p5 and pl9
transcripts. Third, the size of the proteins correspond
very closely to that predicted for p5 and pl9 gene products.
Fourth, recognition of the 70 kd protein was specifically
blocked by competition with the peptide used for
immunizations.
The proteins appear to correspond to the size of
proteins encoded within the unspliced ORFs. These species
were predicted to be the most abundant because the p5 and
pl9 RNAs are accumulated in predominantly spliced form.
Also, genetic analysis indicates an essential role only for
the products encoded by the unspliced RNAs (see chapter I).
Although the study of mutant AAV genomes has clearly
identified critical functions for products encoded by the p5
gene, no function has yet to be assigned to the pl9 gene.
However the data presented in the previous section may
indicate that the pl9 gene may have ability to negatively

124
regulate the activity of heterologous genes in one cell
line. The identification of a putative pl9 gene product
supports a role for the pl9 gene in AAV replication.
Interestingly, the p5 70 kd protein was detected late in
infection while the 44 kd protein was only readily observed
at early times. Thus the production of the two proteins may
be differentially regulated.
It should be noted that due to the poor sensitivity of
these experiments, we cannot conclude whether certain rep
proteins were present in lower undetectable amounts. In
this regard rep proteins encoded by the spliced RNAs may
have been produced but at levels undetectable in these
experiments.
While this work was in progress, another group has used
similar techniques to identify the rep proteins. Using
western blotting they detected four proteins corresponding
to both unspliced and spliced p5 and pl9 ORFs (Mendelson et
al., 1986). These results indicated that the larger
proteins encoded by the unspliced RNA were the most
abundant. Further, the putative p5 proteins were
predominantly nuclear while the pl9 products were largely
found in the cytoplasm. The same study also demonstrated
that the AAV gene products were produced at very low levels
in the absence of helper virus coinfection.
Finally, phosphorylated AAV capsid proteins have been
identified. Phosphorylated versions of all three capsid

125
proteins appear to be produced. Although the smallest
capsid protein, VP3, is the most abundant virion protein as
well as the most highly labeled capsid protein upon pulse
labeling with 3^S-methionine, all three species appeared
equally labeled by orthophosphate. It is not known if this
indicates that the minor virion proteins are highly
phosphorylated, more often phosphorylated, or more stable in
the phosphorylated form. A 75 kd protein was also detected
which may represent a highly phosphorylated variant of the
72 kd VP2 protein.

CHAPTER VI
CONSTRUCTION AND CHARACTERIZATION OF
HYBRID AAV/SV40 GENOMES
Introduction
Although it has been demonstrated that AAV gene
expression occurs in the absence of helper virus, resulting
in the inhibition of activity of heterologous genes, the
level of AAV transcription is very low (chapter III) and
therefore difficult to study. A system was sought which
would amplify the level of AAV gene expression in the
absence of helper virus coinfection. To this end, a hybrid
AAV genome containing the SV40 early promoter, enhancers,
and origin of DNA replication was constructed. It was
predicted that the SV40 origin would allow episomal
replication of AAV genomes in cell lines constitutively
producing the SV40 T-antigen, resulting in a large
amplification of potential transcription templates.
The SV40 early promoter was also used to drive
expression of the AAV p5 gene. This allows several problems
concerning the regulation of AAV gene expression to be
addressed. For example, will sequences within the AAV
genome recognize and regulate the SV40 promoter as an AAV
promoter? This notion arises from the observation made in
chapter III that indicates that all the AAV promoters were
126

127
activated by the AAV rep gene products. Two explanations
are possible for this result. First, each AAV promoter
might contain a regulatory element responsive to rep. A
second mechanism is that a common regulatory element
independent of the AAV promoters, stimulates transcription
of the entire AAV genome in response to activation by rep.
Thus, it is of interest to determine if the AAV rep gene
products are able to activate transcription from an SV40
promoter carried within the AAV genome.
The results presented in this section indicate that the
SV40 promoter is regulated similarly to an AAV promoter in
human cells. Also, recombinant AAV carrying the SV40 early
region are propagated as virus. Thus elements within the
AAV genome appear to regulate the SV40 promoter. The genome
also replicates as a plasmid in cos-7 cells in the absence
of helper virus. However in this case replication is
reduced approximately 10 fold over control plasmids. The
low level of replication appears to be due to a specific
inhibitory effect of a rep gene product.
Results
Construction of AAV/SV40 genomes. The construction and
structure of AAV genomes containing the SV40 early promoter
is described in figure 6-1. The construction of dl03-05/SV
resulted in
deletion of
AAV
sequences
from
base
145
(immediately
downstream
from
the TR)
to
base
264
(immediately downstream from the p5 TATA box). This deletion

128
is expected to completely remove the functional p5 promoter
due to removal of the TATA box and upstream sequences up to
the boundary of the TR. A fragment of SV40 DNA (nucleotides
270-5171 containing the early promoter and origin of DNA
replication (see Tooze, 1981 for review and numbering
system) has been inserted into the site of deletion with the
direction of early transcription in the same orientation as
the p5 promoter. Several variants of this plasmid were also
created including a genome containing insertion of a
frameshift mutation at 32 mu (dl03-05/SVins32). Another
construct used in this study is dllO-37/SV (previously
referred to as dllO-37/EPR in Hermonat, 1984) . This plasmid
contains a deletion of the AAV genome from 10-37 mu and an
insertion of a similar piece of SV40 DNA within the site of
deletion. The structures of the SV40/AAV hybrid genomes are
summarized in figure 6-2.
Expression of dl03-05/SV. The expression of d!03-
05/SV was examined in human cells in the presence or absence
of helper virus coinfection. Either dl03-05/SV or pSM620
were transfected into Ad-2-infected or uninfected human KB
cells. The levels of RNAs accumulated by the plasmids are
shown in figure 6-3. Expression from dl03-05/SV or the wild
type pSM620 genome appeared identical. Neither genome
accumulated significant levels of RNA in the absence of
helper, although large amounts were accumulated in the
presence of helper. Further, the relative levels of the AAV

129
RNAs were unaffected by the SV4 0 promoter and enhancers.
Because the level of the AAV p5 and pl9 transcripts are
affected by the cis-active dep sequences (chapter III), the
levels of 4.2 kb RNA produced by dl03-05/SV may indicate
that the SV40 promoter is regulated by dep.
Replication of AAV genomes in human KB and monkey cos-7
cells. The ability of the AAV genome within dl03-05/SV to
replicate was tested by transfection into KB cells as shown
in figure 6-4. In the absence of helper virus infection
neither dl03-05/SV or pSM620 replicated (i.e. no AAV
specific replication intermediates were accumulated). Upon
Ad-2 infection, both plasmids produced similar amounts of
replicated genomes. Replication of the genomes can be
judged by two criteria. First the hybridization signal
after Ad-2 infection indicates a great amplification of AAV
DNA compared to that observed in the absence of Ad-2
infection. Second, the size of the predominant replicated
DNAs are characteristic of the AAV monomer-duplex (md)
replicative intermediates excised from the plasmid vector.
Replication was also observed when lysates of cells
transfected with dl03-05/SV were used with Ad-2 to coinfect
HeLa cells, indicating the production of virus by the d!03-
05/SV genome. This replicative form DNA was largely
digested with SphI indicating that SV40 sequences were
retained upon packaging into AAV virions.

130
Because dl03-05/SV contains the SV40 origin of DNA
replication, the plasmid should also replicate as an episome
in cell lines which constitutively produce the SV40 T-
antigen, such as cos-7 cells (Gluzman, 1981). Thus, the
cloned genomes were tested for replication upon transfection
into cos-7 cells as shown in figure 6-4B. Very low levels
of plasmid DNAs were detected upon transfection of either
pSM620 or dl03-05/SV in the absence of Ad-2 infection. Only
input plasmid forms were observed indicating that rescue of
the AAV genome had not occurred. Similar amounts of plasmid
DNAs were detected after transfection with either pSM620 or
dl03-05/SV, indicating that the later plasmid had not been
efficiently replicated even though it contained the SV40
ori. However, dllO-37/SV did appear to be replicated after
transfection resulting in a significant amplification of
DNA. Upon addition of Ad-2, a low level of the md
replicative form of pSM620 was detected indicating a low
level of helper-dependent replication in cos-7 cells.
However, no rescued dl03-05/SV md was observed. Although
this result was reproducible, the reason for this difference
was not investigated further.
In order to more clearly analyze the ability of AAV
genomes to replicate as episomes, the transfections were
repeated, and the extracted DNAs were digested with Dpnl.
Dpnl recognizes the sequence GATC only when the G residue is
methylated. Because this methylation occurs only in

131
bacteria, DNA replicated in mammalian cells is resistant to
Dpnl digestion (Peden et al.f 1980). This type of analysis
also eliminates artifacts due to variable recoveries of
input transfected DNA.
As shown in figure 6-5, no Dpnl resistant DNAs were
detected after transfection of pSM620. Further, due to the
low amounts of DNA used in the transfection, no input DNA
was detected in this case. Although transfection of either
dl03-05/SV and dll0-37/SV resulted in the production of Dpnl
resistant DNA, replication by dl03-05/SV appeared to be
approximately 10 fold less efficient than that by dllO-
37/SV. Replication of dJL10-37/SV was as efficient as that
of pSV2cat (containing no AAV sequences). Thus dl03-05/SV
appeared defective for replication in cos-7 cells.
One explanation for this observation is that the poison
sequence within the pBR322 vector (Lusky and Botchan, 1981)
inhibits the replication of dl03-05/SV. This seems unlikely
due to the efficient replication of dllO-37/SV (also cloned
into pBR322). However the poison sequence is known to
affect replication in a somewhat variable manner. Thus, a
poison- version of dl03-05/SV was constructed as shown in
figure 6-1 creating dl03-05/SVori~ (this construction also
resulted in the removal of parts of the AAV TRs) . This
plasmid also replicated poorly in cos-7 cells compared to
pSV2cat (figure 6-5). Thus the inefficient replication of

Figure 6-1. Construction of AAV/SV40 hybrid
genomes. SV40 sequences containing the early promoter,
enhancers and origin of DNA replication are indicated by
the stippled boxes. The AAV TRs are represented as
filled boxes and AAV promoters are indicated with arrows
and promoter designation. Bacterial vector sequences
are represented by dotted lines and AAV sequences by
solid lines. Restriction sites are indicated by small
lines topped with either open circles (BamHI), closed
circles fBelli), filled triangles (BstEII), filled
diamonds (Aval), filled boxes (SacI), perpendicular
lines (EcoRV) or not topped (Smal).
pSVBB was derived from pHM2609 (Hermonat and
Muzyczka, 1984) by insertion of a Bglll linker at the
unique Hindlll site located within the leader of the
SV40 early RNA. The small BamHI/Bqlll fragment of this
plasmid was then inserted into the Bglll site of d!03-
23. The intact rep coding region was then inserted back
into the genome in the following manner. First, the
recipient plasmid, dl03-23/SV was digested with Bglll
and the site made flush by filling in with Klenow enzyme
and nucleotide triphosphates and finally digested with
BstEII (creating noncohesive ends). Second, d!49-94 was
digested with Aval and the ends were made flush as
above. The restriction fragment containing the rep gene
(AAV sequences from 264-2234) was isolated, digested
with BstEII, and finally ligated to the large fragment
of (Í103-23/SV prepared above.
The large Smal fragment of dl03-05/SV was then
ligated into EcoRV-digested pFB69, creating d!03-
05/SVori~. The small internal SacI/BstEII fragment of
ins32ori~ (containing a frameshift mutation at 32 mu)
was inserted into SacI/BstEII-digested dl03-05/SV
creating dl03-05/SVins32.

d»3-O5/SV40ori-

Figure 6-2. Structure of hybrid AAV/SV40 genomes. The various AAV
genomes constructed in this study are drawn schematically. The AAV TRs,
promoters, and sites of polyadenylation are as indicated in previous
figures. The SV40 early promoter region is indicated as a stippled box, and
the location of a frameshift mutation in dl03-05/SVins32 is indicated by a
downward pointing line.

WT( pSM 620)
p5
p 19
p40
r
sve pl9 p40
iwra
sve pl9 p40
dl03-05/SVins 32 -C 1 E—
sve p!9 p40
c*ra»>
sve p40
CIA5ra
sve p40
p5 sve
p5 sve
dllO-37/SV
p40
c

136
12 3 4
Figure 6-3. Expression of AAV plasmids in human cells.
Human KB cells were transfected with 20 ¿ig of either pSM620
(lanes 1 and 3) or dl03-05/SV (lanes 2 and 4) and either
mock infected (lanes 1 and 2) or infected with Ad-2 (lanes 3
and 4) and RNA extracted at 48 hours posttransfection. RNAs
were fractionated and analyzed as described for figure 3-1.
The positions of AAV RNAs are indicated on the side.

137
dl03-05/SV did not appear to be caused by variable effects
of the poison sequence.
Inhibition of replication by the AAV rep gene. One
major difference between dl03-05/SV and dllO-37/SV is that
the former genome encodes functional p5 and pl9 gene
products. In order to determine if the rep gene products
inhibited replication in cos-7 cells, an 8 base frameshift
mutation was engineered into dl03-05/SV40 creating dl03-
05/SVins32. Indeed this mutation allowed efficient
replication (figure 6-6). Further, dl03-05/SV40 inhibited
the replication of dllO-37/SV in trans upon cotransfection,
resulting in approximately equal amounts of replicated DNA
for both genomes. Cotransfection of dl03-05/SVins32 had no
effect on the replication of (3110-37/SV, again indicating
that the rep gene products mediated the inhibition of
replication. Interestingly, upon cotransfection of d!03-
05/SV and pSV2cat, replication of the pSV2cat plasmid was
significantly favored compared to that of dl03-05/SV. Thus
inhibition of replication by rep appears specific for
plasmids containing AAV sequences.
Discussion
In this section the constructjjon and characterization
of a hybrid AAV/SV40 genome has been described. In human
cells, the genome behaved in a manner indistinguishable from
wild type genomes replicating and producing virus in a
helper dependent manner. In addition the levels of RNA

Figure 6-4. Replication of AAV plasmids in various cell lines. A) KB
cells (lanes 1-4 were mock infected (lanes 1 and 3) or infected with Ad-2
(lanes 2 and 4) and transfected with 20 g of either pSM620 (lanes 1 and 2)
or CL103-05/SV (lanes 3 and 4) . A duplicate plate of Ad-2 infected cells
transfected with dl03-05/SV was used to make a virus stock which was used
coinfect HeLa cells along with Ad-2 (lanes 5 and 6) . Low molecular weight
DNA was isolated from the cells at 48 hours postinfection and 20% of the DNA
analyzed by electrophoresis on an agarose gel, blotted to nitrocellulose
and analyzed by hybridization to 32P-labelled AAV virion DNA. The DNA in
lane 6 was digested with SphI prior to electrophoresis. B) Cos-7 cells
were transfected with 20 g either of CT DNA (-) , pSM620 (lanes 1 and 2) ,
d¿03-05/SV (lanes 3 and 4), or dllO-37/SV (lane 5). The lane marked as M
contains marker DNA (9, 6, and 2.4 kb). The cells were either mock infected
or infected with Ad-2 (lanes 2 and 4 only) . Low molecular weight DNA was
isolated and analyzed as in (A). The positions of forms I, II, and III DNA
of input plasmids are shown on the right as well as the position of the md
replicative form DNA.

1 2 3 4 5
139

140
produced by dl03-05/SV in the presence of Ad-2 infection
were similar to that produced by a wild type genome. Thus
the substitution of the SV40 promoter and enhancers did not
have significant effects on AAV gene expression.
This observation is surprising in that the SV40
promoter is thought to be regulated in a manner opposite
that of the AAV p5 and pl9 rep genes. While expression of
the p5 and pl9 genes is apparently induced by an Ela gene
product (Tratschin et al., 1984b), transcription of the SV40
early promoter is inhibited by Ela (Velcich and Ziff, 1985).
However, when present in the AAV genome, the SV4 0 promoter
worked as efficiently as the AAV p5 promoter. One
explanation for this observation is that cis-active
seguences within the AAV genome regulate the heterologous
SV40 promoter, allowing it to both be activated by Ela (or
at least preventing repression of transcription and to be
transactivated by the AAV rep proteins. Although the nature
of the regulation of the SV40 promoter within an AAV genome
remains to be elucidated, the use of the SV4 0 promoter
should provide a useful tool in the identification of
regulatory AAV sequences.
Negative regulation of DNA replication. The data
presented in this chapter also demonstrates that expression
of the AAV rep genes inhibits the T-antigen mediated
replication of AAV genomes containing the SV40 ori in cos-7
cells. The inhibition required an intact rep ORF and was

141
Figure 6-5. Replication of hybrid genomes in cos-7
cells. A) Cos-7 cells were transfected with 1 ¿¿g either
pSM620 (1), dl03-05/SV (2), dllO-37/SV (3), or pSV2cat (4)
plasmid DNAs. Low molecular weight DNA was isolated 48
hours after transfection and analyzed as in figure 6-3
except that pSM62 0 was used as probe so that pSV2cat DNA
would also be detected due to the homologous pBR322
sequences. The DNAs were either undigested (-) or digested
with Dpnl (+) prior to electrophoresis. B) Cos-7 cells were
transfected with 20 ¿¿9 of either dl03-05/SVori~ (1), pSV2cat
(2) , or pSV2cat and dl03-05/SVori~ (3) and low molecular
weight DNA analyzed as in (A) except all samples were
digested with Dpnl prior to electrophoresis. The positions
of forms I, II, and III DNA of pSV2cat are shown on the
left.

142
12 345 6789
Figure 6-6. Inhibition of DNA replication by the AAV
rep gene. Cos-7 cells were transfected with either 0.25 /¿g
pSM620 (lane 1), dl03-05/SV (lane 2), dl03-05/SVins32 (lane
3) , dllO-37/SV (lane 4) or pSV2cat (lane 6) . Some cells
were cotransfected with 0.25 nq dllO-37/SV and 5 ¿¿g of
either dl03-05/SV (lane 4) or dl03-05/SVins3 2 (lane 5) , or
cotransfected with 0.25 /¿g pSV2cat and 10 g of either d!03-
05/SV (lane 8) or dl03-05/SVins32 (lane 9). Low molecular
DNAs were isolated 48 hours after transfection and analyzed
as in figure 6-5. The DNAs were digested with Dpnl (to
remove unreplicated DNA) and PvuII (to linearize the DNAs)
prior to electrophoresis. The positions of linear forms of
dllO-37/SV (A) , d¿03-05/SV and dl03-05/SVins32 (B) , and
pSV2cat (C) are indicated.

143
mediated in trans by cotransfection, strongly suggesting a
role for the p5 and/or pl9 gene products.
The inhibition does not appear to be due to a decrease
in T-antigen synthesis for the following reasons. First, T-
antigen is extremely stable in SV40 transformed cells with a
half life over 24 hours (Edwards et al. , 1979; Rawlins et
al., 1983). Thus, a decrease in T-antigen levels due to an
inhibitory effect of T-antigen expression is doubtful during
a transient assay.
Second, expression of the rep genes does not appear to
significantly inhibit the replication of pSV2cat in
cotransfection assays. Thus the machinery required to
utilize the SV40 ori appears intact after transfection with
the AAV genomes. This observation implies that the
inhibition of replication was specific for replicons
containing AAV sequences. However the nature of such
sequences is not known at this time.
Thus in addition to transactivation of AAV transcription
and DNA replication and negative regulation of heterologous
gene expression, an AAV rep gene product(s) appears to have
a negative regulatory function for DNA replication. We
propose that this function may be analogous to the function
of the lambda repressor in the maintenance of a latent
infection. In the absence of an inducer of AAV replication
(e.g. helper virus coinfection) the rep proteins may bind an
AAV sequence and prevent high levels of replication, perhaps

144
only allowing the AAV genome to be passively replicated as
an integrated provirus. Upon induction by helper virus
coinfection, the function of the rep proteins could then be
altered to that of a positive activator of DNA replication.
This type of a model is appealing in light of two
recent observations. First, the AAV genome can be replicated
in the absence of helper virus under certain conditions of
cell stress (see chapter I) . Thus the only viral gene
products apparently absolutely and perhaps directly
essential for AAV growth are the AAV gene products
themselves. Second are the direct and indirect observations
that the AAV rep gene products are produced in the absence
of helper virus functions in many cells. The rep gene
products produced in the absence of helper are clearly
functional having effects on both gene expression and DNA
replication. Thus the question of why AAV does not
replicate normally in the absence of helper virus
coinfection becomes an interesting one.
In light of the observations made here, it is suggested
that AAV has developed systems both for the maintenance of a
silent latent state, and for the activation of a lytic state
after specific types of induction. The maintenance functions
would include a negative regulatory function of DNA
replication and perhaps of gene expression, although the
latter has not yet been investigated. The activation step
includes both transactivation of gene expression and DNA

145
replication. This activation step does not appear to
require different AAV proteins, but rather appears to
require a modification of their function.
Other laboratories have recently reported that a system
for negative control of replication is also encoded by BPV
(Berg, et al., 1986; Roberts and Weintraub, 1986). In this
case, it was proposed that this function prevented
uncontrolled amplification of the BPV genome in transformed
cells, thus preventing the killing of the host cell. This
system has several similarities to the regulation of
replication by AAV. In both cases, a viral gene product as
well as cis-active sequences were required for negative
regulation. Thus both AAV and BPV may share mechanisms for
the maintenance of a latent infection.

CHAPTER VII
CONCLUSION
In this dissertation, we have examined the ability of
the AAV genome to regulate its own gene expression and to
effect the expression of heterologous genes. The results
presented here indicate that the regulation of AAV gene
expression and interactions between AAV and heterologous
genes are complex.
The AAV p5 and pl9 rep genes are of central importance
in all the regulatory effects of the AAV genome. The rep
genes have now been shown to be activators of AAV
transcription and DNA replication in helper virus infected
cells. Conversely, in the absence of helper virus the rep
genes have been shown to act as negative regulators of both
gene expression and DNA replication. These observations
indicate that the rep proteins are the elements which allow
AAV to exist as either a silent provirus or as an actively
replicating virus. In conclusion, our initial hope that
AAV would provide a useful and interesting model of
eukaryotic gene expression certainly appears to be true.
146

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BIOGRAPHICAL SKETCH
Mark Aron Labow was born on February 6, 1960, in
Elizabeth, New Jersey. He is the son on Kenneth Arthur and
Audrey Labow who also have one daughter, Nina. He attended
public schools in Middlesex, New Jersey, and Hudson, Florida.
He graduated from Hudson High School in June 1978. He
attended local community colleges before entering the
University of Florida, Gainesville, Florida, in 1979. There
he received a Bachelor of Science degree in microbiology in
1981. He then entered graduate school at the University of
Florida in the Department of Immunology and Medical
Microbiology. He conducted his doctoral research in the
laboratory of Dr. Kenneth I. Berns.
After completion of his degree, he will continue
research in a postdoctoral position in the laboratory of Dr.
Arnold J. Levine.
162

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
I
, Chairman
Professor of Immunology and Medical
Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
ss B. Flanegan
s^ociate Professor /
Medical Microbiology
Immunology and
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Professor of Immunology and Medical
Microbiology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Edward J. Siden
Assistant Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
William B. Gurley
Associate Professor
and Cell Science
icrobiology
This dissertation was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate School and was accepted
as partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
Y-/°r/r7
Dean, Graduate School

UNIVERSITY OF FLORIDA
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