Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant negative phenotype for DNA replication.

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JOURNAL OF VIROLOGY, Apr. 1990, p. Vol. 4 0022-538X/90/041764-07$02.00/0

Mutation of

a

Consensus

Purine

Nucleotide

Binding Site in the

Adeno-Associated Virus

rep

Gene

Generates

a

Dominant Negative

Phenotype for DNA

Replication

NOR CHEJANOVSKYAND BARRIEJ. CARTER*

Laboratory of Molecularand CellularBiology, National InstituteofDiabetes andDigestive andKidneyDiseases,

Building8, Room304, Bethesda, Maryland 20892

Received6September1989/Accepted 21 December 1989

Adeno-associated virus(AAV) containsamultifunctionalnonstructural gene, rep, which isrequired forAAV DNA replication and has pleiotropiceffects on positiveandnegative regulation of geneexpression. All of the parvovirus nonstructural genes contain a region of highly conserved amino acid homology. Within this conservedregionis the consensussequence forapurine nucleotidebinding site.We constructed a mutant AAV having a mutation in this site by converting lysine 340 to histidine. The resulting mutant AAV genome, pNTC23, overproduced themutantRep proteins, indicating that theseproteins areautoregulated. Further-more, the mutant gene was unable to replicate butwas able toinhibitin transwild-typeAAV DNAreplication. Thus, pNTC23 representsa dominant negative mutant of AAV. Theseresults suggest thatrephas separate functional domains important forDNAreplication.

Site-specific mutagenesis canbe used readily togenerate mutations in specific genes. Generally,this results in inacti-vation of the gene product. However, some mutations may resultin overexpression of a mutantpolypeptidewhich can inhibit the activity of the wild-type gene product. Recently,

it was pointed out that such dominant negative mutations may be especially useful for the analysis of regulatory

proteinsor enzymeswhich have severalfunctionaldomains oractivities (22). We have constructedadominantnegative

mutationinthe adeno-associated virus (AAV) rep gene. AAV, like all other parvoviruses, contains a

multifunc-tionalgene, rep,which is required for AAV DNA replication

(7, 8, 21, 31, 33, 35) and has pleiotropic effects on positive

andnegative regulation of gene expression (25, 26, 36, 38). The AAV rep gene uses two transcription promoters to encodefouroverlapping Repproteins:Rep78 and Rep68 are

encodedby transcripts fromtheP5promoter,and Rep52 and

Rep4Oareencoded by transcripts from the P19 promoter (29,

38). Rep78 and Rep52 are encoded by unspliced mRNAs, whereas Rep68 and Rep4O have altered carboxyl termini,

reflecting splicing ofthetranscripts (29, 38). Rep78 or Rep68 isrequired for AAV replicating-form (RF) DNA replication

apparently by bindingtothereplication origin (1, 24).Rep52

andRep4Oarenotrequired for RF DNA replication but may

be required for the accumulation of single-stranded DNA

(ssDNA) and infectiousparticles (8).

Allofthe parvovirus nonstructural genes contain a region ofhighlyconservedamino acid homology (2, 9, 32). Notable within this region is the consensus sequence G(X)4GKT/

S(X)5_6I/L/V

for a purine nucleotide binding site present in a variety of other eucaryotic and procaryotic proteins,

includ-ingthelargeTantigens of simian virus 40 and polyomavirus (2,

5,

9, 13, 16, 19). In simian virus 40 and polyomavirus an ATPase activity that is associated with this domain is required for viral DNA replication (10-12).

In AAVtheputative purine nucleotide binding site (2, 33) is common to all four Rep proteins. In this work, we constructed a mutant AAV having a mutation in this site.

* Correspondingauthor.

Thecodonforlysine340in theconsensusnucleotidebinding

sequence was changed to histidine. The resulting mutant AAVgenome (pNTC23)overproducedthemutantRep

pro-teinsbutwasunabletoreplicate.Themutantgenewasable to inhibit in trans wild-type AAV DNA replication. Thus,

pNTC23 representsa dominantnegative mutantof AAV.

MATERIALS ANDMETHODS

Cells andviruses. An established humanembryonic kidney

cellline transformedbyadenovirus 293-31(293 cells; 18)was grown at 37°C in 5% CO2 in monolayer cultures in Eagle

minimalessentialmediumsupplementedwith antibiotics and 5% fetal calfserum. Human adenovirus type 5 and AAV type 2 (AAV-2) were grown and assayed as described previously (6).

Plasmid construction and mutagenesis. Plasmid construc-tion andmutagenesis wereperformed asdescribed in detail elsewhere (7, 8). Plasmid pNTC244 contains the

wild-type

AAV-2genome inserted viaBglII linkers(27)atthe BamHI

sitein thepolylinkersequenceof thephagemid pTZ19U

(8).

pNTC3 is an amber mutant of pNTC244 having a point

mutation in the rep geneatmapposition22(7).pNTC23was

generated from pNTC244 by oligonucleotide site-directed mutagenesis of thewild-typeAAV-2 genomewiththe

oligo-nucleotide 5'-ACTACCGGGCACACACCAACATC-3' by

use of the Muta-gene system (BioRad Laboratories, Rich-mond, Calif.) as previously described (7, 8). pNTC41 was

produced by deleting the SstI fragment between the SstI sites in the polylinker of pNTC244 and at nucleotide 814 (mapposition 17.4)of the AAV-2 sequence(8).Thisdeletion

removes theP5 transcription unit but leaves the P19

tran-scriptionunit intact. pNTC41awasgenerated frompNTC41 by introducing the same amber mutation as in pNTC3 (8). pNTC8 was constructed from pNTC23 by deleting the p5 transcription unit by use of the SstI sites as described for pNTC41. pTS18 isaRep-p40-catconstruct(36,37) grown in Escherichia coli HB101 andpurifiedby standardprocedures

(28, 36). Stocks of pTZ19U and the pNTC plasmids were grownin E. coli MV1190 and purifiedby alkaline lysis and

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Gly GGG

1338 1340

1 1

Pro Ala Thr Thr Gly Lys Thr Asn lle

CCT GCA ACT ACC GGG MG ACC AAC ATC

., ,~~~~~

I I

CAC

-- His -'

5 19 40 "

2 10

120

30

1,4148

50

Rp I 1 I . 1,

Rop78 -11*

60 70 80 90 , 100

I I I

I

96

'9 4.2kb

Rep68 :|

Rep52

Rep4O

I ;'993 ; 2183

* II

aI II

993 ' 1 2252

1

.1907

I 3.9kb

*, 3.6kb

" 3.3kb

pNTC244

M*.

pNTC23

E.-pNTC3

pNTC41

pNTC41a

pNTC8

a

FIG. 1. AAV rep gene structure and consensuspurine nucleotide binding site sequence. The AAV-2 genome (33) is shown on a scaleof 100mapunits(1unit is approximately 47nucleotides).Control regions are shown asstippledboxes (DNAreplication origins;20) andsolid circles(p5,Plg,andP40transcription promoters). Heavy arrows show p5 orp19mRNA,with theintron (mapunits41to48) shownbythe carets and thepolydenylation siteatmapunit 96 shownby the arrowheads.The

p4o

transcriptswhich codeforcapsid proteins andthe minor RNA species derived by alternate splicingareomittedfor clarity. The coding regions forthefourproteins, Rep78, Rep68, Rep52,andRep40 (29, 38),areshownbyopenrectangularboxes. Theputative purine nucleotide binding regionwith consensusamino acids underlined(2)atmap position28.6is indicatedatthetop, as arenucleotides 1338 and 1340, which were altered to convert the consensuslysine340codonAAG to ahistidinecodon,CAC. Inthelowerportionareshown theregions ofAAV-2 DNAcontainedin severalplasmids. pNTC244, pNTC23, andpNTC3 containtheentireAAV-2genome. InpNTC41, pNTC41a,andpNTC8,nucleotides 1to814 of AAV-2aredeleted. pNTC23and pNTC8 contain the mutation in the purine nucleotide binding site indicated by x. pNTC3 and pNTC41a contain a nonsense mutation (indicatedby "a") atnucleotide1033generated by changing a serine codon, UCG,to an ambercodon, UAG(7). Thenucleotidenumbering scheme is thatofSrivastavaetal. (33). kb, Kilobases.

pZ523columns(5Prime-3Prime, Inc., West Chester, Pa.) in accordance with manufacturerprotocols (7, 8, 28).

DNA transfectionandviralDNAreplication. 293cells(5 x

105/35-mm

dish)were transfected by thecalciumphosphate

precipitation method (35). For AAV replication, the cells were infected with adenovirus type 5 (5 PFU per cell) 1 h prior to transfection as described before (27, 35). For indi-vidual transfection experiments, the total amount ofDNAin each transfection mixture was adjusted to the same level with control plasmid pTZ19U.

IntracellularAAV DNAreplication forms wereextracted

40 h aftertransfection bythe modified Hirt sodiumdodecyl sulfate-high-salt lysis procedure (27, 35). The DNA forms were analyzed by agarose gel electrophoresis followed by

blotting to nitrocellulose filters and hybridization with

[32P]AAV

probes radiolabeled by the random priming pro-cedure (17). The amount of radioactivity hybridized was

quantitated byscintillationcountingof individual bands from theblots.

Proteinanalysis. Cell lysates foranalysis ofRepproteins

wereprepared 48 h aftertransfection ofadenovirus-infected

293 cells and electrophoresed in 12% polyacrylamide gels. AAV Rep proteinswere detected with anti-Repantiseraby

the immunoblotting procedure (29, 38) exactlyasdescribed

previously (8).

CAT assays. 293 cells

(106)

were transfected with 1

jig

of

pTS18,aRep- AAVp40-catvector(36, 37),and upto10 ,ug ofacomplementing

plasmid.

Cell extracts were

prepared

at 48 h after transfection and assayed for

chloramphenicol

acetyltransferase (CAT)

activity

as described

previously

(36).

RESULTS

Mutagenesisof theputativenucleotidebindingsite in therep reading frame. The structure of the AAV genome and the

organization ofthe rep gene are shown in

Fig.

1. The rep gene is transcribed fromtwodifferent promoters,

designated

atp5andP19(at map

positions

5 and 19,

respectively).

Four Repproteins were identified with

specific

anti-Rep

antibod-ies (29, 38). The predominant

proteins

were

Rep78

and

No

1 fth4 1 1 1 . --l-

p-"i-)

1

-

u--_

>s 2252

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c\j

Rep78_

Rep68-

Rep52-

Rep4O-1

2 3 4

5

6 .0

CO '- :

c'ij

-to~

<

...

MP

.1

,A

[image:3.612.366.514.72.289.2]

."p

FIG. 2. Production of Rep proteins by mutant AAVgenomes.

Adenovirus-infected 293 cells (5 x 105 cellsper35-mmdish) were

transfected with the AAV plasmidsdesignated by the numbersatthe top(thepNTC designation is omitted). Ad indicates control adeno-virus-infected cells. Proteinextractswereanalyzedby immunoblot-ting withananti-Rep antibody.

Rep52, whereas Rep68 and Rep4O were produced at much lower levels (29, 38; Fig. 2).

The consensus purine nucleotide binding site in the rep

reading frame (2) was at map position 28.6 and was thus

common to all four Rep proteins (Fig. 1). To produce a

mutation in the putative nucleotide binding site, we

intro-duced atwo-base change in the lysine 340 codon (Fig. 1).

Thus, the codon AAG was convertedto CAC (nucleotides 1338 and 1340), coding for histidine, to generate mutant pNTC23.

SeveralothermutantAAVplasmidsare alsodiagrammed

in Fig. 1. pNTC3 contains an amber mutation at map

position 22. pNTC41, pNTC41a,andpNTC8 areanalogous

topNTC244, pNTC3, and pNTC23, respectively, but retain onlytheP19transcription unit.

Repprotein production by AAVmutants. The expression ofthe Rep proteins from mutant and wild-type AAV ge-nomes wasdetermined by immunoblot analysis ofextracts prepared fromadenovirus-infected 293 cellstransfected with the appropriate plasmids. pNTC23 produced all four Rep proteins at a higher level than did the wild-type AAV plasmid pNTC244, suggesting that the production of Rep proteins is autoregulated (Fig. 2). It is possible that the overproductionby pNTC23representsanincreasedstability

of Rep proteins. However, we have previously shown by

pulse-chaseanalysisthatRep proteinsarehighlystable(30).

Figure 2 also shows, as wereportedpreviously for pNTC41

andanalogous plasmids (8, 29, 38), that mutationordeletion

of the p5promoterortheamino terminus ofRep78orRep68

resulted in the overproduction of Rep52 and, to a lesser

extent,ofRep4O.Incontrast,pNTC8, which is analogousto pNTC41 but which has thesame mutation intheconsensus

nucleotide binding site as pNTC23, did not overproduce Rep52. This resultsuggeststhat the expression ofRep52 (or Rep4O) may be independently autoregulated in addition to

being regulated by Rep78 or Rep68. Elsewhere we showed

thatpNTC3and pNTC41a,whichhaveanamber mutationat

mapposition22, produced no Repproteins (7, 8).

Blockage of AAV DNA replication by a mutation in the

consensus nucleotide binding site. The Rep proteins are

essentialfor AAVDNAreplication(7,8, 21,24, 31, 35). The effectof themutation intheputative nucleotide binding site of rep was analyzed by isolating intracellular replicating

AAV DNA from adenovirus-infected 293 cells transfected with pNTC23 or pNTC244. pNTC23 was defective for

[image:3.612.104.256.73.200.2]

23

244

FIG. 3. Replication of AAVgenomes. Adenovirus-infected 293 cellsweretransfected withpNTC244orpNTC23 DNAasfollows. Intracellular AAV DNA was extracted, electrophoresed in an agarose gel, and detected by Southern blotting with an AAV [32P]DNA probeasdescribedin Materials and Methods. Lanes: 1, 6 jigofpTZ19U;2,0.1 ,ug of pNTC23;3, 1 jigofpNTC23; 4, 6 jig of pNTC23; 5, 0.1 ,ug of pNTC244; 6, 1 ,ug of pNTC244. All transfec-tion mixtureswereadjustedto6 ,ug with control plasmidpTZ19U. RFd, RFm, and SS indicate replicating dimer DNA, replicating

monomerDNA, andprogenyssDNAs,respectively.

replication,and the normal AAV DNA intracellularspecies (4, 34), includingmonomeranddimer RFs, (RFm andRFd,

respectively) andprogenyssDNA, were notobserved(Fig.

3). We reported elsewhere that pNTC3, pNTC41, and pNTC41a were all unable to replicate AAV DNA (7, 8). Similarly, pNTC8, which is both an ori mutant and a rep

mutant, like pNTC41 and pNTC41a, didnotreplicateAAV DNA(datanot shown).

Inhibition of AAV DNA replication bymutantRepproteins. Detection of AAV DNA replication in adenovirus-infected 293 cells is a sensitive assay, and RFs resulting from transfectionof less than 10ngofanAAV-containing plasmid canbe observed(unpublished results).We used thisassayto determine if the mutant Rep proteins from pNTC23 could inhibit wild-type DNA replication. AAV DNA replication

was easily detected when 50 ng of wild-type AAV, pNTC244,wastransfectedintoadenovirus-infected293cells (Fig. 4, lane 1). Increasing amounts of wild-type AAV plasmidincreased theamountof intracellular DNAsynthesis (Fig. 4,lanes 2to4). However,when50ngofpNTC244was

cotransfected with increasing amounts ofpNTC23, AAV DNA replication was severely inhibited (Fig. 4, lanes 8 to 10). Indeed, significantinhibitionby pNTC23 wasseenwith

only a 1:1 ration of pNTC23 to pNTC244 (Fig. 4, lane 8). When the amber mutant pNTC3 was cotransfected with pNTC244 (Fig. 4, lanes 5 to 7), RF DNA levels were

decreasedbyno morethan2-fold,evenata20-foldexcessof themutant.Sincethe amberplasmid pNTC3 suppliesnoRep

protein, the drastic inhibition by pNTC23 must have been

duetothemutantRep proteinsandnot totheeffect ofadding

additional AAVtemplates containing replication origins. Additionalexperiments(Fig. 5, lanes 7to12) showed that inhibitionby pNTC23wasnotobserved when theamountof

RFd

RFm

Ss

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12 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 12

RFd

RFm4

Ss

-.4~~~~~~~~~~~~~~~~~~~~~~~~

3 23

FIG. 4. Inhibition of wild-type AAV DNA replication by a trans-dominant mutant. Adenovirus-infected 293 cells were trans-fected with0.05 jigof pNTC244(lane 1) and cotransfected with an additional0.05, 0.1, or 1jigof pNTC244(lanes 2 to 4), pNTC3 (lanes 5 to 7), or pNTC23 (lanes 8 to 10), respectively. Lane 11 shows adenovirus-infectedcellstransfected withthe controlplasmid alone. Alltransfection mixtureswereadjustedto 1 ,ugwithcontrol plasmid pTZ19U. AAV DNA was extracted and analyzed as for Fig. 3. RFd, RFm, and ssDNA species are indicated. The additional species in lanes 7 and 10 areinputpNTC3 andpNTC23DNAs,respectively. mutantplasmid was decreased to about one-tenth that of the wild-type plasmid. Similarly, inhibition could be overcome when the amount of wild-type plasmid was increased 10-fold (Fig. 5, lanes 2 to 5). Also, pNTC23 could be complemented forreplicationwhen wild-typerep was suppliedby

cotrans-fectinga10-to20-foldexcessofanOri- AAV plasmid (data not shown).

Themutation in pNTC23 affected both the Rep78 or Rep68 and Rep52 or Rep4O reading frames. Thus, it was conceiv-able that the mutant Rep52 or Rep40 protein alone might inhibitwild-typeAAV DNAreplication.This possibility was tested by cotransfecting the wild-type AAV plasmid with

pNTC8, aswellaswith pNTC41 and pNTC41a(Fig. 6). No

significantinhibition of AAV DNAreplicationwasobserved with any of these plasmids, even at a 20-fold excess over the wild-type plasmid, pNTC244 (Fig. 6).

The datafrom

experiments

similartothose inFig.4and 6 werequantitated by cutting the blots afterhybridizationand determining theamountof

[32P]DNA

probe annealedto the RFmspecies. A 1:1ratio ofpNTC23topNTC244 produced

20-fold inhibition, whereas even a 20:1 excess of plasmids

pNTC3, pNTC41, pNTC41a, andpNTC8producednot more than2- to2.5-fold inhibition(Fig. 7). This lower inhibitionby

the latter plasmids may reflect simply a dilution effect of

providing a large number of substrates (i.e., replication origins) thatpotentially bind Rep proteins. This appears to be thecase evenforpNTC3,whichcanbecomplemented by

RFd-

RFm-em.

w

a

.6

pNTC244

pNTC23

FIG. 5. Effect of relative concentration ofwild-type and mutant plasmidsonAAV DNAreplication. Adenovirus-infected 293 cells weretransfected with DNA as follows: lane 1, 0.1 jigofpNCT23; lanes 2to5,0.1 jig of pNTC23 plus0.1jLg(lane2), 0.5 ,ug(lane 3), 1jig(lane4), or 2 ,ug(lane5)ofpNTC244;lane 6,pTZ19U control DNA; lane 7, 0.05 jig of pNTC244; lanes 8 to 12, 0.05 jig of pNTC244 plus0.1 jig(lane 8), 0.05 ,ug(lane9), 0.01 jig (lane 10), 0.005jig(lane11),or0.001 ,ug(lane 12) ofpNTC23.Alltransfection mixtureswereadjustedto 2jigof total DNA with pTZ19U DNA. AAV DNAreplicationwasanalyzedasfor Fig.3.

the wild-type plasmid to generate mutant genomes that competefor, but donotsupply, Rep proteins. Data similarto those inFig. 7werealso obtained when the radioactivityin RFd or ssDNA was quantitated, except that ssDNA was moresensitivetoinhibition thanwasRFd DNA(e.g.,Fig.4, lanes 5to 7). Thisprobablyreflectsacascade effect in that decreased ssDNA accumulation may result both directly

fromdecreased RFd DNA synthesis andperhaps indirectly

from decreased wild-type protein synthesis. Both capsid proteins and the Rep52 or Rep4O protein are apparently required for efficientssDNA accumulation(8, 21, 35).

trans-regulation by pNTC23-encoded Rep proteins. Previ-ous studies showed that in uninfected 293 cells the Rep proteinsexert pleiotropic effects on the level ofexpression fromchimericp40-catconstructs(36, 37).Thus,Repproteins increasedbytwo- tothreefold the accumulation ofchimeric

P40 RNA

transcripts

but inhibited the

expression

of CAT

activity (37). The ability of the mutant Rep proteins from

pNTC23 to trans-regulatep40-cat expression was analyzed

with thechimeric Rep-p40-catconstructpTS18(Fig. 8) (36, 37). Cotransfection ofwild-type pNTC244 with the reporter

plasmidresulted inafourfoldinhibitionof CATactivity (Fig.

8). In contrast, with pNTC23 there was about a twofold

stimulation, whereas the rep amber mutant

pNTC3

had a null effect (Fig. 8). This result suggests that the mutant

pNTC23may haveretainedthetrans-activation property but

apparentlyhas lostthe negative regulatoryeffect of rep.

DISCUSSION

Alterationof the

putative purine

nucleotide

binding

site in the AAV rep gene resulted in a mutant,

pNTC23,

which

overproduced all four Rep

proteins

but was defective for AAV DNA replication. Furthermore, the mutant

proteins

SS-l

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41..8_._ . ...

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41 41ta 8

FIG. 6. Replication of wild-type AAV DNA in thepresenceof

mutant AAV genomes producing only p19-encoded Rep proteins.

Adenovirus-infected 293 cells were transfected with 0.05 ,ug of

pNTC244 (lanes 1 to 4 and 6 to 14) and cotransfected with an

additional 0.05, 0.1,or1,ug, respectively, of pNTC244 (lanes 2to4), pNTC41(lanes 6to8),pNTC41a (lanes 9to11),orpNTC8 (lanes 12

to 14)or1 p.gof controlplasmid pTZ19U (lane 5). All transfection

mixtureswereadjustedto1 pLgwith the controlplasmid. AAVDNA replicationwasanalyzed asfor Fig. 3.

wereinhibitory for wild-typeAAVDNAreplicationintrans.

However, this inhibitionwas overcomebya 10-fold excess

ofwild-type AAVgenomes.Presumably, thisresult reflects

theneedfor increased wild-type Rep proteininthepresence

ofoverproduced mutantprotein. pNTC23fulfills the criteria foradominant negativephenotype (22), suggestingthat the putative purine nucleotide binding site may be important

both for AAV DNA replication and for some of the

pleio-tropic properties ofrep.

TheRep78orRep68 proteinisnecessary(7, 21, 31,35, 36) andsufficient (8) forinvivo amplificationof AAVduplexRF DNA. Consistent with this, Rep78 or Rep68 binds

specifi-cally to the AAV palindromic termini which constitute the replication origins (1, 24). Furthermore, recent evidence from in vitro assays indicates that the Rep78 or Rep68

proteinappears tocontain an ATP-dependent, site-specific,

strand-specific nuclease activity involved in the resolution of theAAVDNA termini(32a). In in vitroassays,resolution of

thetermini butnotbinding of Rep78orRep68tothe origins requires ATP (24; Snyderet al., in press). Thus, the DNA-negative phenotype ofpNTC23 provides in vivo evidence consistentwiththepossibility thatafunctional ATP binding

site inRep78 orRep68is requiredforRepfunction inAAV

DNA replication. That the DNA-negative phenotype of pNTC23 isdominant intrans suggeststhatotherfunctional domainsarestill presentin the mutantproteins.

The most obvious explanation ofthe dominant negative phenotype of pNTC23for AAV DNA replication is that the mutantRep78 and Rep68 proteins have retainedtheir origin-binding ability but havelost their enzymatic activity requir-ingATP. Thus,themutantproteinscouldcompete withthe wild-type protein. However, inhibition could also be medi-ated by protein-protein interactions between mutant and wild-type Rep proteinsorbyinteraction withcellularfactors

5

1

la

pNTC23

100 1000

AddedDNA(ng)

FIG. 7. Effect of cotransfectedmutant AAVgenomesonAAV DNAreplication. The data werederived from experiments

analo-goustothose inFig. 4 and 6. Theamountofradioactivityhybridized

tothe RFmspecies afterblottingwasdeterminedby cutting the filter and liquid scintillation counting. The effect of cotransfecting

ge-nomesis shownby plotting theamountsofradioactivitypresentin the RFm band as a percentageof the control amount present in adenovirus-infected293 cells transfected with 0.05,ugofpNTC244.

important forAAV replication. It is also possible that the dominantphenotype of pNTC23 could be mediated by the mutantRep52orRep4O proteinthatwasalsooverproduced. This possibility is less likely, however, because a 20-fold

excessofpNTC8,whichproducedmutantRep52andRep4O proteins but not Rep78 and Rep68 proteins, showed no

specificinhibitoryeffect.However, pNTC8didnot

overpro-duce the mutantproteins, so evenatahigh excess ofinput

genomesit maynotgenerate sufficient mutantprotein. The overproduction of the Rep proteins by pNTC23 providesthe first directevidence that theexpressionofthese proteins is autoregulated. This regulation is complex. We previously reported that Rep78 orRep68 apparently

down-regulates the expression ofRep52 or Rep 40 (29, 38). The

present report suggests that Rep78 and Rep68 may also down-regulate their own expression. Preliminary evidence (B. Antoni and B. Carter, unpublished data) suggests that this negative regulation is mediated at least in part at the level of RNA accumulation. Recent evidence also suggests thatRep proteinsdownregulatetheexpressionof CATfrom thep5 or P19 promoter and the expression ofRep mRNA

from a simian virus 40early promoter (3). In contrast, in adenovirus-infected KB cells, Rep proteins apparently

in-1 2 3 4 5 6 7 8 9 10 11 12 13 14

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Aa *cilc]p,uo

1p3s

:; ; LITERATURE CITED

I2~t %'i ~ C w

s1.

tot !Ashktorab, H.,C and A.Srivastava.1989. Identification of nuclear proteins that specifically interact with adeno-associated virus type 2 inverted terminal repeat hairpin DNA. J. Virol. 63: 3034-3039.

2. Astell, C. R., C. D. Mol, and W. F. Anderson. 1987. Structure andfunctional homology of parvovirus and papopavirus poly-peptides.J. Gen. Virol.68:885-893.

3. Beaton, A.,P.Palumbo,and K.I. Berns. 1989.Expressionfrom * * *^ * the adeno-associated virus p5 and p19 promoters is negatively W W regulatedin transbythe repprotein.J. Virol.63:4450-4454.

4. Berns, K. I., and W. W. Hauswirth. 1984. Adeno-associated virus DNA structure and replication, p. 1-31. In K.Berns(ed.),

5 5 ^ 5^ ^ Theparvoviruses. Plenum Publishing Corp.,New York.

@

___w_ 5. Bradley, M. K., T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster. 1987. Consensus topography in the ATP * * * * * ,,, binding site ofthesimian virus40andpolyomaviruslarge tumor

antigens. Proc. Natl. Acad.Sci. USA84:4026-4030. .4Q.

lis 6. Carter,B.J., C. A.Laughlin,L. M.de laMaza,and M.Myers.

1979. Adeno-associated virus auto-interference. Virology 92: FIG. 8. trans-Regulation by rep of expression from a p40-cat 449462.

plasmid (pTS18) in uninfected293 cells. Cells were transfected with 7. Chejanovsky, N., and B. J. Carter. 1989. Replication of a human pTS18(1 ig)and10,ugofwild-type (wt) DNA (pNTC244) or 2.5 or parvovirus nonsense mutant in mammalian cells containing an 10 ,ugofmutant DNA (pNTC23 and pNTC3) or 10 ,ug of control inducible amber suppressor. Virology 171:239-247.

plasmid pBR322 (c). CAT activitywas measured 48 h after trans- 8. Chejanovsky, N., and B. J. Carter. 1989. Mutagenesis of an fection. The percent acetylation in each reaction is shown at the AUG codon in the adeno-associated virusrepgene: effects on

bottom. viral DNA replication. Virology 173:120-128.

9. Chen, K. C., B. C. Shull, E. A. Moses, M. Lederman, E. R. Stout,and R.C. Bates.1986.Complete nucleotidesequenceand genome organization ofbovine parvovirus. J. Virol. 60:1085-creased the transcription of mRNA from all three AAV 1097.

promoters(26). Furthermore, we previously showed that for 10. Clark, R., D. P. Lane, and R. Tjian. 1981. Use of monoclonal

acatgeneexpressedfromanAAVP40promoterin 293 cells, antibodies as probes of simian virus 40 T antigen ATPase

Rep79 orRep68 simultaneously mediateda modest activa- activity. J. Biol. Chem. 256:11854-11858.

tion of two- tothreefold inp40-cat mRNA expression but a 11. Clark, R., K. Peden, J. M. Pipas, D. Nathans, and R. Tjian.

fivefolddecrease in netCAT activity, apparently because of 1983. Biochemical activities of T-antigen proteins encoded by

a moredrastic10-to20-foldposttranscriptional inhibition of simian virus 40 A gene deletion mutants. Mol. Cell. Biol.

CAT

more y h eprmn n i.8sgessta e 3:220-228.

CAT activity. The experiment in Fig. 8 suggests that Rep 12. Clertant, P., and F. Cuzin. 1982. Covalent affinity labeling of

proteinsfrompNTC23 may have lost the negative regulation periodate-oxidized[a-32P]ATP of the large-T proteins of poly-but have retained the positive activation of p40-cat expres- oma and

SV40

viruses. J. Biol. Chem. 257:6300-6305.

sion. 13. Clertant, P., andI. Seif.1984. A common function for polyoma

In summary,ourresults, together withthoseofothers (24, viruslargeT andpapillomavirusEl proteins.Nature(London)

32a), suggest that the Rep proteins may have separate 311:276-279.

functional domains, including an ori binding domain and 14. Cole, C. N., J. Tornow, R. Clark, and R.Tjian. 1986. Properties

probablyanATPbindingdomain. Apreliminary analysis(J. of the simian virus 40(SV40)large T antigens encoded bySV40

Trempe,R. Owens,N. Chejanovsky,andB. Carter, unpub- mutants with deletions in gene A. J. Virol.

57:539-546.

lisheddata)suggeststhatthemutantRep78protein produced 15. Denhez, R., B. Heiman, L. d'Anriol, T. Graf, M. Coquilland,J.

bypNTC23isafunctionalAAV oribindingprotein. Defin- Coll, F. Galibert, K. Moelling, D. Stehelin, and J. Ghysdase.

by p

oTC23

iS a

tunctilonal

AAV

ourifinding

protein.

Defin-

1988. Replacement ofIys622in the ATP binding domain of

pl00

itiveproofofthis will require purification and characteriza- gag-mil abolishes the in vitro autophosphorylationof the protein tion of mutant Rep proteins. Nevertheless, these observa- and biological properties of thev-miloncogene of MH2 virus. tions indicate that rep may beanalogous to other viral genes EMBOJ. 7:541-546.

such as the simian virus 40 T-antigen gene, which has 16. Doolittle,R. F. 1986. Proteinsequencedata banks: the continu-separatedomainsmediatingoribinding andATPbindingor ing search for released structures. Protein Eng. 1:15-27. ATPase (10-14, 19). Finally, we note that in a variety of 17. Feinberg, A. P., and B. Vogelstein. 1982. A technique for

protein kinases,notdirectlyrelatedtoRep butcontainingan radiolabeling DNA restriction fragments to high specific activ-ATPbinding site, mutation of the conservedlysine specifi- ity. Anal. Biochem.

132:6-13.

caTPy

b.'. . c eo. 18. Graham, F. L., J. Smiley,W. C.Russell, andB. Naiva. 1977. cally alters biological activity. For instance, conversion of Characteristics of a human cell line transformed by DNA

lysine721 ofthe humanepidermal growthfactor receptorto adenovirus type 5. J. Gen. Virol. 36:59-72.

alanine abolished tyrosine kinase

activity,

altered receptor 19. Griffin, B. E. 1981. Structure and genomic organization ofSV40

recycling,and abolishedsignal transduction(23). Similarly, and polyoma virus, p. 61-124. In J. Tooze (ed.), Molecular the conversion of lysine 623 of the retroviral mH2 v-mil

biology

ofDNAtumorviruses. ColdSpringHarborLaboratory, oncogene protein,

plOOga-"',

to methionine abolished Cold Spring Harbor, N.Y.

serine-threonine in vitro autophosphorylationandbiological 20. Hauswirth, W. W., and K. I. Berns. 1977. Origin and

termina-properties of the protein (15). tion of adeno-associated virus DNA replication. Virology 79: 488-499.

21. Hermonat, P., M. A. Labow, R. Wright, K. I. Berns, and N. ACKNOWLEDGMENTS Muzyczka. 1984. Genetics of adeno-associated virus: isolation andpreliminarycharacterization of mutants of adeno-associated Wethank N. Muzyczkaand K. Bernsforuseful discussion and virus type 2. J. Virol. 51:329-339.

communicationofdatapriortopublication. 22. Herskowitz,I. 1987. Functional inactivation ofgenesby

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.612.57.299.71.251.2]

(7)

CHEJANOVSKY AND CARTER

nantnegative mutations. Nature(London)329:219-222.

23. Honegger, A. M., T. J. Dull, S. Felder, E. Van Obberghen, F.

Bellot,D. Szapary, A.Schmidt, A. Ullich, and J. Schlessinger. 1987. Point mutation atthe ATPbinding site of EGF receptor

abolishes protein-tyrosine kinase activity and alters cellular routing. Cell51:199-209.

24. Im, D.-S., and N. Muzyczka. 1989. Factorsthatbindto adeno-associated virus terminalrepeats. J.Virol. 63:3095-3104. 25. Labow,M.A.,L. H. Graf,Jr., andK. I.Berns. 1987.

Adeno-associated virusgeneexpression inhibits cellular transformation

byheterologousgenes. Mol.Cell. Biol. 7:1320-1325.

26. Labow, M. A., P.L. Hermonat,andK. I. Berns. 1986.Positive andnegative autoregulation of the adeno-associated virustype2

genome. J.Virol.60:251-258.

27. Laughlin, C. A., J.-D. Tratschin, H. C. Coon, and B. J. Carter.

1983. Cloning of infectiousadeno-associatedvirus genomes in

bacterialplasmids. Gene 23:65-73.

28. Maniatis, T., E. F. Fritsch,and J.Sambrook. 1982. Molecular cloning:alaboratorymanual. Cold Spring Harbor Laboratory,

ColdSpring Harbor, N.Y.

29. Mendelson, E., J. P. Trempe,andB.J. Carter. 1986. Identifi-cation of thetrans-acting Repproteins of adeno-associatedvirus by antibodiestoasynthetic oligopeptide. J. Virol. 60:823-832.

30. Redemann, B. E., E. Mendelson, and B. J. Carter. 1989. Adeno-associated virus Repproteinsynthesis during productive infection. J. Virol.63:873-882.

31. Samulski, R. J., L. S. Chang, and T. Shenk. 1987. A

recombi-nant plasmid from which aninfectious adeno-associated virus

genome can be excised in vitro and its use to study viral replication. J. Virol. 61:3096-3101.

32. Shade, R.O., M. C. Blundell, S. F. Cotmore, P. Tattersall, and C. R. Astell. 1986. Nucleotide sequence and genome organiza-tionof human parvovirus B19 isolated from the serum of a child during aplastic crisis.J.Virol.58:921-936.

32a.Snyder, R. O., R. J. Samulski, and N. Muzyczka. 1990. In vitro resolution ofcovalently joined AAV chromosome ends. Cell 60:105-113.

33. Srivastava, A., E. W. Lusby, and K.I.Berns. 1983. Nucleotide sequenceandorganization of adeno-associated virus2genome. J.Virol. 45:555-564.

34. Straus, S. E., E. D. Sebring, and J. A. Rose. 1976. Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virusDNAsynthesis. Proc. Natl.Acad. Sci. USA 73:742-746.

35. Tratschin,J.-D., I. L. Miller, and B. J. Carter. 1984. Genetic analysis of adeno-associated virus: properties of deletion mu-tantsconstructedin vitroand evidence foranadeno-associated virusreplication function. J. Virol. 51:611-619.

36. Tratschin, J. D., J. Tal, and B. J. Carter. 1986. Negative and positive regulation in trans of gene expression from adeno-associatedvirus vectorsinmammaliancellsbyaviralrep gene product. Mol. Cell. Biol.6:2884-2894.

37. Trempe, J. P., and B. J. Carter. 1988. Regulation of adeno-associated virusgeneexpression in293cells:control of mRNA abundance and translation.J. Virol. 62:68-74.

38. Trempe, J. P., E.Mendelson, and B. J. Carter. 1987. Charac-terization ofadeno-associated virusrepproteinsin humancells byantibodies raised against repexpressed inEscherichia coli. Virology 161:18-28.