Proc. Natl. Acad. Sci. USAVol. 91, pp. 7311-7315, July 1994Biochemistry

The Vpr protein of human immunodeficiency virus type 1 influencesnuclear localization of viral nucleic acids in nondividing host cells

(AIDS/preintegratlon complex transport)

NINA K. HEINZINGER*, MICHAEL I. BUKRINSKYt, SHERYL A. HAGGERTY*, ANNA M. RAGLAND*,VINEET KEWALRAMANI*, MAY-ANN LEEt, HOWARD E. GENDELMAN*, LEE RATNER§, MARIO STEVENSON*¶,AND MICHAEL EMERMANt*Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-5120; *Program in Molecular Medicine, FredHutchinson Cancer Center, Seattle, WA 98104; 1Picower Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030; and §Division ofHematology and Oncology, Washington University, St. Louis, MO 63105-2298

Communicated by Harold Weintraub, March 24, 1994 (received for review February 22, 1994)

ABSTRACT The replication of human I uo yvirus type 1 (HIV-1) in nondividing host cells such as those ofmacrophage lineage is an important feature of AIDS patho-genesis. The pattern of HIV-1 replication is dictated, in part,by the nucleophilic property of the viral gag matrix (MA)protein, a component of the viral preintegration complex thatfacilitates nuclear loItion of viral nucleic acids in theabsence of mitosis. We now identify the accessory viral proteinVpr, as a second nucleophilic component that influences nu-clear localization of viral nucleic acids in nondividing cells.Reverse transcription and nuclear localization of viral nucleicacids following infection of cells by viruses lacking Vpr orviruses containing mutations in a gag MA nuclear localizationsequence were i ishable from the pattern observed incells infected by wild-type HIV-1. These viruses retained theability to replicate in both dividing and nondividing host cellsincluding monocyte-derived macrophages. In contrast, intro-duction of both gag MA and Vpr mutations in HIV-1 attenu-ated nuclear localization of viral nucleic acids in nondividingcells and virus replication in monocyte-derived macrophages.These studies demonstrate redundant nucleophilic determi-nants of HIV-1 that independently permit nuclear localizationof viral nucleic acids and virus replication in nondividing cellssuch as monocyte-derived macrophages. In addition, thesestudies provide a defined function for an accessory geneproduct of HIV-1.

The ability to replicate in nondividing host cells (1) distin-guishes human immunodeficiency virus type 1 (HIV-1), alentivirus, from the animal onco-retroviruses such as murineleukemia virus (2, 3). The onco-retroviruses require mitosisfor entry of viral DNA into the host cell nucleus (2, 3).Localization of HIV-1 nucleic acids in the nucleus, on theother hand, is not dependent on mitosis (4, 5). The ability ofHIV-1 nucleic acids to access the nucleus in the absence ofmitosis is influenced by nucleophilic viral components asso-ciated with viral nucleic acids in the context of a highmolecular weight nucleoprotein preintegration complex. We(6) and others (7) previously identified the matrix (MA)protein of the gag gene as a component of the preintegrationcomplex whose nucleophilic signal is important for infectionof nondividing cells (8). In the current study we extend ouranalysis of viral proteins that influence the infection ofnondividing cells by HIV-1. Of the four accessory proteinsencoded by the genome of HIV-1, Vpr is incorporated intovirions through interaction with gag p6 and is also localizedin the nucleus in HIV-1-infected cells (9, 10). This wouldpredict that Vpr may influence early events in the replication

cycle of HIV-1 by a mechanism similar to that described forgag MA (8). Our studies define a role for Vpr in mitosis-independent nuclear localization of HIV-1 DNA and specif-ically in permissiveness of monocyte-derived macrophagesto productive HIV-1 infection.

MATERIALS AND METHODSIsolation and Characterization of HIV-1 Nucleoprotein Pre-

integration Complexes. HIV-1 MFA (11) infected cells weresuspended at 106 cells per ml in hypotonic buffer (12) withprotease inhibitors (10 mg of leupeptin per ml, 20 mg ofaproptinin per ml, 1 mM phenylmethylsulfonyl fluoride) andlysed by Dounce homogenization as detailed previously (6).Homogenates were centrifuged (500 x g, 5 min), pellets(nuclear fraction) were extracted with high salt buffer (12)and supernatants were adjusted to 100 mM KCI/20 mMHepes/0.5 mM MgCl2, membranes were pelleted (100,000 xg, 40C, 30 min), and supernatants (cytoplasmic fraction) wereharvested. Cytoplasmic and nuclear extracts were fraction-ated on nonionic density gradients (6). Gradient fractions (1ml) were dialyzed and integration activity in each fractionwas examined in an in vitro integration assay (13). In vitrointegration products were extracted and analyzed by PCRusing HIV-1 long terminal repeat (LTR) U5 (5'-G%50TCT-GTTGTGTGACTCTGGT) and LTR U3 (5'-G9126TGGTAG-ATCCACAGATC) primers and 30 cycles of amplification(950C/30 sec of denaturation; 560C/30 sec of annealing;720C/60 sec of extension). Association of viral DNA withviral proteins was assessed by immunoprecipitation of viralDNA using protein G-agarose and antibodies to HIV-1 Vpr(10) or HIV-1 capsid (CA) orMA (American Biotechnologies,Cambridge, MA) followed by detection of viral DNA inimmunoprecipitates by PCR. Dialyzed nuclear or cytoplas-mic gradient fractions were clarified by conjugation to non-immune mouse IgG or rabbit serum (2 h, 40C) in the presenceof 0.1% Triton X-100 and protease inhibitors and immuno-precipitated with protein G-agarose (10 mg), 1 h at 40C. Aftercentrifugation (10,000 x g, 60 sec), 10-15 Mg of monoclonalanti-MA or anti-CA or 0.01 vol of anti-HIV-1 Vpr antibodywas added to the supernatant and incubation was continuedovernight at 40C. Immune complexes were collected byaddition of 10 mg of protein G-agarose (1 h, 40C). Immuno-precipitates were washed (three times) with 0.1% TritonX-100/10 mM Tris HCl, pH 8.0/140 mM NaCl/0.025% so-dium azide and eluted with 50 mM Tris-HCl, pH 8.5/2 mMEDTA/1% SDS/15 pg of salmon sperm DNA per ml at room

Abbreviations: HIV-1, human immunodeficiency virus type 1; Vpr,viral protein R; LTR, long terminal repeat; MA, matrix; CA, capsid;NLS, nuclear localization signal.ITo whom reprint requests should be addressed.

7311

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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7312 Biochemistry: Heinzinger et al.

1250

1000-

E 750,

C

cm' 500-C" *a. o-o0I..AIVPR-

-OLAINLS-/ VPR--

0 2 4 6 8 10 12Days After Infection

FIG. 1. HIV-1 variants containing single and double mutations ingag MA NLS and/or in Vpr elicit an efficient spreading infection inproliferating CD4+ MT4 cell cultures. Proliferating MT4 cells (20)were infected with the indicated HIV-1 variants at a multiplicity ofinfection of 0.1 for 2 h. Cells were washed twice and resuspended infresh medium at 1 x 106 cells per ml. Virus production in culturesupernatants was determined at 2- to 3-day intervals by gag p24 (CA)ELISA.

temperature for 10-15 min. DNA was extracted from eluatesusing guanidune isothiocyanate (Iso-Quick nucleic acid ex-traction kit, Bothell, WA) and analyzed by PCR with LTRR/gag primers.

Preparation of HIV-1 Molecular Clones Contai Muta-tions In gag MA and In Vpr. To examine the phenotype ofHIV-1 gag MA and Vpr mutants in CD4+ T cells, mutationsin MA and/or Vpr were engineered in pLAI, an infectiousmolecular clone that has complete reading frames for allaccessory proteins (14). The LAINLs variant contains gag

Proc. NatL Acad. Sci. USA 91 (1994)

MA Lys-26,27 -- Thr substitutions as described (8). LAIVPRcontains a frameshift at codon 40 of Vpr. LAINL./VpR- is avariant containing both mutations. Since T-celi-adaptedHIV-1 variants do not replicate in monocyte-derived macro-phages, the phenotypes of single and combined gag MAnuclear localization signal (NLS) and Vpr mutations in mono-cyte-derived macrophages were engineered into pNLHX-ADA-SM, an infectious molecular clone containing macro-phage tropic determinants derived from HIV-1 ADA (15).The MA coding regions (720-bp BssHII-Sph I fragment) ofparental NLHXADA-SM clones p121 (Vpr+) and p130 [Vpr,containing a frameshift at codon 63 ofVpr (15)] were replacedby a similar fiagment from HIV-1 MFD (11) to derive ADAGEand ADAvpR., respectively, or with a similar fragment fromHIV-1 MFDMl7 (containing Lys-26,27 -- Thr substitutions)to derive ADANLS and ADANLS-/vpR., respectively. Thisreplacement of the MA coding region of HIV-1 ADA withHIV-1 MED was to ensure the presence of a functional NLSin ADAwt since the parental NLHXADA clone lacks aconsensus gag MA NLS motif (16).

Analysis of Viral DNA Synthesis by PCR. Late reversetranscription products of HIV-1 were amplified by PCR withprimers to HIV-1 LTR R (5'-G45GGAGCTCTCTGG-CTAACT) and gag (5'-G912GATTAACTGCGAATCGTTC)regions resulting in the production of a 447-bp PCR fiagment(6). One-LTR-circle forms of extrachromosomal HIV-1DNA, which are formed only after synthesis of full-lengthviral cDNA and its transport to the nucleus (17), wereamplified using primers to HIV-1 gag and nef (5'-C9"CTCAGGTACCTTTAAGACC) regions of the viral ge-nome (6). Primers are numbered according to the HIV-1HXB2 sequence (18) and for LTR primers, only 3' LTRcoordinates are given. HIV-1 cDNA standards and one-LTR-

-90 h -42 h

ISOLElJCINE BLOCK

virus cell cycleadded analysis

O h ?4 h

MIMOSINE ARRLST

HIV-1 VARIANT LAl WT LAI VPR- LAINLS- LAI

TIME POST-INFECTION (h) - co N m t

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Fio. 2. Synthesis and nuclear import of viral DNA by gag MA NLS and Vpr mutants of HIV-1 in growth-arrested CD4+ MT4 cell cultures.MT4 cell cultures were synchronized by' isoleucine deprivation for 48 h and then released into complete medium or into medium containingmimosine (400 pM), a plant amino acid that arrests the cell cycle at the G1/S boundary (21). Cells were infected 42 h after release from isoleucinedeprivation. At the indicated times postinfection, cell aliquots were harvested (2-3 x 106 cells) for isolation of total cellular DNA (Iso-QuickDNA extraction kit) and analysis of viral and cellular DNA by PCR. Viral DNA was analyzed using primers to late products of reversetranscription (LTR R/gag) and to one-LTR-circle forms of viral DNA (gag/nef) that are formed specifically in the nucleus. Equivalent amountsof cellular DNA (lower panels) in each sample are demonstrated by PCR with primers to tubulin (22). For analysis of cell cycle parameters,cells (1 x 106) were removed at the indicated intervals and cellular DNA was stained with propidium iodide at 40C for 1 h. Cells were analyzedby fluorescence-activated cell sorter analysis and the number of cells in G1, S, and G2/M phases is shown as a percentage ofthe viable cell totalusing Cell-Fit software (Becton Dickinson).

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.

0 h PI

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STANDARDS

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Proc. Natl. Acad. Sci. USA 91 (1994) 7313

circle standards were generated by PCR on serial 2-folddilutions of 8E5 cells [each containing a defective proviralgenome (19)] or HIV-1-infected CD4+ MT4 cells, respec-tively. Viral and cellular DNA was amplified by 30 cycles ofPCR as outlined for in vitro integration products.

RESULTSWe have previously demonstrated that an NLS in the gagMA protein of HIV-1 is important for nuclear localization ofviral nucleic acids and virus replication in nondividing cells(6). In those studies we used an HIV-1 IIIB-derived molec-ular clone, HIV-1 MFD (11), which, like several HIV-1 IIIBvariants, encodes truncated versions of the Vpr protein (ref.18; M.E., unpublished data). Because Vpr is a virion-associated protein with nucleophilic properties (9, 10) weasked whether Vpr played a role in infection of nonprolifer-ating cells by HIV-1 in the context of proviral clones pos-sessing or lacking a functional gag MA NLS. HIV-1 variantscontaining single or combined gag MA NLS and Vpr muta-tions elicited a spreading infection in dividing CD4+ MT4cells with kinetics indistinguishable from that of wild-typeHIV-1 (Fig. 1). In addition, the quantity of virus particlesproduced from HeLa cells transfected with wild-type andMA/Vpr mutant molecular clones of HIV-1 was indistin-guishable. This indicated that virus viability was not com-promised by the single or combined gag MA and Vprmutations. We next examined the effects ofgag MA and Vprmutations on viral cDNA synthesis and nuclear localizationin dividing CD4+ MT4 cells and in MT4 cells arrested at theG1/S boundary of the cell cycle by the action of mimosine(21). In proliferating MT4 cultures, viral cDNA synthesis(LTR R/gag) and circularization of viral DNA in the nucleus(gag/nef) were equivalent (not shown). In growth-arrestedcells, nuclear forms of viral DNA in cultures infected with anHIV-1 variant containing combined MA NLS and Vpr mu-tations were detectible but in greatly reduced quantitiesrelative to wild-type HIV-1 or to viruses containing singleMA NLS or Vpr mutations, while the abundance of viralcDNAs (LTR R/gag) was equivalent for each of the virusvariants (Fig. 2). This pattern of DNA synthesis and circu-larization was observed in three independent experiments.The low level of one-LTR-circle (gag/nef) products ob-served in cells infected with the double mutant most probablyrepresented virus infection of a subpopulation of cells thatwere still in cycle (3%) in those cultures. In addition to thepredicted 1008-bp gag/nef PCR product, a band was ob-served at 24 h with cultures infected with the double mutantand this most likely represents an aberrant product of viralDNA synthesis not associated with the nucleus since it wasabsent at later time intervals.Terminally differentiated macrophages are natural nondi-

viding cell targets of HIV-1 in vivo (23-25). Thus, we ana-lyzed the ability of monocytotropic HIV-1 variants contain-ing single and combined gag MA NLS and Vpr mutations toelicit a spreading infection in monocyte-derived macro-phages. HIV-1 variants either containing a gag MA NLSmutation or lacking functional Vpr demonstrated almostidentical replication kinetics in monocyte-derived macro-phages and were able to elicit an efficient spreading infection,although the rate was reduced when compared to wild-typeHIV-1 (Fig. 3). In contrast, a virus containing both gag MANLS and Vpr mutations was greatly attenuated in its abilityto elicit a spreading infection in monocyte-derived macro-phages (Fig. 3). The inefficient replication of the combinedgag MA NLS/Vpr mutant in monocyte-derived macro-phages was determined at the level of nuclear localization ofviral DNA. Viral cDNA synthesis (LTR R/gag primers) incells infected with wild-type HIV-1 or with single and com-bined MA NLS and Vpr mutants (Fig. 4) was equivalent (no

viral DNA synthesis, as evidenced by PCR and LTR R/gagprimers after exhaustive Dpn I digestion, could be detectedin macrophage cultures at 5 or 8 h postinfection). However,when monocyte-derived macrophages were analyzed for thepresence of nuclear forms of viral DNA using PCR andgag/nef primers, there was a complete absence of circleforms of viral DNA in macrophage cultures infected with thecombined MA NLS/Vpr mutant. This indicated a defect innuclear localization of viral DNA (Fig. 4). Taken together,these results indicate that nucleophilic functions associatedeither with HIV-1 gag MA or with Vpr are sufficient fornuclear localization of viral DNA in the absence of mitosisand for replication of HIV-1 in nondividing cells. As aconsequence, mutation ofboth gagMA and Vpr nucleophilicfunctions greatly attenuates nuclear localization ofviralDNAand replication of HIV-1 in nondividing monocyte-derivedmacrophages.

In previous studies MA was found associated with viralnucleic acids in the preintegration complex (6, 7), and byvirtue of an N-terminal NLS, MA influenced nuclear local-ization of viral nucleic acids after infection of nondividingcells. Thus, we asked whether Vpr was also associated withviral preintegration complexes. Nucleoprotein preintegrationcomplexes of HIV-1 were obtained from density gradient-fractionated nuclear and cytoplasmic extracts of acutelyinfected CD4+ MT4 cells, and association of viral proteinswith viral nucleic acids in these complexes was determinedby immunoprecipitation ofviral cDNA with antibodies to gagproducts including MA CA and to the accessory viral proteinVpr. The immunoprecipitation PCR approach was necessaryto distinguish de novo synthesized viralDNA associated withnucleoprotein preintegration complexes from preexisting vir-ion-associated viral DNA introduced with high-titer virusstocks (27, 28). These preexistingDNA forms were dispersedin all gradient fractions (and in nuclear fraction 8 afteroverexposure of the blot) (Fig. 5, upper panel) as evidencedby direct PCR of gradient fractions. Preintegration com-plexes were located predominantly in fractions 5-7 of cyto-

2.0- ADAWTo-o ADAVPR-

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FIG. 3. Replication of HIV-1 gag MA NLS and Vpr mutants inmonocyte-derived macrophage cultures. Monocytes were isolatedby countercurrent centrifugal elutriation of mononuclear leukocyte-rich cell preparations obtained from normal HIV-1 and hepatitis Bseronegative donors after leukapheresis (26). Monocytes were cul-tured in 96-well plates as adherent monolayers in medium containing1000 units of monocyte-colony stimulating factor per ml (GeneticsInstitute, Cambridge, MA). Monocyte-derived macrophages wereinfected with the indicated HIV-1 variants 7 days after plating. Equalamounts of virus [based on reverse transcriptase (RT) activity andgag p24 content] were added to wells for 3 h and then cultures werereplaced with fresh medium. Reverse transcriptase activity in su-pernatants (averaged from triplicate wells) was measured at 2- to3-day intervals.

Biochemistry: Heinzinger et al.

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7314 Biochemistry: Heinzinger et al.

HIV-1 VARIANT ADA WI

TIME POST-INFECTION (h) i m v x

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ADA VPR- ADA NI S- ADA NI S-/VPR-

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FIG. 4. Synthesis and nuclearimport ofviralDNA by wild-type, gagMA NLS, and Vpr mutants ofHIV-1 in monocyte-derived macrophages.Macrophages were established in 24-well culture plates and at the indicated times postinfection, cells from individual wells were harvested byscraping followed by DNA isolation. Prior to PCR, residual plasmid DNA remaining from transfection was removed by exhaustive Dpn Idigestion, which cleaves bacterially derived plasmid DNA but does not digest viral cDNA synthesized de novo within HIV-1-infectedmacrophages. Viral cDNA synthesis and circularization were assessed by PCR with primers to LTR R/gag and gag/nef, respectively.

plasmic and fraction 8 ofnuclear extracts as evidenced by thepresence of in vitro integration activity in those fractions(Fig. 5). Antibodies to HIV-1 Vpr immunoprecipitated viralDNA predominantly from fractions 5-7 of cytoplasmic andfractions 8 and 9 of nuclear extracts (Fig. 5). Immunopre-cipitation of gradient-fractionated extracts using antibodiesto HIV-1 MA, demonstrated previously to be containedwithin viral preintegration complexes (6, 7), gave a pattern ofDNA distribution in cytoplasmic and nuclear extracts similarto that observed for Vpr immunoprecipitates (not shown).The specificity of the immunoprecipitation PCR was ad-dressed by immunoprecipitation PCR analysis of gradient-fractionated cell extracts using antibodies to HIV-1 CA. Inthis instance there did -not appear to be any correlationbetween those fractions containing CA immunoprecipitableviral DNA and fractions containing in vitro integration ac-tivity (Fig. 5). Thus, in agreement with previous observations(6, 7), HIV-1 CA is either not associated or only loosely

CYTOPLASM145 F

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Fraction

PCR(LTR U3/gag)

in vitrointegrationactivity

I.P. PCRVpr

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associated with viral preintegration complexes. Taken to-gether, these studies are consistent with the association ofVprbut notCA with viral nucleic acids in the context ofa highmolecular weight nucleoprotein preintegration complex.

DISCUSSIONWe show here that HIV-1 possesses two signals that inde-pendently influence nucleophilic properties of the viral pre-integration complex and that independently facilitate nuclearlocalization of viral nucleic acids in the absence of mitosis.We have demonstrated previously that HIV-1 MF variants,which lack functional Vpr, are competent for nuclear local-ization of viral DNA in nondividing cells (4, 8). Undersingle-cycle infection conditions, analysis of nuclear importdid not reveal any differences between the single HIV-1 MAor Vpr mutants and wild-type virus (for example, Figs. 2 and4), suggesting that the gag MA and Vpr sials may be

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FiG. 5. Antibodies to HIV-1 Vpr protein, but not CA protein, immunoprecipitate viral cDNA from gradient-fractionated nuclear andcytoplasmic extracts of HIV-1-infected CD4+ cells. High-titer viral supernatants (0.5-1.0 gg of gag p24 per ml) from chronically infected MT4cells were cleared by low-speed centrifugation and filtered (0.8 AM). MT4 cells (4 x 10 were infected by resuspending cells in high-titer viralsupernatants at a density of 1 x 108 cells per ml. At 1 h postinfection, infected cells were resuspended in fresh medium and incubated at 37"C.At 5 h postinfection, cells were harvested for hypotonic lysis and gradient fractionation of cytoplasmic and nuclear extracts (see text). Onehundred microliters of each resultant fraction was used for analysis of density and 100 JA was subjected to direct PCR analysis of viral DNA(top panels). The remaining fraction (800 p4) was dialyzed and analyzed for in vitro integration activity (100 id) and by immunoprecipitation(I.P.) (200 pi) prior to PCR using antibodies to HIV-1 Vpr and CA (lower panels) or HIV-1 MA (not shown).

Proc. Nad. Acad. Sci. USA 91 (1994)

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Proc. NatL Acad. Sci. USA 91 (1994) 7315

completely redundant. However, analysis ofthese mutants inthe context of a spreading infection in monocyte-derivedmacrophages, where subtle differences are amplified overmultiple rounds of infection (Fig. 3), indicates that the twosignals may be additive. This phenotype is in agreement withprevious studies (29) in which the replication of single Vprmutants in monocyte-derived macrophages was reduced rel-ative to wild-type HIV-1. Presumably the additive effect ofgag MA and Vpr would improve the stoichiometry of nu-cleophilic components associated with the viral preintegra-tion complex and facilitate its nuclear import characteristicssince increasing the number of nucleophilic motifs associatedwith the particle improves the rate and extent of its nuclearimport (16).The distribution of HIV-1 in terminally differentiated and

nonproliferating cells of macrophage phenotype (23-25) is animportant feature in AIDS pathogenesis and is directly re-flected by the ability of HIV-1 to replicate within primarymacrophages in vitro (1, 30). The fact that HIV-1 containsredundant signals for nuclear localization of its DNA in theabsence of mitosis argues that there is a strong selectivepressure for this function. This is further supported by thedegree of conservation observed in NLS sequences in MAand Vpr among HIV-1 and HIV-2 strains and in many strainsof simian immunodeficiency virus (18). Selection for mac-rophage-tropic strains in primary HIV infection may resultfrom selective transmission of virus to cells of macrophagelineage (31, 32) and this could also account for the selectivepressure to maintain an efficient mechanism of integrationinto nonmitotic cells.

N.K.H. and M.I.B. contributed equally to this research. We thankS. Malli, L. Regen, L. Lukas, N. Sharova, P. Westervelt, and Y.-L.Lu for excellent technical assistance and C. Bowers for manuscriptpreparation. S.A.H. is a Scholar of the American Foundation forAIDS Research. This work was supported by the National Institutesof Health (M.S., M.E., M.I.B., H.E.G., L.R.), the American Foun-dation for AIDS Research (H.E.G., M.I.B.), the American CancerSociety, and the Department of Defense (L.R.). H.E.G. is a Carter-Wallace Fellow.

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