(NAS Colloquium) Genetic Engineering of Viruses and Viral Vectors

142 Pages·1996·4.2 MB·English

by  Proceedings of the National Academy of Sciences

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origiublic
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are truersion ACADEMY OF SCIENCES OF THE
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riginal ohic errp GPeanpeertsi cf reonmg ain Neeatriionnga lo Af cvaidreumseys o af nSdci eonfc vesir Cuos llvoeqcutiourms:: GAe pneretifca Ecnegineering of Viruses and Viral Vecto rs 11287
he gra Peter Palese and Bernard Roizman
m tpo Site-specific integration by adeno-associated virus 11288–11294
y
d froe tm R.Michael Linden, Peter Ward, Catherine Giraud, Ernest Winocour, and Kenneth I.Berns
eeatd so Oncogenic potential of the adenovirus E4orf6 protein 11295–11301
es crd, an AdMenaorvyi rMuso-moreed, iNatoebdu ion tHerolreiukkoisnh-i1, 2a ngde nTeh tohmeraasp Sy hfeonr kmetastatic colon carcinoma 11302–11306
m XML file retaine MMialntoune lF Cinaerugsoold, ,K Shaiveimo LP.hCa.mW-Noog,u aynedn ,S Yhuo-kH-Lsiaam C Khewnong, Bisong Xu, Ken-Ichiro Kosai,
ed fronnot b TheB feurnncatrido nR ooifz hmearpnes simplex virus genes: A primer for genetic engineering of novel vectors 11307–11312
sa
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mper, The application of genetically engineered herpes simplex viruses to the treatment of experi- 11313–11318
ov mental brain tumors
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n rho Samita S.Andreansky, Bin He, G.Yancey Gillespie, Liliana Soroceanu, James Markert,
eeg, Joany Chou, Bernard Roizman, and Richard J.Whitley
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as atti Replication-defective herpes simplex virus vectors for gene transfer in vivo 11319–11320
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work c for PWeigllgiya mM Far.Gcoonini,s D, Davaivdi dK Jr.iFskinyk, ,T ahnodm Jaoss eOplhig Cin.Go,l oPriieotsroo L.Poliani, Ramesh Ramakrishnan,
original g-specifi A dteivleet idoune mtou ata fnati liunr teh ien h auumtoarne gcuyltaotmioengalovirus gene encoding IE1491aa is replication defec- 11321–11326
the ofinesett HuEmdawn acrydt oSm.Megoaclaorvsikriu, sG UeoSr3g iem Wpa.Kirse mtrbanles,p Joorht na nMd .m Laytluer,a atinodn Roifc mhaarjdo rF h.Gisrteoacvoemspatibility 11327–11333
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ntatioher ty cTohmomplaesx Rc.lJaossn eIs h, eEamvym cahnauienls J.H.J.Wiertz, Lei Sun, Kenneth N.Fish, Jay A.Nelson, and
esed ot Hidde L.Ploegh
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na TABLE OF CONTENTS ii
nal; liation
origiublic Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety 11341–11348
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e to thof this ApBpleicrnaatirodn Ms oofs spox virus vectors to vaccination: An update 11349–11353
aks are trunt version NegEPaenttzievore PP-saatolrealsenetdt,i HRoNnAg yvoirnugs eZsh: eGnegn, eOtitch menagr iGne. eErinngge lahnadr datp, pSltiecpathiaonn sPleschka, and Adolfo 11354–11358
e brehe pri García-Sastre
ag te Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorpo- 11359–11365
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ng files.Please u rMKa.taRetdtoh seieaffsi cJi.eSncthlyn eilnl,t oL vinirduas Bpuarotnicolceosre, Evelyne Kretzschmar, Erik Johnson, and John
settied. Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the 11366–11370
ypesert HIV-1 envelope spike protein
ginal tally in Teshome Mebatsion and Karl-Klaus Conzelmann
orient Alphavirus-based expression vectors: Strategies and applications 11371–11377
e d Ilya Frolov, Thomas A.Hoffman, Béla M.Prágai, Sergey A.Dryga, Henry V.Huang, Son-
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m tac dra Schlesinger, and Charles M.Rice
on
ot frbee Early events in poliovirus infection: Virus-receptor interactions 11378–11381
ne Vincent R.Racaniello
k, av
booy h Efficient transfer, integration, and sustained long-term expression of the transgene in adult 11382–11388
er ma rat brains injected with a lentiviral vector
papors Luigi Naldini, Ulrike Blömer, Fred H.Gage, Didier Trono, and Inder M.Verma
riginal ohic errp Usep eosfv viriurisoens DNA as a cloning vector for the construction of mutant and recombinant her- 11389–11394
he gra S.Monroe Duboise, Jie Guo, Ronald C.Desrosiers, and Jae U.Jung
m tpo Development of HIV vectors for anti-HIV gene therapy 11395–11399
y
d froe tm Eric Poeschla, Pierre Corbeau, and Flossie Wong-Staal
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eatd s A stable human-derived packaging cell line for production of high titer retrovirus/vesicular 11400–11406
es crd, an sDtoanmiealt iSti.sO vriyru, sB Gev persleyu dAo.Ntyepuegseboren, and Richard C.Mulligan
m XML file retaine CelAl-.sDurufsatcye M reiclleeprtors for retroviruses and implications for gene transfer 11407–11413
ed fronnot b Immcoumnibziantaiotino nw, iitnhd DucNeAs p vraocteccintievse e inmcmoduinnigt yg ilny caonpirmoatel imn oDd eolrs g olfy choeprproetse sinim Bp,l eaxlo vnier uosr- 2in 11414–11420
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ompover, c dWisiellaisaem L.McClements, Marcy E.Armstrong, Robert D.Keys, and Margaret A.Liu
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n rho Fusigenic viral liposome for gene therapy in cardiovascular diseases 11421–11425
eeg, Victor J.Dzau, Michael J.Mann, Ryuichi Morishita, and Yasufumi Kaneda
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na GENETIC ENGINEERING OF VIRUSES AND OF VIRUS VECTORS: A PREFACE 11287
nal; liation
origiublic This paper serves as as introduction to the following papers, which were presented at a colloquium entitled “Genetic Engineering of
e p Viruses and of Virus Vectors,” organized by Bernard Roizman and Peter Palese (Co-chairs), held June 9–11, 1996, at the National
e to thof this Academy of Sciences in Irvine, CA.
are truersion Genetic engineering of viruses and of virus vectors: A preface
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Pag tse PETER PALESE* AND BERNARD ROIZMAN†
ng files.Please u B.KoG*vDlieverep Vamritrema al e Ofnitrn mcoof s lopMgoiytc orLonab wbioohlrioacgthoy rt,io eM ss,to aTunhndet, USaninndia vIie wSrsciilhtly om ooolf voCefh tMihceae gdeoaic,r it9nh1e.0, 5Etahs tA 5v8etnhu Set raete 1t 0C0htihc aSgtore, eItL, N60e6w3 7York, NY 10029; and †The Marjorie
settied. ANrecahrliym etwdeos centuries ago, Jenner used a live virus of another species to combat smallpox—one of the most lethal human pathogens
peert known. In the intervening years, science has provided the tools to produce by design in the laboratory other live viruses capable of protecting
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orient herpesviruses account for a very significant portion of the total costs. While efforts designed to eliminate other infectious diseases from human
he cid society continue, other uses for viruses emerged. They stem from four considerations.
m tac First, viruses attack cells they recognize by specific receptors that are present on cell surfaces.
on Second, viruses evolved by borrowing and modifying cellular genes. Yet, all viruses depend on specific cellular functions for their
ot frbee replication or survival in their hosts. Some of the functions required by viruses for their replication are expressed in most cells, some only in
ne dividing cells, and some only in highly differentiated cells.
k, av Third, viruses form two groups (those that infect organs at or near a portal of both entry and exit), multiply efficiently, and ultimately are
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boy eliminated by the immune response, and those that remain after infection are in a latent state for the life of the host.
er ma Last and perhaps foremost, for the past two decades, molecular and genetic tools became available to construct novel viruses that never
papors existeTdh beseefo creo nasnidd,e rinat imonoss t sienrsvtaen acse st,h lea cfko utnhde aetvioonlu otifo nthaer yi daedav athnatat giet ss thhoaut lwd obuel dp opsesrimbliet tthoe mco tnos tsruurcvti vheig ihnl yn amtuorde.ified, attenuated, viruses that
riginal ohic errp otiannrccgleue tdt ehs peteihrce i tfpaisockt ec neislt lisad loa tnnoed ,s etoolre ictntoit rvoeesdltyua cbdelei ssinthrt oolyi ft ehclaeon ntcager rgl eactteeeldnls c cybe ylcl s“o hndiceto-saminreidtd-a rnfuutn nw”c ivttiihor nutssh eeds e etlhxibapetr reiansts etihloyins i nioncfs otara pncocerelal utweldoar ui lngdte obn eeth eenl eivmcierisansla atgreeydn fbooymr tethshe.e T imshuemrsvueiv nfaeul ns oyctfsi toethnmes
he gra infected cell. As the accompanying reports indicate, the development of magic bullets is far along, but we are not there yet.
m tpo A decade ago, reports on genetic engineering of viruses would have focused on the development of better vaccines to prevent infections by
d froe tym owuer anraet ubreagli nennienmgi etos —thitnhke voifr ouuser sa anncdie nmt ifcoreoso ragsa onuisrm frsi etnhdats .prey on us. It is a reflection of the development of virology over the last decade that
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na SITE-SPECIFIC INTEGRATION BY ADENO-ASSOCIATED VIRUS 11288
nal; liation
origiublic This paper was presented at a colloquium entitled “Genetic Engineering of Viruses and of Virus Vectors,” organized by Bernard
e p Roizman and Peter Palese (Co-chairs), held June 9–11, 1996, at the National Academy of Sciences in Irvine, CA.
hs
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peert allowed us to conclude that the recombination event is directed by cellular DNA sequences. Recombinant junctions isolated from our
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orient The human parvovirus, adeno-associated virus (AAV), has aroused considerable interest as a potential vector for human gene therapy.
he cid Among favorable properties of the virus are its lack of association with any human disease (1), the wide range of cell lines derived from
m tac different tissues which can be infected (2), and the ability of the virus to integrate into the genome of the infected cell to establish a latent
on infection (3). The latter property appears to be unique among mammalian viruses for two reasons. The first is that integration can occur in
ot frbee nondividing cells (4, 37), albeit at a lesser frequency than in dividing cells. Second, AAV integration occurs at a specific site in the human
ne genome, on the q arm of chromosome 19 between q13.3 and qter (5–9). For several years our laboratory has studied the mechanism underlying
k, av the site specificity of AAV DNA integration.
oh
boy To date, a number of experiments have been initiated to address the feasibility of gene transfer with AAV. In preliminary experiments
er ma nondividing cells [hematopoetic progenitor cells (10), neurons (11), photoreceptor cells (12), etc.] were shown to be stably transduced by these
papors frreocmom wbiinldan-tty pAeA AVA vVec. toTrhs.e sTeh ev emctaojrosr itdyo onf ovte cintotersg ruastee di nh aav es ictoen-stpaienceifdi ct hme ainnvneerrt.e dK nteorwmliendagl er eopfe atthse ( ImTRec)h aasn itshme so nlelayd ginegn ettoic sinitfeo-rsmpeactiifoinc
riginal ohic errp i1n6t)e.g RrIanat tichoeenlrl ,m ctuahylet ulvereiar duA stA op Vae n sdeutopreaestre inos ort tco lu atnhsdse e onrfgu oAc lApeurVosd- bwuachsteeivrdee v tiehncefte ovcritsri.oaln guennloemsse t hise ruen icso aa tceodi n(Kfe.cIt.iBo.n awndit hS .a Ahdellpere,r uandpeunbol-i s(h1e3d, 1d4a)ta o).r Lheitrtplee svviirrauls g (e1n5e,
he gra expression occurs, and that which does serves to repress further viral gene expression and to inhibit most viral DNA synthesis. In place of
m tpo productive viral infection, the viral genome is integrated to establish a latent infection (2, 3, 17). The virus is maintained in the latent state
d froe tym aincdtievfainteiste ltyh,e tvhiursa l pgeerpneotmuaet,i nlega dthineg v tiora lv igreanl egteicn ei nefxoprmreastsiioonn. aHnodw toev reers, csuuep earnidn freecptiloicna toiof nt hoef ltahtee nvtilrya li ngfeencotemd ec ewlli thw istuhb asedqeuneon- to pr rohderupcetisovnir uosf
eo viral progeny (18). Cells in culture can also be made permissive for AAV-productive infection in the absence of helper adenoor herpesviruses
eatd s by exposure to genotoxic chemicals or radiation (19–21). Because of its usual dependence on a helper virus for productive infection, AAV was
es crd, an ionrfigoirnmaalltyio nc obnys itdheer eedst atbol ibshe mdeenfet cotfi vlea.t eOncuyr. cWurhreenn tt hme ocdeelll ios fs ttrhees sreedp, ltihcea tAioAn Vo fg ethneo mvier uiss aicst itvhaatte dA tAo Vpr ohdaus ceev noelvwe dp rotog epneyr ptoet uleaatvee i tths eg ceenlel ttioc
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osca 35-mer which was repeated in tandem 10 times. This minisatellite sequence is found at about 60 sites in the human genome, all of which occur
mper, on the q arm of chromosome 19 (22). However, none of these features of AAVS1 was sufficiently distinctive to indicate uniqueness of the
ov preintegration site within the the human genome. The possibility existed that the unique aspect of the specific site of integration was not in the
ce
ew sequence but in some higher order structure of the chromatin structure of 19 q.
n rho To resolve the question of whether the specificity lay in the sequence, we have moved AAVS1 to another site in the cell. Our assumption
eeg, was that if the determining factor was the sequence, the viral genome would integrate regardless of the location of AAVS1. To move the
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work c for pv2ir2a0l .g2e nise dperpoednudcet nret qouni rtehde top riensietinactee roefp ltihcea tEiopns taeti no-rBiPa.r rA AorVigSi1n woaf sD inNsAer treedp ilnictaot itohne s(houritPtl)e tvheact tofur,nctions in the presence of EBNA1, the only
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na SITE-SPECIFIC INTEGRATION BY ADENO-ASSOCIATED VIRUS 11289
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origiublic which was then transfected into human C17 cells (293 cells which constitutively express EBNA1). Pools of hygromycin-resistant C17 clones
e p containing the shuttle vector (hygromycin serves as a selective marker for p220.2) were isolated and grown up. On average the clones
e to thof this ctroanntsafiencetded 5 0in–t1o0 E0. ccoopliie. sT ohfe tohne lsyh cuottlloen vieesc ttoor fpoerrm c ewlle. rTe hteh ocsleo nceodn tcaeilnlsin wg etrhee isnefleecctteadb lwe imtha ArkAeVr o afn tdh ea fstheur t4tl8e hvre pctloasr m(aimd pDRN wAh wicahs iiss oclaartreiedd a bnyd
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ag te proteins; the ORF in the left half of the genome encodes four regulatory proteins, Reps 78, 68, 52, and 40, with overlapping amino acid
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peert genes, most by down regulation (26). In the presence of helper virus (i.e., the permissive state) Rep 68/78 is required for AAV gene expression
ys and transactivates expression of the structural proteins; it is also required for viral DNA replication and rescue of the integrated viral genome.
ginal tally in IntereTshtien gAlyA,V it ginehniobmitse ecxopnrteasinsiso ann o IfT thRe ohfe 1lp4e5r natd e(Fniogv.i r2u)s. Teahrel yf igrsetn 1es2 5(M nt. Aco.Lnasbtiotuwte a annd oKv.eI.rBal.l, upnalpiunbdlriosmheed i ndtaetrar)u.pted by two smaller internal
orient palindromes of 21 nt, one immediately on either side of the overall axis of symmetry. When folded on itself to optimize potential base pairing,
e d the palindromic sequence forms a T-shaped structure. The long stem of the T-shaped structure contains an RBS. When Rep binds to the ITR it
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om tn ac i(n2t7e)r.a Actfst ewr inthic akti nlgea, sRt eopn eis ocfo tvhael ecnrtolyss b aorumnsd otof tthhee 5T(cid:1) (soidr es mofa tlhl ein nteicrkn aaln pda lciannd rfoumncet)i oann da sc aa nh emliackaes ea ( 2si8te).- sTpheec iIfTicR n iisc kth bee ctwise-aecnt invte 1s2ig4n aanl din 1 t2h5e
not fre bee enxopnrpeesrsmioinss, isveer vsetsa taes fthoer othrie fonre gDaNtiAve rerpegliuclaattiioonn, aonfd gise nree qeuxirperde sfsoiro nre sacnude oDfN thAe vreirpalli cgaetnioonm. eI nfr othme thpee rimntiesgsirvaete ds tsattaet et.he ITR enhances gene
k, av Thus, it was of interest to note that the 510 nt of AAVS1 sufficient to direct site-specific integration contained both an RBS and TRS in the
oh appropriate orientation with a comparable spacing between them. A third signal of potential interest was a hexanucleotide homologous to an
er bomay eRnBhSan acnedr oTfR mSe) ioatti co ngee neen dco onfv ethrsei oAnA (MV 2g6e)n ionm feis. sBioino cyheeamsti c(2al9 )e.x Tpheirsim seeqnutse nhcaed wshaso walns ot hparte sReenpt a6t8 a pcporuolxdi mbiantde layt ththee s RamBeS pions iAtiAoVn S(1re alantdiv eth taot
papors oligomeric complexes of Rep could link the ITR of AAV to the corresponding sequences in AAVS1 (30). It was also shown in vitro that bound
riginal ohic errp Rep could nick the sequence
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the ofinesett FIG. 1. AAVS1-derived target sequences cloned into p220.2 are graphically displayed. Boxes represent different fragments
n p from AAVS1. Gray boxes highlight the 510-bp fragment sufficient as a target for integration. Average recombination
ntatioher ty farne qAuAenVc-iesps eacrief iicn dpircoabtee.d Tfohri se afrcahc tsiuobnf rwagasm ceonnt.s Tidheeryed w teor er ecparlecsuelnatte tdh ea sf rtehqeu fernaccyti own iothf Ew.h cicohli AcoAloVn iheas dw ihnitcehg rhaytebdri diniztoe dt htoe
esed ot shuttle vector. The data were taken from ref. 24.
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origiublic at AAVS1 TRS and initiate unidirectional DNA synthesis (31). To directly demonstrate that the putative signal sequences in AAVS1 were
e p actually required for site-specific integration, a number of genetic analyses were performed (32). These are summarized in Fig. 3. Insertion of
e to thof this DspNecAif ifcr aignmtegenratsti orenp; rMes2e6n twinags tnhoots.e I np ofrutritohnesr oefx ptherei m51en0t sn,t ccroeanttiaoinn inogf mthuet aTtiRonSs ainndv oRlvBinSg i n2t oo rt h3e n st hiunt tRleB vSe octro Tr RasSs awye rwee srheo rweqnu tiore bdl ofcokr ssiittee--
are truersion psapdroedccitieifsoiscn. aIilnt etiesx gporefa rtiiinmotneer.n etTsst h wtuose ,n howateve e th hfaaotvu tenh deds iterhe sacittg lany a3ld3se -imnnt o tonhlseit grcoaotnneutdec xlteth ooattfi d Adee AcfioVnne ctdao innssiingtigntu attlhe e stseheqe u tmwenionc iesmsig aanlr aoel rsirgeeiqqnuu oeirnfe cDde Nst oAis drsieurpefflciitcc aisetiintoetn -tsop edcirifeicct tihntee ginrtaetgiornat. ioInn
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orient FIG. 2. An AAVITR is shown. The figure represents the T-shaped structure resulting from the palindromic sequence folded
e d on itself to optimize potential base pairing. The stem contains a Rep binding site (RBS) and a terminal resolution site (TRS).
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om tn ac Although the M26 sequence does not seem to play a direct role in site-specific integration, there are data which suggest that M26 may play
ot frbee ath ge esnheurattll er ovlee citno rd elesdta btoil irzeianrgr aAnAgeVmS1e natn odf ththues vmeacyto cr osnetqriubeuntcee isn dinir ethctel yC t1o7 t hcee lilns tewghriacthio nw aresa icntidoenp e(3n2d)e.n It nosfe rtthioen p oref stehnec 5e 1o0f- nAt AfrVag minefenct tiinotno.
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k, av Deletion of the sequences containing M26 has led to stabilization of the shuttle vector in C17 cells. Whether M26 has a comparable effect at the
oh chromosomal level is unknown; there is at least one fragile site on the q-arm of 19 and it has been extremely difficult to map this region,
er bomay becauSster ubcotuthr ecso osmf aid ns uamndb eyre aosft tahreti frieccioalm cbhirnoamntoss opmroedsu cheadv eu bsienegn tvheer ys huuntstltea bvlee.ctor assay have been determined (Table 1) (33). About 20% of
papors the recombinants contained an intact AAV
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work hc form Bnuomxebse r[sg rinayd ic(Uatpedp earr)e agnidv eenm wpittyh (rLeospweecrt) ]to i nAdAicVaSte1 . kMno2w6,n e DnhNaAnc esri gonfa mls eiidoetinct igfieende wcoitnhvine rsthioen 5 i1n0 f-ibspsi osneq yueeanscte; .C NRuEc,l ecoytcildiec
al cifi AMP response element; Sp1, transcription factor Sp1 consensus sequence; TRS, terminal resolution site; RBS, Rep binding
origing-spe sciatlec.u l(aLtoewd ears) dSesycnrtihbeetdic i no tlhigeo tneuxct laeso twideells acsl ionn Fedig .i n1t.o D tahtea wsheurett ltea kveenc ftroorm p r2e2f0. .322. .Recombination frequencies indicated are
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origiublic genome in which the junctions with vector DNA occurred within the ITR. In every case, a portion of the ITR had been deleted. In 2 instances
e p (out of 20) the integrant was more than unit length AAV, stretching from one partial ITR through the ITR at the other end of the genome which
e to thof this rheacdo mbebeinn aenltos nwgearteed q utoit ef oinrmte reas thineagd, -btoec-taauils ej uwnhcetino nth we irtehc oam pboinrtainotn polaf sma isde cwoansd trvainraslf egcetendo mineto t h2a9t3 ecxetlelsn dinedfe ctote dm wapit hp oadsietnioonv i5ru. sT, hthees eA tAwVo
are truersion signeetqneuogemrnaenc.et Iwenxa mste ronesdstec duc aefsdre oasm ntdh aer eAppolAiicnVatt eswdei qtthuoie nmn catehksee mpinriotsesgriennnagyl wvAeiArrieoV nf srso.e mMqu otehsnte c oepf a ttrhhtr eoo rufe gmthha eit nhAdeA eIrVT o Rfg etahnteo mtrheeec oe3mn(cid:1) cbeoinnddain nogtsf tchtohene tR agEienPne odgm elenes,es s w.t hhTaihnce ha iafnugtlealgi-nlrea nhtegadtdh D bvNeireAanl
e breaks he print v eaofrlbfeo oqrnvougeleal)nit.ne tTlgdy h ctenoi re lcfaaolrrer g mRere B npaSu lih;mc ebabatueditor- nttooh.f -e Ot iaonfittl he pjgeaurrrn atjcinucttiunsol cnawtri oi atnnnhod bht etee htawwedna-e tseoe nxt-h ttaveaiintlr daojeuln dnae cn itjndiuo navncnestoc isottuohngre rgbD eesNstewtqsAue asee nncs ceivreeci mrutaolela dmra nitanodpt envpreemovcseetiortdi rioo ansctee c5q,u uwr( eawhnsi cicitehnhs i cntaho leAuw lAtadwVy bosSe 1if .ntu hvPlelor- ollrevebnesigdnut glhtA toAihnfVe sa Ser 1rleti csmso edimqteeubsdcei nrnfiaocbnreemtsds,
age t with oligonucleotides from the upstream AAVS1 sequences demonstrated that in a majority of cases the AAVS1 sequence immediately upstream
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y
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eatd s folded ITR containing the free 3(cid:1) OH group. Alternatively, the TRS can be nicked by Rep (IIB), allowing elongation from the
es crd, an nick creating a structure similar to step I.
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hm FIG. 5. Model for AAV site-specific DNA integration. Parallel lines in the AAV molecules indicate that the sizes of DNA
work c for structures in the figure are not drawn in their actual proportions. A thick grey line indicates the newly synthesized strand; the
al cifi dashed line indicates the displaced strand of AAVS1. (I) Complex formation between AAV and AAVS1 is mediated by Rep
origing-spe f6a8c/t7o8r.s .( (IIII)I )I nDtrNodAu cstyinotnh eosfi sa b syt rsainndg-lesp-setcraifnicd ndiicspkl aacte tmhee nTt RorSig iinn aAtiAngV Sa1t tbhye TreRpS 6i8s /f7o8l loanwde da sbsye mtebmlyp loatfe csetlrlaunldar s wreiptclihc aotnioton
the ofinesett Athfet edri sspylnatcheeds isst roafn dA. A(IVV ) DAN sAec soenqdu estnrcaensd as wthiticrdh toecmcuprlsa toe nsttor aAndA Vsw cirtcehat ibnagc ka loinnkto bAetAwVeSe1n rAeAsuVlSts1 iann ad sAeAcoVn ds elqinuke nbceetsw. e(eVn)
ntation her typ rveisraull tsa nind ihnotesgt rDatNedA c osepqieuse nocf eAs.A (VV ID) NRAep waiirt hoifn DAANVAS 1s.t rTuhcitsu rfeigs ucroen itsa tiankinegn fnroonmc ormefp. l3e2m.entary strands by cellular enzymes
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origiublic A model for the integration process has been developed (32). Because of the involvement of Rep proteins, RBS and TRS, it appears likely
e p that viral DNA replication and localized DNA replication within AAVS1 are involved in integration. AAV DNA replication involves a single
are true to thersion of this ssstrhetwtrrpeaai ltnnifccddihra s tdittisie o smdfnpua lpluacllycgoae hmtcemtosepe prl( nei3esxt5 td mr),ta .oe nth Rcdseh ew aap3nsil(cid:1)ti iccesthamhn tedi t o( eonFnmfei giwptn.hl ia4ettti )eean m (teefswpostr lrlaaf yartn eo dsr myes( vni .ititesheh. we,e a s,blIi sTzysoee R dest whrisneetirtf ca.tu hhn3neid4dn )fegc.or a Allntyde iemmfndogp al sdljatom aotrete nefsc e)hiw.at asthIunetilri cfsei hms ao ns fsde iuAnr gvb AgeethesgVse ita n ersg ed sepb ynltonihecttrahhaat te titsiothohinesne oiosoifr n fita t hra aeisnnb eadsecb iroctrihnl aietdntey tnp n odAreifewmA nt hceVseyrt .r eaDWolnofNd hn,aAeg tna hR tpiitsenah prgte-ti immstcetleermea dsunpi sadlaita nnettogdde
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ag te parental strand.
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peert adenovirus-infected cells is to enhance the ability of the elongating DNA strand to remain on the original template. Use of uninfected cell
ys extract leads to premature strand switching and the consequent interruption of the normal replication process with the synthesis of defective
ginal tally in DcoNinAfe cmtioolne cpullaeyss (a3 m5)a. jWor er oblee liine vthe et hinatte gthrea tieonnh apnrocceeds sp.robability of strand switching during DNA synthesis in the absence of a helper virus
orient A model for the integration process must take into account the following properties. (i) Involvement of RBS and TRS. (ii) Rearrangement
e d of the AAVS1 sequences at one junction. (iii) Presence of head-to-tail AAV junctions, (iv) Despite the requirements for very distinct integration
hci
m tac signals (RBS and TRS) a model must account for the observation that integration junctions observed are scattered within ca. 1 kb of AAVS1
on downstream of RBS and TRS. A simplified model which can account for these features is shown in Fig. 5. An oligomeric complex of Rep
not fre bee pbirnodtesi nto ( Rtheep )R mBeSd ioant eAd AaVliSg1n manendt toof tthhee RreBcSom inb itnhaet iAonA pVa rItTneRr,s tAhuAsV l iannkdin Ag AaV cSi1rc, uinlaitriiazteidn gd tuhpel enxo nAhAomV omloogloeucus lree ctoo mAbAiVnaSt1io. nT hevise nret porbesseernvtes da.
k, av Rep then introduces a nick into the AAVS1 TRS. DNA synthesis initiates, displacing a single strand of AAVS1. The extension of replication
oh determines the location of a junction with AAV subsequently formed (see requirement iv mentioned above). It should be noted that the
er bomay dexistpenlascieodn sthineg leel osntrgaantidn gis sctriarcnudl asrw bietcchaeuss et oR tehpe ids icspolvaacleedn tlsyin bgoleu nsdtr atnod t haes 5th(cid:1) ee ntedm apnlda tep;r ensoutme etdh atto cboep ysitnilgl boofu tnhde tdoi stphlea cReBd Sc.i rAcuftlearr lAimAVitSed1
papors sequence leads to inversion of the sequence. When it reaches the end of the displaced strand (close to the RBS as observed in the shuttle vector
riginal ohic errp mtpe2mo2dp0el.l2a t seiny, sttthheeem es)lh,o untthgtleae t eivnleogcn tsgotarr atsinnydgs t reesmtarc)a.hn Eedsv aeRgnBatuSina lwlsyhw etihrtece h Rseiesn pgt leiesm- sbptorlaauntnedds ,ga annpdo witnh veoo nslttvoria nntghd et ha gec aiirincnsu eslrawtrei tdAc hAAeAVs Vt oD saNe qnAue.ew nA ctefetm earpn ldsay ttenh teoh nien sAviseA rpVterSdo1 cA e(AeadlVtseS r1on nast eitqvhueele ynA coAen tVios
m the ypogra GreepnaBirTaehndek. ,Up lrniokdpeoeluyrb etcexodpnlljyau,in ntchintiisgo mnth ooed fae plR piBsa Srsei mnatnp pdlir feTiseRednS, cb eru eotq fwu oiern ebldye laifeo svri eni gnthlteea gts riitat etiis of conor nisssp iepsctreiefnsitec n wAti AtohnV mly ia nnotyeng corefa ttiihnoe n tf.h eHea toduwareteasv eocrbo,s nRecrBevrSend ihn iagns AtbhAeee Vnh uinnmotteaegndr aagttei onmnou.mlteip ilne
d froe tm csietlelsu lianr cthoeu nhtuemrpaanrt goef ntohme Re;e ipn p1ro4t/e1i5n cwaistehs paonsasliybzlee dre, gitu laaptopreya rfsu ninc titohne s5, (cid:1)w-uhnitcrha nasllsaot erde croeggnioizness oRfB cSh.a Irna catderdiizteiodn g iet nseese;m tsh elirkeefolyre t,h aint dAicAatVin hga as
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eatd s evolved to take advantage of one copy of this recognition signal sequence.
es crd, an mechOanuirs mcu, rmreanyt khneolpw ilne dthgee doef stihgen roefq iumirpermoevnetds AfoAr Vsi-tbea-sspeedc vifeicct oArAs fVo rD gNenAe itnhteergarpayti. oAn,t tthoigse tphoeirn tw iitt hca onu br ep croopnocsluedde md othdaetl tfhoer Rane pi nptreogtreaitni oins
m XML file retaine tnaaner cgaeebstTsseoahdrli uysi nt,ew to eorrge rerqkavu tewiinorea ndms. eRessuniarptta hpfboeolrerr, t, t eihfdto e ir bs ss yiptt aeoab s slsgpeir,eb acllnoeiftn itgcfhri attoeytm r imon ftt hegAege ArnNietVya d toDieoflNin vtahAeler yIiInn.TsteRtigt uriastet io oonnfl.y AF rilenlqeaurlgliyrye, daw nfedo rpI rneoffpfeiocctsiieeo nutths ar eDt stichsueeea f suoelfsl iA(nAtAeIg2Vr2a 2ItTe5dR1 ) p maronavdyi rnbuoyst e asb ,e ga rn afenuctne csfrstioaomrny ntfhooert
d fronot b NWaitniostnoanl FIonustnitduattei oonf. General Medical Sciences (GM50032). R.M.L. was supported in part by a fellowship from the Norman and Rosita
en
sa 1. Blacklow, N.R., Hoggan, M.D., Kapikian, A.Z., Austin, J.B. & Rowe, W.P. (1968) Am. J. Epidemiol. 88, 368–378.
oc
ompver, 23.. BCehrenusn, gK, .AI..,K P.i,n Hkeorgtognan, ,T M.C..D, T., hHoamuasws, iGrth.F, .W &. WHo. g&g aBne, rMns.,D K. .(I1. 9(17958) 0V)i rJo. lVoigryo l6. 83,3 5, 5763–95–6704.8.
ecwe 4. Podsakoff, G., Wong, K.K., Jr., & Chatterjee, S. (1994) J. Virol. 68, 5656–5666.
n rho 5. Kotin, R.M. & Berns, K.I. (1989) Virology 170, 460–467.
eeg, 6. Kotin, R.M., Siniscalco, M., Samulski, R.J., Zhu, X.D., Hunter, L., Laughlin, C.A., McLaughlin, S., Muzyczka, N., Rocchi, M. & Berns, K.I. (1990)
as battin 7. Kotin, PRr.Moc.., NLaintld. eAnc, aRd..M Sc. i&. U BSeArn 8s7, ,K 2.2I.1 (11–929221)5 E.MBO J. 11, 5071–5078.
hm
work c for 89.. KSaomtiunl, sRk.iM, R.,. JM., eZnhnui,n Xge.,r ,X Ji.Cao.,, WX.a, rBdr, oDo.kC, .J &.D .B, eHrnosu,s mK.aIn. ,( 1D9.9E1.,) EGpesnteoimn,i cNs .1 &0, H83u1n–te8r3, 4L..A. (1991) EMBO J. 10, 3941–3950.
nal ecifi 1110.. KZhaopuli,t tS, .MZ..,G C.,o Lopeoern,e S, .P, .K, Sanamg, uLls.Yki.,, RR.uJg.,g Xieiraio, ,L X., .,H Pefiamfff,e Dld.,W S..,, SOr’iMvaasltlaevya,, KA..L &. & B Drouxrminegy,e Mr, .HJ.. E(1. 9(9149)9 4N)a Jt.. GExepn.e Mt. 8ed, .1 14789–,1 15846.7–1875.
the origiofing-spesett 111342... AHAtolcig,h gRisa5.on9Rn,4 .,M,. RR..DeWi.c,.h ,B eClla,a cMsktol.oB, wB., ,. TCNh.. rR&a.s Hh&ea rRm, oAmw.oJe.n,, , LWWe.v.P i(.n 1(s91k69i,56 )R6 )S. JcP.i,re Konccin.e nN 1oa4nt9l,. , C A7.5c, a4Kd–a.7 nS5uc6gi..a U, NSA., 5H5u,n 1t4, 6D7.–M1.4 &71 B. hattacharya, S.S. (1996) Hum. Mol. Genet. 5, 591–
n p 15. Buller, R.M., Janik, J.E., Sebring, E.D. & Rose, J.A. (1981) J. Virol. 40, 241–247.
ntatioher ty 16. Weindler, F.W. & Heilbronn, R. (1991) J. Virol. 65, 2476–2483.
esed ot
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