Expression from
the Adeno-Associated Virus
p5
and
p19 Promoters
Is
Negatively Regulated in
trans
by
the
rep
Protein
ANN
BEATON,
PAUL PALUMBO, AND KENNETHI. BERNS*Hearst Microbiology Research Center, Department of Microbiology, Cornell UniversityMedicalCollege, New
York,
New York 10021The
leftward
twopromotersof the adeno-associated virus(AAV)
2genomewerefused toreporter
genes,andthe
constructs wereusedtotransfect HeLacells. The promoters functionedconstitutively
butwererepressed
intrans
by
the
AAVrepgeneproduct(s).
Therepression
wasrelievedby
adenovirusinfection. Evidencewhichindicated
anenhancer function for the inverted terminal repeat of theAAV-2genomewasalsoobtained.The
genomeof the human dependovirus
adeno-associatedvirus
(AAV)
2is
alinear single-stranded
DNA molecule of4,680
bases withinverted, palindromic
terminal repeats of145
bases (19). Promoters have
been identified at mappositions 5, 19,
and40
(6, 13).
When the intact genome isintroduced into continuous human
cell lines inculture,
transcripts from all three
promoters are difficult to detectunless
there is concomitant expression
ofgenes fromhelper
virus
(either adenovirus
orherpesvirus (2, 4).
A fundamentalquestion is
whether thedifficulty
indetecting AAV-specific
gene
expression
inthe absence ofhelper
functionimplies
aninherent
lack
ofability of
the promoters to function undersuch
conditions
orwhether the virus itselfnegatively
regu-lates its
own geneexpression
under these conditions. Sup-portfor the latterpossibility
comesfrom earlierexperiments
which
have demonstrated the ability of
AAV rep geneproducts
toinhibitthe expression
ofgenesunder
thecontrolof the simian virus
40
(SV40) early
promoter in transient transfection and under the control of several viral and cellularpromotersinpermanenttransformation(11, 12,
21).
More
directly,
the AAV promoter(p40)
which controlstranscription of RNA species from which the
AAV coatproteins
aretranslated has been shown
tofunction
constitu-tively
in humanHeLa
cells(21).
In
this
paper we reportexperiments which demonstrate
that boththe AAVpromoters atmap
positions 5
and 19canfunction
constitutively
inHeLa cells
inthe
absence of helper
function.
However, these
constructsaresusceptible
toneg-ative
regulation
by
AAVrepgenefunction,
andthe
negative
regulation
canbe reversed by
adenovirus infection. Thus,
these constructs
behave
in a manneranalogous
tothat of
AAV.
Finally,
wepresentevidence that the inverted
termi-nalrepeats can
function
asenhancers.
Inthepresence
of
helper virus coinfection, AAV
repgenefunction
isrequired in
transfor
the optimal accumulation of
AAV-specific
transcripts
(3, 23). Wewished
totestwhether the AAV rep geneproduct
might inhibit
geneexpression
from the p5 and p19 promoters
in
theabsence of
helper virus.To do this,
we created plasmid constructs which containedeither
the p5
orthe
p19 TATAbox
and 60or 30more upstream
bases, respectively,
fused to thechloram-phenicol
acetyltransferase
(CAT) gene as a reporter gene(Fig. 1). When either of
theseconstructswastransfected into HeLacells, CAT activity
wasreadily
detected (Fig. 2 and3,*Corresponding author.
tPresentaddress: DepartmentofPediatrics, University of
Med-icine and Dentistry of New Jersey, New Jersey Medical School,
Newark,NJ 07103.
Table 1). Hence, both
promoters wereable
tofunction
constitutively in HeLa cells in the absence of helper virus
gene
expression. Cotransfection of
pSM620 (17), which
contains
anintact AAV
genome(Fig. 1),
inhibitedexpres-sion of either
p5 CAT
orp19 CAT (Fig. 2 and 3, Table
1).
Thus,
in the absence ofahelper
virus therewasexpressionof
rep,but the
neteffectof
suchrepexpression appeared
tobe inhibition of further expression from either p5
orp19.
That
thenegative
effects observed in cotransfectionwith
pSM620
weredue
to rep was demonstratedby
cotransfec-tion of plasmids with mutacotransfec-tions in either the
repgene orthe
capgene(coat proteins) (Fig. 2).
In the absence of adenovi-rusinfection, AAV plasmids
with anintact
repgene and adeletion
in thecapgenestill inhibited expressionby
eitherp5
CAT or
p19
CAT,
whereas a deletion or a frameshiftmutation in
repabolishedthe
inhibitory
effect(Fig.
2).
Ataconstant input concentration of the
p5
CAT construct,increasing
amountsof therepconstructin thecotransfection
mixture led to decreased amounts of CAT
activity
in the absence ofhelper
viruscoinfection and also decreasedthe
ability
ofhelper
viruscoinfectionataconstantmultiplicity
ofinfection
toreversetheinhibitory
effect(Fig. 3,
Table1).
In thecaseof
thep19
CATconstruct,asimilardose-dependent
inhibition ofrepwas
observed,
butthedifferences
atthetwohigher
concentrations ofrepwereminimal and there seemedto be a
slight
reversalof theinhibitory
effectatthehighest
concentration ofrep
input
(Fig. 3;
Table1).
In the
absence
of the repproduct,
adenovirus infection
had a
modest effect
on CATexpression. Expression of
p5
CAT was increased
by
1.5-fold in one instance(Table 1,
experiment 2).
Variableeffects
were seenwithp19 CAT:
in one caseexpression
was increasedthreefold
(Table 1,
ex-periment 3),
but inanother(Table 1, experiment 4),
expres-sion was decreased
by 2.5-fold.
In no case was a moremarked stimulation or
repression noted.
By
contrast,aden-ovirus infection hadamuchmoremarked
stimulation
of bothp5
andp19
CATexpression
inthe
presenceof
rep(Fig. 3;
Table1, experiments
1and2).
Inthe
presenceof 5
igof
rep,p5
CATexpression
wasrestored50-fold
toits original
level(Table 1, experiment 2). The
resultswith p19 CAT
wereless
impressive, although in the
presenceof
2,ug of
rep, adeno-virus infectionincreased
p19CAT expression 12-fold in
experiments
inwhich CATexpression
wasunder control of
the
SV40 early
promoter(Table
1,experiment
1).Similar
experiments
wereperformed with SV2
CAT (Table
1,exper-iment
3) (5).
Very modest effects of
rep or repplus
adeno-virusinfection
onSV2
CAT
expression
were observed. Whether the actualdifferences
observed
aresignificant is
questionable.
Although these experiments
were transfec-44500022-538X/89/104450-05$02.00/0
Copyright
©D
1989, American SocietyforMicrobiologyon November 10, 2019 by guest
http://jvi.asm.org/
NOTES 4451
<1
+
o 0
Ns N
tD N
(C to C
CL Q
-C. C.)
CN N4 Nl
C, U, Cl c
++i
;+
I + I + I + I
+ + ++ + ++ +
+ +
I I + I
a] cn 04 +
C.
PS Pe
om_*
y_wm~
P4O
--o
-0---a
--o
-4
--4
-0---a
-4 ti*t$;wt
dO0O3;titr*W di0.O;OSntr7SW di00; SushIt,SV
do"&, dltrw Sv
doO-,trto,SV
d9&4--0;tetr V o0O05,SS-1I trir; SW
a ttr;wt smi trtrnwt
do-os,9Sw00;tr,wt
FIG. 1. Structure of wild-type AAV (A), p5andp19CAT
con-structs (B), andterminal repeat (tr) deletion/pS replacement
con-structs(C). (A),trsof AAVarerepresented byopenboxesatboth endsof the genome. The three promoterregions areindicated by
right-angledarrows at map positions 5, 19, and 40.The two viral
open reading frames designated REP and CAP are indicated. (B)
StructureofCATgeneconstructs.CATgene(Pharmacia, Inc.)was
cloned into the HindlIl site of pBluescript (thin lines). The p5
promoterregionderived fromanMvaI-FnuDIIdigest (bases144to
310) ofan AAVclone containing only the PstC fragment of AAV (17) was blunted and ligated into the SmaI site of the Bluescript polylinker. The poly(A) site,a235-base-pair fragment derived from aSnaBI-PstIdigestion of pSM620 (17)wasblunted and inserted into
the bluntedXhoIpolylinker site. For p19 CAT, an SstI-BclI
frag-ment(nt 814to964) isolated from pSM620grownin dam-negative
bacteriawasligatedtoSstI-BamHI-digested pCAT. The poly(A) site
was inserted as for p5 CAT. (C) Structures of terminal repeat
deletions andSV40promoterp5 replacements. Open boxes
repre-sentterminalrepeats.Wtindicatesthat the unalteredp5region from
AAVispresent.Closed black boxes and thedesignation SVonthe
rightrepresentreplacement of theAAVp5region (d103-05) with the
entireSV40regulatory region (nt 5171-5243-270) oriented such that theSV40 earlypromoterdrivesp5 geneexpression (10). Restriction enzymedesignationsontheright indicate partial deletion of thetrs,
i.e., sequences outside of those restriction sites were deleted.
Deletions of0to05or95to100orbothindicatecomplete deletions
ofone orbothtrs (1).
tions and thusnot
physiological
in the same senseasvirioninfection,
theresultsdo mimictoalarge
extentthoseseeninvirion infections
(AAV plus
orminushelper).
The situation isrenderedmorecomplex
becauseof theinhibitory
effects ofrep on adenovirusgene
expression,
which isgreatly
depen-dent on the relative
multiplicities
of infection ofthe twoviruses
(3).
From these data we conclude:(i)
the AAVp5
and
p19
promoterscanfunctionconstitutively
in HeLacells,
and
(ii)
in the absenceofhelper
virus, AAVcannegatively
regulate
geneexpression
fromthep5
andp19
promoters inHeLacells.
Ithad been
previously reported
thatCATexpression
fromthe
p19
promoter within the context of the AAV genome*
-.A:
1 2 3 4 5 6 7 8 9 10 11 12 13
FIG. 2. The effect ofreporcap orbothonexpression from the
p5 promoter in the presence or absence of adenovirus. HeLa
monolayer cellswere transfected (24) with 5 ,ug ofp5 CAT alone
(lanes 1 and 2)orp5CAT in thepresenceof5 pug ofoneof several
AAV constructs: lanes 3 and 4, wild-type AAV; lanesSand6,ins
32, a frameshift insertion which results in inactivation of rep,
pHM326 (9); lanes 7 and 8, d13-23 a deletion which results inrep
inactivation, pHM1515 (9); and lanes 11 and 12,capdl pLB101 (9),
whichisadeletion frommapunit63to86 but leavesrepintact.In
lanes 9 and 10, 1.25 or 2.5 jig of p5 CAT, respectively, were
transfected, and lane 13representschloramphenicol treated with a
commercially available preparation of CAT enzyme(control). All transfections were equalized to 20 ,ug of total DNA by using
high-molecular-weight Escherichia coli DNA ascarrier.
Precipita-tion of DNA for transfection was by the CaPO4 technique (24). Transfections wereharvestedat48h; cellextractswere prepared,
andCAT activitywasassayed (5).
could be
significantly
inducedby adenovirus
infection(20)
and that aminimal amount of
constitutive
CAT expression
froma
p19
CATconstructcould beobserved
in HeLacells.
In the latter case, adenovirus infection increased CAT
activity
fourfold(22).
These dataareincontrast tothemorelimited effect of
adenovirus observed
with thep19
CATconstruct used in these
experiments
and suggest additionalsignals
for theregulation
ofp19 expression beyond
the 30 bases upstream from the TATA box that were in ourconstruct.From
previous
work inwhichgenomereplication
was studied, it was shown the AAVterminal
repeats alsoplay
a role as a target sequence forregulation by
rep geneproducts
(10).
Toinvestigate
apossible regulatory
role of theterminal repeats in AAV
transcription,
weengineered
aseriesof constructs in which various
lengths
ofthe terminal repeatsequencesweredeleted andthesubsequent
effectonaccumulation of AAV
transcripts
wasassayed. Previously,
we havereported
construction ofahybrid AAV/SV40
ge-nomein which theSV40
regulatory
sequences(nucleotides
[nt] 5171-5243-270)
were substituted for AAV sequencesfromnt144to264
(10).
In humanKBcells,
thephenotype
ofthe
hybrid
construct(dl03-05/SV40)
wasindistinguishable
inthat
study
from that ofwild-type AAV; i.e., transcript
accumulationwas notdetectable inthe absence of
adenovi-rus.Inadenovirus-infected KB
cells,
thehybrid
genomewasA.
PS pig P40
B.
PSCAT'
psc^T ~ CA
PA
i-..
P19 CAT-s
"cr,
VOL.63, 1989
on November 10, 2019 by guest
http://jvi.asm.org/
[image:2.612.317.558.76.305.2] [image:2.612.64.295.76.347.2]P5 CAT P19CAT REP _A4_
2ug -
-2ug - 2ug
2ug - 5ug
-2ug - 2ug +
2ug - 5ug +
NJ
4
CD
- -4
8 -i
t roz
Ivs
...dm
.0 2ug 14ug
2ug 2ug 2ug
- 2ug 5 ug
2ug 2ug +
2
2u g 5ug +
- 2ug 4ug +
c
FIG. 3. Effectsofvariouslevelsofreponexpressionfromp5or
p19in the presenceorabsence of adenovirus. Transfections were
carriedoutwith2 p.gofp5CATorp19 CAT alone (lanes1and7)or inthepresenceof 2,5,or14pLgoftherep-containing plasmid
LB101
(d163-86; see reference 9) in the absence (lanes 2, 3, 8, and 9) or
presence(lanes4to6and lanes 10to 12)of adenovirus. Lane 13 is
acontrol.
biologically
active and virions were produced. Thus, atthe
levelofa
full-length
genome oritsequivalent, thecontrol ofgene
expression
by the hybrid genome is indistinguishablefrom that ofthe
wild
type. However, removal of125bases
frombothends
of the
genome (i.e., deletion of thepalindro-mic
regions
oftheterminal
repeat) haddifferentialeffects. Inthe case ofthe
wild-type
AAV genome, the terminaldele-tions still
allowed
readily detectable AAV transcriptaccu-mulation in
adenovirus-infected
KB cells (Fig. 4).Such
transcript
accumulation
in adenovirus-infected KBcells
could barely, if at all, be detected when either 103 or 125
baseswere
deleted
fromtheAAV/SV40
hybridgenome(Fig.4)butwere
detectable
whenonly55basesweredeletedfrombothtermini. Thus the critical regionappears toliebetween
nt 56 and 102. However, in hybrid constructs from which
eitherthe
right
orthe left terminal repeat had been deletedbut the other
terminal
repeat was present, transcriptaccu-mulation was
readily
detected with adenovirus coinfection.Thus, some sequence within the terminal repeat was
re-quired
fortranscript
accumulation in the presence ofaden-ovirus, butthe
positive
effect wasposition and (apparently)orientation
independent.
Therefore, under these conditionstheterminal repeat fulfilled the criteria ofan enhancer, but thiseffectwasonlyobservable under thoseassay conditions
when the
SV40
sequence was substituted for the p5 pro-moter. Within the context of the AAV genomes, the abilityof AAV
expression
from the leftward-most promoter torespond
toadenovirus
helperfunction inatransfectionassayappears to be mediated by at least two very different
sequences, one corresponding to the p5 promoter (map
positions
3 to 5) and the other in the terminal repeat. To detecttranscription,
either site appears to be sufficient.However, these data do suggest the potential for multiple
layersof
regulation
ofAAVgene expression. Regulationatposttranscriptional
andtranslationallevels have beenprevi-ously
described
(12, 23).We have
presented
data which indicate that undernon-TABLE 1.
Expression
from both the p5
andp19 promoters
Trans- Amtofplasmid transfected
%
fection Virusai
Acetyl-no.
pS
CATpl9
CAT SV2 CATpSM620
pLB101
ationb
Expt
11
2,u
g - 952
2 pg 2l.g - 123
2,u
g5,ug
- 0.84
,ug
2
2pg + 885
2,ug
5pg + 856
,ug
2
Fg
14 + 137
2,ug
- 26.58
2,ug
2,ug
- 0.89
2,ug
S,ug
- 3.710
,ug
2 2pg + 9.911
2,ug
5pg + 2.412
2,ug
14 .g + 4.1Expt2
1
2,ug
- 11.92 2pg + 18.3
3
2,ug
S,ug
- 0.24
2,ug
S,ug
+ 11Expt
31
2,ug
-
5.32
2,ug
2,ug
-
2.83
2p.g
2,ug
+ 12.34
2,ug
+ 7.85
2
tg-
2.5Expt 4
1 2
.g
+ 19.22
2,ug
- 47a
-, No
adenovirus infection; +, adenovirus
infection.
b %
Acetylation was
calculated by dividing the
amount
of 14C
radioactivity in theacetylatedform ofchloramphenicol
bythesumtotalofradioactivityinthe
acetylated
andnonacetylatedforms.permissive
conditions AAV
can
negatively
regulate
gene
expression
from
its
own
promoters.
The
data help
toexplain
the
unique
behavior
of AAV
in
cell culture infections
and
serve to
illustrate the
complex
autoregulation
of
the
replica-tion of this virus. The
multiple
layers
of regulation that
areinvolved
are
also
suggested by
the
finding
that the terminal
repeats
can
function as
enhancers of
transcription, in
addi-tion
to
their demonstrated roles in DNA replication
(10,
20),
integration
into
other DNA
sequences
(7,
8), and
rescuefrom
the
integrated
state
(18).
We
have
demonstrated
previ-ously
that
in
monkey
cos
7
cells,
which
constitutively
synthesize
the
SV40
T
antigen,
AAV
can
negatively
regulate
the
replication
of
an
AAV/SV40
hybrid
genome (10). In that
case,
the
replication
of
an
SV40
replicon
was
inhibited by
the
rep gene
product
in
trans and
the AAV
terminal
repeat was
the cis-active
target
sequence.
Although
the latter data
areindirect
with
respect
to AAV DNA
replication,
we
have
hypothesized
that under
nonpermissive
conditions in human
cells,
the
AAV
genome
inhibits its
own
replication as well.
Our
current
model
is
that AAV
negatively
regulates
its
owngene
expression
and DNA
replication
under
nonpermissive
conditions
in order to enhance its
ability
to
integrate into the
cellular
genome
to
establish a
latent
infection.
In this way,
the survival of the viral
genome
is
optimized.
This model
is
supported by
the
following
observations:
the
plasmid
pSV-neo,
which contains the
SV40
origin
of
replication,
replicates
in
cos 7
cells but
does
not
integrate
into
cellular
DNA to
stably
transform the cells to
neomycin
resistance (M.
La-bow,
A.
Levine,
and K.
I.
Berns, unpublished
experiments).
In
contrast,
an
AAV/SV40
hybrid
plasmid
in
which the same
SVneo
sequences
as in
pSVneo
were engineered
into a
on November 10, 2019 by guest
http://jvi.asm.org/
[image:3.612.317.558.89.369.2]NOTES 4453
>->;bX> >
0u
-E
3
0 00 ~0
PS
>P 19
:>P40
la 2a 1 2 3 4 5 6 7 8 9 10 11 2 13 14
FIG. 4. Effect ofterminalrepeat(tr)deletions orreplacementof p5 with the SV40 regulatory region on accumulation of AAV-specific transcripts. KBcellsweretransfected by the DEAE dextran technique (14) with 20 p.gof wild-type AAV plasmid(lanes 1 and la [shorter exposure])orwith plasmids withawild-typep5 region and completedeletions of thetrs(nt 0to145)(lanes 3 and 4) deletions in
asfarasnt 121(BalI restriction site) (lane 13),orinasfarasnt 55 (SmaI restriction site)(lane 14). Transfectionswerealsocarriedout
with plasmids in whichthe AAV p5promoterregion(map units 03 to05)wasdeleted and replaced with theSV40regulatoryregion(nt 5171-5243-270) inaconstruct containing complete trs, (lanes 2 and 2a),notrs,(lanes 5,6,and12),ordeletions inasfarastheSmaIsite (lane7), BalI site (nt 121) (lane 8), orBssH2site (nt 105) (lane 9). Deletionsof the righttronlyorlefttronlyfrom theseconstructsare shown in lanes10and11, respectively(1). Lanes 1, la, 2, 2a, 3, 5, and7to14depict transfections in whichadenovirusinfection ofthe cellswascarriedout 1h priortotransfection. RNAwasisolatedat
48 hpostinfection,electrophoresedon a1.3%agarose-formaldehyde
gel, blotted onto nylon, and probed with an AAV-specific probe (12).
deletion inthe AAV cap genedid notreplicate well incos 7
cells but did integrate to stably transform the cells to
neomycin resistance. Thus, at least in the case of AAV, replication of the viral genome and integration into the
cellular genome seem to be mutually exclusive. Similar behaviorhas been observed inthecase of bovine papilloma virus (16).
Parallels canbe drawnbetween the AAVrepgeneandthe
adenovirus ElA gene. Not only do products of each
nega-tively and positively regulate gene expression, but both
types of regulation may be exerted on the same gene. Similarly, products of both regulatory genes affect a
wide
rangeof heterologouspromoters. In thecaseof AAV, there
areat leastfour rep gene products
with overlapping amino
acid sequences (15), so that it has been difficult to sort out
the specific functions of each. To make matters more
dif-ficult,theapparent phenotype ofagivenrep gene mutant ishighly dependent on the cellinfected. The functional
diver-sity of the AAV rep gene products
exceeds
thatof ElA
products in that AAV repgene productsmay
directly
affect
AAVDNAreplication negatively as well as
positively.
Thepleiotropyof therepgeneresults in aviruswhose
life cycle
maybe thought tobe theparadigm ofa
successful
parasite.
Whenthehostcell ishealthy, the AAVgenome
establishes
a
latent infection with
no apparentill effect
onthecell.
But when the cellis exposed to toxic agents, stressed, or infectedby
alytic nuclear DNA virus, the
AAV genomeis
activated
so
that
virions
maybe
produced to continue the cyclein
a new host.We thank N. Cortez for able technical assistance and R. Kotin
and M. A. Labow for helpful discussionsand critical reading of the
manuscript. Some preliminary experiments were performed by A.
Greenberg.
The research was funded by Public HealthService grantAI-22251
from the National Institutes of Health and grantNP00588 from the
American Cancer Society. One oftheauthors(A.B.) wassupported by AmericanCancer Society fellowship PF 2731.
LITERATURE CITED
1. Berns, K. I., R. M.Kotin, and M. A. Labow. 1988. Regulation of
adeno-associated virus DNA replication. Biochim. Biophys. Acta 951:425-429.
2. Buller, R. M. L., J.Janik, E. D. Sebring, and J. A. Rose. 1981.
Herpes simplexvirus types 1 and 2completely help adenovirus-associated virus replication. J. Virol. 40:241-247.
3. Carter, B. J., C. A. Laughlin, L. M. De La Maza, and M. W.
Meyers. 1970. Adeno-associatedvirus auto-interference.
Virol-ogy92:449-462.
4. Carter, B.J., C. A. Laughlin, and C. J. Marcus-Sekura. 1983.
Parvovirus transcription, p. 153-207. In K. I. Berns (ed.), The
parvoviruses. PlenumPublishing Corp., New York.
5. Gorman, C. M., C. F. Moffat, and B. H. Howard. 1982. Recombinant genomes which express chloramphenicol
acetyl-transferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051. 6. Green, M., and R. G. Roeder. 1980. Definition of a novel
promoter for the major adenovirus-associated virus mRNA.
Cell 22:231-242.
7. Grossman, Z., K. I. Berns, and E.Winocour.1985. Structure of
simian virus 40-adeno-associated virus recombinant genomes. J. Virol. 56:457-465.
8. Grossman, Z., E. Winocour, and K. I.Berns. 1984. Recombina-tion between simianvirus 40 andadeno-associated virus: virion
coinfection compared to DNA cotransfection. Virology 134:
125-137.
9. Hermonat, P. C., M. A. Labow, D. Wright, K.I.Berns, and N.
Muzyczka. 1984. Genetics ofadeno-associated virus: isolation and preliminarycharacterizationofadeno-associatedvirustype
2 mutants. J. Virol. 51:329-339.
10. Labow, M. A., and K. I. Berns. 1988. The adeno-associated
virus rep gene inhibitsreplication of anadeno-associated virus/
simian virus 40 hybrid genome in cos-7 cells. J. Virol. 62:
1705-1712.
11. Labow, M. A., L. H. Graf, and K. I. Berns. 1987.
Adeno-associatedvirus geneexpression inhibitscellulartransformation
byheterologous genes. Mol. Cell. Biol. 7:1320-1325.
12. Labow, M. A., P. L.Hermonat, and K. I. Berns. 1986. Positive
andnegativeautoregulation oftheadeno-associated virustype 2 genome. J. Virol. 60:251-258.
13. Lusby, E. W., and K.I. Berns. 1982. Mapping of5' termini of
twoadeno-associatedvirus RNAs in theleft half ofthe genome. J. Virol. 41:518-526.
14. McCutchan, J. H., and J. Pagano. 1968. Enhancement ofthe
infectivityofSV40DNAwith DEAEdextran. J. Natl. Cancer
Inst. 41:351-357.
15. Mendelson, E., J. P. Trempe, and B. J. Carter. 1986. Identifi-cation of the trans-active repproteinsofadeno-associated virus
by antibodies to a synthetic oligopeptide. J. Virol.
56:823-832.
16. Roberts, J. M., and H. Weintraub. 1986.
Negative
control ofDNA replication in composite SV-40 bovine
papilloma
virusplasmids. Cell 46:741-752.
17. Samulski, R. J., K.I. Berns,M. Tan, and N.
Muzyczka.
1982.Cloning ofadeno-associated virusinto
pBR322:
rescue of intactvirus from therecombinant plasmidin human cells. Proc. Natl. VOL.
63,
1989on November 10, 2019 by guest
http://jvi.asm.org/
[image:4.612.62.297.79.285.2]Acad. Sci. USA79:2077-2081.
18. Samulski, R. J., A. Srivastava,K. I. Berns,and N. Muzyczka.
1983. Rescue ofadeno-associatedvirus from recombinant plas-mids: genecorrection within the terminalrepeatsof AAV. Cell 33:135-143.
19. Srivastava, A., E. Lusby, and K. I. Berns. 1983. Nucleotide
sequence and organization of the adeno-associated virus 2
genome.J.Virol. 45:555-564.
20. Tratschin,J.-D., I. L. Miller, and B. J. Carter. 1984. Genetic analysis ofadeno-associated virus: properties of deletion
mu-tantsconstructed in vitro andevidence foranadeno-associated
virus replication function. J. Virol. 51:611-619.
21. Tratschin, J.-D., J. Tal, and B. J. Carter. 1986. Negative and positive regulation in trans of gene expression from
adeno-associated virusvectorsin mammaliancellsbyaviralrepgene
product. Mol. Cell. Biol.6:2884-2894.
22. Tratschin,J.-D.,M.H. P.West,T.Sandbank,and B.J. Carter. 1984. A humanparvovirus, adeno-associated virus,as a
eukary-oticvector: transient expressionandencapsidationofthe
pro-caryoticgeneforchloramphenicol acetyltransferase. Mol. Cell.
Biol.4:2072-2081.
23. Trempe, J. P., and B. J. Carter. 1988. Regulation of adeno-associated virusgeneexpressionin 293 cells: control ofmRNA
abundance andexpression.J. Virol.62:68-74.
24. Wigler, M., A.Pellicer, S. Silverstein, R. Axel, G. Urlaub, and L. Chasin. 1979. DNA-mediated transfer of the adenine phospho-ribosyl transferase locus into mammalian cells. Proc. Natl.
Acad. Sci.USA76:1373-1376.