Simian 40-Based Replication of Catalytically Inactive Human Immunodeficiency Virus Type 1 Mutants in Nonpermissive T Cells and Monocyte-Derived Macrophages

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Citation Lu, R., N. Nakajima, W. Hofmann, M. Benkirane, K. Teh-Jeang, J. Sodroski, and A. Engelman. 2003. “Simian Virus 40-Based Replication of Catalytically Inactive Human Immunodeficiency Virus Type 1 Integrase Mutants in Nonpermissive T Cells and Monocyte- Derived Macrophages.” Journal of Virology 78 (2): 658–68. https:// doi.org/10.1128/jvi.78.2.658-668.2004.

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41482903

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA JOURNAL OF VIROLOGY, Jan. 2004, p. 658–668 Vol. 78, No. 2 0022-538X/04/$08.00ϩ0 DOI: 10.1128/JVI.78.2.658–668.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Simian Virus 40-Based Replication of Catalytically Inactive Human Immunodeficiency Virus Type 1 Integrase Mutants in Nonpermissive T Cells and Monocyte-Derived Macrophages Richard Lu,1 Noriko Nakajima,1,2† Wolfgang Hofmann,1,2‡ Monsef Benkirane,3§ Kuan Teh-Jeang,3 Joseph Sodroski,1,2,4 and Alan Engelman1,2* Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute,1 Department of Pathology, Harvard Medical School,2 and Department of Immunology and Infectious Diseases, Harvard School of Public Health,4 Boston, Massachusetts 02115, and Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208923 Downloaded from

Received 13 August 2003/Accepted 7 October 2003

Integrase function is required for retroviral replication in most instances. Although certain permissive T- lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following , we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin

(oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen http://jvi.asm.org/ 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration- competent genotype. Adding Tag to integrase mutant-oriT yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies. on October 4, 2019 by guest Integration of retroviral cDNA into a host cell circles (5). Homologous DNA recombination yields circles is a key step in the viral cycle. Integration inextricably links containing one copy of the LTR (1-LTR), and circles contain- the virus to the host, such that it persists as cellular DNA for ing two tandem copies of the LTR (2-LTR) form by nonho- the remainder of the infected cell’s life. Integration is mediated mologous DNA end joining (32). by the viral integrase (IN) protein, the proteolytic cleavage Functional IN protein is required for productive retroviral product of the carboxyl-terminal portion of the Gag-Pol replication in most instances. Certain highly permissive trans- polyprotein. IN enters the cell as a component of the infecting formed T-cell lines, however, supported replication of catalyt- virion. ically inactive class I human immunodeficiency virus type 1 After entry and uncoating, the virion enzyme reverse tran- (HIV-1) IN mutant viruses under conditions that restricted scriptase (RT) copies genomic RNA into linear double- growth of pleiotropic class II IN mutants (38). This finding led stranded cDNA containing a copy of the viral long terminal to the suggestion that, under certain conditions, HIV-1 might repeat (LTR) at each end, and this DNA form is the substrate productively replicate from unintegrated DNA templates. for IN-mediated DNA recombination (see reference 5 for a However, since host cell DNA recombination machinery non- review). A variety of circular DNA products also form during specifically integrates exogenous DNA at a low frequency (11, the early phase of retroviral infection. Whereas some of these 21), it is important to consider the potential role of host- result from IN-mediated autointegration of linear viral cDNA mediated or illegitimate DNA recombination (17, 29) in IN into itself, host recombination enzymes form two other types of mutant viral replication. For example, since the recombination frequency of a class I IN mutant virus was greater than mutant viral titers (relative to that of the wild type [WT]) across cell * Corresponding author. Mailing address: Department of Cancer lines, it seemed likely that illegitimately integrated Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney St., contributed to mutant viral replication in permissive T-cell Boston, MA 02115. Phone: (617) 632-4361. Fax: (617) 632-3113. E- mail: [email protected]. lines (38). In this study, a novel strategy was developed to † Present address: Department of Pathology, National Institute of investigate productive HIV-1 replication from unintegrated Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan. DNA. ‡ Present address: Novartis/CIBA Vision Diagnostic Lens SBU, Unintegrated HIV-1 DNA is lost from cells through one of 63762 Grossostheim, Germany. two different means. Nascent linear cDNA as part of the re- § Present address: Laboratoire de Virologie Mole´culaire et Trans- fert de Gene, Institut de Ge´ne´tique Humaine, CNSR UPR1142, Mont- verse -preintegration nucleoprotein complex is pellier, France. degraded via a proteosome-dependent process (7, 45). In con-

658 VOL. 78, 2004 HIV-1 IN MUTANT REPLICATION IN NONPERMISSIVE CELLS 659 trast, 1-LTR and 2-LTR circles are relatively stable, their num- Single-round viruses containing deletions in were used to initiate infections bers diminishing only following cell division (7, 43). Uninte- for real-time quantitative PCR (RQ-PCR) assays. Whereas env deletion-contain- ing variants of pNL43/XmaI and IN mutant pNLX(N/N) were previously de- grated DNA circles may therefore fail as viral replication scribed (33), pN/N.Tag-oriT⌬env was made by swapping the 2.3-kb env deletion- templates simply because they lack a functional origin of DNA containing EcoRI/BamHI fragment from pNLX⌬env for the corresponding replication. To address this, the Epstein-Barr virus (EBV) or- fragment in pN/N.Tag-oriT. Expression vectors for vesicular stomatitis virus igin of replication (oriP) was inserted into a class I IN active- G (VSV-G) and WT and D116A IN mutant Gag-Pol, Rev, and site mutant vector and gene expression was measured in cells HIV-1NL4-3 gp120- were previously described (34, 38). Single-round expression vectors carrying the gene for puromycin acetyltrans- expressing the EBV nuclear antigen 1 (EBNA-1) protein. Sim- ferase (pac) were constructed as follows. WT and GKH ClaI/XhoI Tag-oriT ilarly, by incorporating the simian virus 40 (SV40) origin of fragments isolated from pN/N.Tag-oriT and pN/N.GKH-oriT, respectively, were replication (oriT) and infecting T-antigen (Tag)-expressing ligated to ClaI/XhoI-digested pBluescript II SKϩ (Stratagene). The NotI site in cells, we assayed whether amplifying the level of unintegrated the resultant was mutated to an MscI site by QuikChange mutagenesis. DNA overcame the block to IN mutant replication in nonper- Tag was amplified with XbaI- and NotI-tagged primers, digested, and ligated to XbaI/NotI-digested pIRES (Clontech Laboratories, Inc.). The resulting IRES- missive cells. Whereas oriP did little to enhance IN mutant Tag-oriT fragments were amplified with NotI-tagged primers, and digested

gene expression in EBNA-1-expressing cells, oriT greatly en- were ligated to NotI-digested pHI.puro (38), generating pHI.puro.Tag- Downloaded from hanced mutant infectivity in Tag-expressing cells. Moreover, oriT and pHI.puro.GKH-oriT. full-length IN-defective strains carrying both oriT and Tag Cells. 293T (42), HeLa (18), CV-1 (25), CV1/EBNA (36), and COS-1 (20) cells were grown in Dulbecco’s modified Eagle medium containing 10% fetal calf replicated in nonpermissive Jurkat T cells and primary mono- serum, 100 IU of penicillin per ml, and 100 ␮g of streptomycin per ml (DMEM). cyte-derived macrophages (MDM). Our results demonstrate Jurkat (51), Jurkat TagC15 (39), 1D Jurkat, and Raji B lymphoblast cells (44) HIV-1 replication in primary human cells in the absence of engineered to express CD4 and the HIV-1 coreceptor CCR5 (Raji T4/R5; a functional IN enzyme, indicating a potential strategy for atten- generous gift from Hyeryun Choe, Children’s Hospital, Boston, Mass.) were uated viral gene expression in the absence of efficient integra- grown in RPMI 1640 medium containing 10% fetal calf serum, 100 IU of penicillin per ml, and 100 ␮g of streptomycin per ml (RPMI). MDM were tion. derived from peripheral blood mononuclear cells as previously described (38).

Derivation of Tag-expressing 1D Jurkat cells. Jurkat cells (3 ϫ 106) trans- http://jvi.asm.org/ fected with 3 ␮g of pCMV-Tag by using Lipofectamine (Invitrogen Corp., Carls- MATERIALS AND METHODS bad, Calif.) were treated with G418 sulfate (0.8 mg/ml) 2 days posttransfection. Plasmids. HIV-1 vector pHI.GFP.oriT, which carried oriT downstream of After 2 weeks, cells were seeded in 96-well plates and individual G418-resistant green fluorescent protein (GFP), was built by amplifying oriT (345 bp) from colonies were expanded and assayed for Tag expression by Western blotting and pMAMneo (Clontech Laboratories, Inc., Palo Alto, Calif.) with NotI-tagged cell surface CD4 by fluorescence-activated cell sorting (FACS). primers and Pfu DNA (Stratagene, La Jolla, Calif.), followed by Western blotting was performed essentially as previously described (8). Cells enzyme digestion and ligation to NotI-digested pHIlib.GFP (38). To construct a (5 ϫ 106) washed in phosphate-buffered saline (PBS) were resuspended in 1 ml minimal oriP, the 20 tandem EBNA-1 family-of-repeat binding sites in oriP were of Nonidet P-40 (NP-40) lysis buffer (1% [vol/vol] NP-40, 10% [wt/vol] glycerol, amplified with 5Ј CGATGCTCGAGTGCAAAGGATAGCACTCCC (XhoI site 137 mM NaCl, 20 mM Tris-HCl [pH 8.0]) containing 10 ␮g of aprotinin per ml highlighted in bold) and 5Ј CGTGCGGCCGCTCGCTGAGAGCACGGT and 0.5 mM 4-(2-aminoethyl)benzenesulfonylfluoride. Tag was immunoprecipi- on October 4, 2019 by guest GGGC (NotI site in bold), and the four EBNA-1 dyad symmetry binding sites tated from clarified supernatants with monoclonal antibody PAb101 (8) (a gen- were separately amplified with 5Ј ACTGCGGCCGCTCAGCCACTGCCCTTG erous gift from James DeCaprio) and protein A-Sepharose beads (Amersham TGAC (NotI site in bold) and 5Ј CGATGCTCGAGATAAGGCCCCCTTGTT Pharmacia Biotech Inc., Piscataway, N.J.). Immune complexes were washed AACC (XhoI site in bold). The two PCR products were digested with NotI, three times with NP-40 lysis buffer, twice with 0.5 M LiCl–20 mM Tris HCl (pH ligated, and then amplified with the XhoI-tagged primers. pNL4-3oriP 8.0), and once with NP-40 lysis buffer. Following fractionation through 7.5% was built by digesting this product with XhoI and ligating the resulting 922-bp polyacrylamide gels and electrotransfer to Hybond-C extra (Amersham Pharma- family-of-repeat–dyad symmetry-containing fragment to XhoI-digested pNL4-3 cia Biotech, Inc.), membranes were reacted with a 1:50 dilution of PAb101, (1, 24). Plasmid pHI.GFP.oriP was built by amplifying oriP from pNL4-3oriP followed by a 1:1,000 dilution of peroxidase-conjugated sheep anti-mouse im- with BsrFI-tagged primers, digesting it with BsrFI, and ligating it to BsrFI- munoglobulin G (Sigma Aldrich, St. Louis, Mo.). Proteins levels detected with digested pHIlib.GFP. the ECL Western blotting kit (Amersham Pharmacia Biotech, Inc.) were quan- Plasmids pCMV-Tag (8) and pSG5-K1 (56), encoding WT and E107K Tag, tified by densitometry (IS-1000 Digital Imaging System; Alpha Innotech Corp., respectively, were generous gifts from James DeCaprio (Dana-Farber Cancer San Leandro, Calif.). Institute). Plasmids encoding replication-defective U19 (26, 41) and transforma- For cell surface CD4 detection, PBS-washed cells were incubated with phy- tion-defective (td) D402H (35) Tag, referred to here as pCMV-U19 and pSG5- coerythrin-conjugated OKT4 antibody (Ortho-McNeil Pharmaceuticals, Raritan, D402H, respectively, were also obtained from James DeCaprio. The C105G N.J.) for 20 min at 4°C in the dark. Following two washes with PBS, cells were change was introduced into pSG5-K1 by QuikChange mutagenesis (Stratagene). fixed with 2% formaldehyde and analyzed by FACS. The GKH triple mutant carrying C105G, E107K, and D402H changes was Viruses and infections. Virus stocks were generated by transfecting 293T or similarly built with pSG5-D402H as the template. HeLa cells with calcium phosphate as previously described (15, 38); HeLa cells

To generate replication-competent HIV-1NL4-3 carrying oriT and Tag, over- were used for Tag-oriT-containing viruses to circumvent Tag-expressing 293T lapping PCR (15) was used to mutagenize p83-10 (19) into p83-10⌬nefMCS-1 by cells. Virus stocks for RQ-PCR assays generated with FuGENE 6 (Roche Mo- replacing nef nucleotides 8785 to 9017 (37) with unique ClaI, NotI, and XhoI lecular Biochemicals, Indianapolis, Ind.) were treated with DNase I prior to sites. Plasmid p83-10⌬nef.oriT was built by amplifying oriT with NotI- and infection essentially as previously described (10, 33).

XhoI-tagged primers, digestion, and ligation to NotI/XhoI-digested p83- Whereas full-length HIV-1NL4-3 was derived from one plasmid, two plasmids ⌬ 10 nefMCS-1. The WT and U19 mutant Tag proteins amplified from pCMV- were required for env deletion-containing HIV-1NL4-3, and GFP- and pac-ex- Tag and pCMV-U19, respectively, were digested and ligated to ClaI/NotI-di- pressing vectors required the transfection of three or four different plasmids, gested p83-10⌬nef.oriT, yielding p83-10⌬nef.Tag-oriT and p83-10⌬nef.U19- depending on the identity of the (Env), as previously described ϩ oriT. These plasmids were digested with PmlI and NheI, and the oriT-containing (38). Transfected cell supernatants assayed for Mg2 -dependent 32P-labeled RT fragments were ligated to PmlI/NheI-digested pNL43(N/N) (38), yielding IN activity (15, 38) were filtered through 0.45-␮m-pore-size filters prior to infection. mutant plasmids pN/N.oriT, pN/N.Tag-oriT, and pN/N.U19-oriT. Plasmids pN/ CV-1, CV1/EBNA, and COS-1 cells (2 ϫ 105) plated in six-well plates 24 h N.E107K-oriT, pN/N.D402H-oriT, pN/N.C105G/E107K-oriT, and pN/N.GKH- prior to infection were infected with 3 ϫ 106 RT cpm of VSV-G-pseudotyped, oriT were built by amplifying Tag from the relevant pSG5-based plasmid, diges- GFP-expressing virus for 1.5 h at 37°C, washed three times with serum-free tion, and ligation to ClaI/NotI-cut pNN.oriT. Macrophage-tropic variants of DMEM, and cultured in 2 ml of DMEM. Cells were trypsinized, fixed with 2% oriT-containing viruses were made by swapping the 2.7-kb EcoRI/BamHI env formaldehyde, and analyzed for GFP expression by FACS. Jurkat and Jurkat fragment from pNL(AD8) (16) for the corresponding fragment in T-cell-tropic TagC15 cells (2 ϫ 106) were infected with 107 RT cpm of GFP-expressing viruses clones. in 0.5 ml for 60 min, washed once, and plated in 5 ml of RPMI. Raji T4/R5 cells 660 LU ET AL. J. VIROL.

(2 ϫ 105) were similarly infected with 107 RT cpm of VSV-G pseudotypes. GFP Ϫ values from cells mock infected with Env viruses were subtracted from VSV- G-mediated infections. For assays of viral spread, T cells (2 ϫ 106) were infected with 107 RT cpm of WT (approximately 1.5 ␮g of p24, equating to an approximate multiplicity of infection of 0.04) (34, 38) or IN mutant HIV-1NL4-3 for18hat37°C, washed three times with serum-free RPMI, and resuspended in 5 ml of RPMI unless otherwise noted. MDM infected with 18 ϫ 107 RT cpm for 18 h at 37°C were washed two times with serum-free RPMI and resuspended in 1.5 ml of macroph- age-driving medium (30% [vol/vol] RPMI 1640 medium containing 25 mM HEPES, 45% [vol/vol] serum-free RPMI, 15% [vol/vol] L-929 cell [14] condi- tioned medium, 10% [vol/vol] human serum type AB [BioWhittaker Inc., Walk- ersville, Md.], 0.055 mM ␤-mercaptoethanol, 2.5 U of macrophage colony-stim- ulating factor [PeproTech Inc., Rocky Hill, N.J.]) per ml. Southern blotting and PCR. Infected 1D Jurkat cells were lysed and fraction- ated, and DNA samples were analyzed by Southern blotting and inverse PCR as

previously described (38). Levels of unintegrated HIV-1 DNA were quantified by Downloaded from PhosphorImager with ImageQuant version 1.11 (Molecular Dynamics, Sunny- vale, Calif.). For sequencing of passaged viruses, infected cells were lysed by the Hirt method as previously described (38). Whereas the region of N/N.oriT was molecularly cloned and sequenced as described for mutant N/N in reference 38, N/N.Tag-oriT pol and oriT PCR products were sequenced directly following FIG. 1. Tag-expressing cells support high levels of transient gene expression from D116A.oriT. (A) CV-1 cells infected with the WT (ᮀ) purification from polyacrylamide gels (38). छ E For RQ-PCR, Jurkat cells (6 ϫ 106) infected with 6 ϫ 106 RT cpm of virus or the D116A ( ) or D116A.oriT ( ) mutant virus were ana- ϫ lyzed for GFP expression at the indicated times. (B) CV1/EBNA1 cells VSV-G-pseudotyped viruses by spinoculation (40) at 530 g for2hat25°C were ᮀ छ F washed and cultured in 5 ml of RPMI. Approximately 1.2 ϫ 106 cells were infected with the WT ( ) virus or the D116A ( ) or D116A.oriP ( ) removed at each time point. When the culture was reduced to 2 ml, 2 ml of fresh mutant virus were analyzed as described for panel A. (C) COS-1 cells infected as described for panel A. (D) Raji T4/R5 cells infected as RPMI was added, followed by the addition of fresh RPMI (2 ml) every 24 h. http://jvi.asm.org/ DNA was extracted from cells with the Qiagen DNeasy Tissue kit (QIAGEN, described for panel B. Similar gene expression profiles were obtained Valencia, Calif.), and 10 ␮l of DNA-containing eluate was analyzed by RQ-PCR. in three independent sets of experiments. Three different HIV-1 amplicons, late reverse transcription (LRT) products, 2-LTR circles, and integrated proviruses, were analyzed by RQ-PCR. Primers,

PCR conditions, and plasmids for generating HIV-1NL4-3 LRT and 2-LTR standard curves were as described previously (33). content was quan- containing episomes (27), SV40 uses Tag protein to replicate tified with HIV-1 and Alu primers as described previously (6, 28). The and amplify oriT-containing amplicons (12). standard curve for Alu RQ-PCR was prepared essentially as previously described (6), with genomic DNA from cells stably transduced with HIV-1 expression Our preliminary approach was to insert oriP or oriT into a ϫ 6 class I IN mutant GFP and assay infectivity vector pHIlib.GFP. Jurkat cells (6 10 ) infected with VSV-G-pseudotyped on October 4, 2019 by guest pHIlib.GFP (1.8 ϫ 107 RT cpm) were cultured for 40 days, GFP expression was in cells expressing the various viral proteins. Three related stimulated with tumor necrosis factor alpha at 10 ng/ml, and stimulated cells African green monkey cell lines, CV-1 (25), CV1/EBNA (36), were sorted to 97 to 98% GFP positivity by FACS. Genomic DNA prepared from and COS-1 (20), were initially analyzed. Whereas CV1/EBNA expanded cells with the Qiagen DNeasy tissue kit was adjusted to 50 ng/␮l, and a 1:2 dilution series was used to generate the Alu standard curve by normalizing cells stably express EBNA-1, COS-1 is a CV-1 derivative line the resulting genomic LRT and Alu RQ-PCR signals to a plasmid-generated that stably expresses Tag. Cells infected with equal RT activity LRT standard curve. of WT or IN mutant VSV-G-pseudotyped virus were assayed Levels of LRT, 2-LTR, and Alu RQ-PCR products were normalized to either for GFP expression at various times postinfection. cellular mitochondrial cytochrome oxidase (plasmid DNA containing a cloned copy of human cytochrome oxidase was a generous gift from M. D. Miller, Merck In repeated experiments, 10 to 20% of the CV-1 cells in- Research Laboratories) or endogenous 3 (ERV-3) (55). Normalized fected with the WT expression vector were GFP positive 3 days values were expressed in arbitrary units. postinfection (dpi) (Fig. 1A and data not shown). At this time, Isolation of puromycin-resistant colonies. Puromycin-resistant colonies were 3 to 5% of the cells infected with IN active-site mutant D116A isolated following infection with single-round pac-expressing viruses essentially (2, 38, 52) were GFP positive (Fig. 1A). Neither oriT (Fig. 1A) as previously described (38), with the exception that cells were infected by spinoculation at 530 ϫ g for2hat25°C prior to incubation at 37°C. nor oriP (not shown) altered the pattern of D116A mutant gene expression in CV-1 cells. About 45% of the WT-infected CV1/EBNA1 cells were RESULTS GFP positive at 3 dpi (Fig. 1B). In this case, about 8% of the SV40-based amplification of HIV-1 IN mutant gene expres- D116A-infected cells were positive (Fig. 1B) and oriP did not sion in infected cells. Although certain highly permissive T-cell appear to alter this expression pattern. In repeated experi- lines supported the continual replication of HIV-1 in the ab- ments, D116A.oriP and D116A supported similar levels of sence of IN catalytic function, the majority of cell lines, as well GFP expression and the rate of GFP loss was, for the most ϩ as primary human CD4 cells, failed to support IN mutant part, unaffected by oriP (Fig. 1B). growth (38). Since unintegrated HIV-1 DNA is significantly In contrast, oriT greatly enhanced IN mutant infectivity in less stable than integrated proviruses (7, 43), we incorporated Tag-expressing cells. Whereas 20 to 30% of the WT-infected different origins of DNA replication into IN mutant HIV-1 to COS-1 cells were GFP positive at 3 to 6 dpi, about 3% of the investigate the relationship between unintegrated DNA repli- D116A-infected cells were positive at 6 dpi (Fig. 1C). Notably, cation and mutant virus growth in nonpermissive cell types. almost 60% of the D116A.oriT-infected cells were positive at 3 The replication mechanisms of two different DNA viruses, dpi (Fig. 1C). In contrast to the WT infection, the D116A.oriT EBV and SV40, were analyzed. Whereas EBV uses its infection was transient, as only a small number of cells re- EBNA-1 protein to stably maintain the copy number of oriP- mained GFP positive at 9 dpi (Fig. 1C). Enhanced GFP ex- VOL. 78, 2004 HIV-1 IN MUTANT REPLICATION IN NONPERMISSIVE CELLS 661

FIG. 2. SV40 Tag and oriT yield high levels of transient IN mutant expression in nonpermissive human T cells. (A) Jurkat cells infected ᮀ छ E with the WT ( ) virus or the D116A ( ) or D116A.oriT ( ) mutant FIG. 3. N/N.oriT replication in 1D Jurkat cells. Supernatants of virus were analyzed for GFP activity at the indicated times. (B) Jurkat cells infected with the WT virus (ᮀ) or the N/N (ϫ) or N/N.oriT (छ) TagC15 cells infected as described for panel A. Nearly WT levels of mutant virus were analyzed for RT activity at the indicated times. D116A.oriT expression in Jurkat TagC15 cells were observed in two Similar results were observed in three independent experiments. Downloaded from independent experiments.

Class I IN mutant replication in Tag-positive Jurkat cells pression required oriT as part of the D116A virus, as COS-1 requires oriT. We next analyzed the effect of SV40 sequences cells infected with virus made from a D116A construct carrying on the spreading replication of a class I IN mutant. Full-length oriT in the plasmid backbone instead of the viral mutant N/N carrying amino acid substitutions D64N and supported GFP expression levels indistinguishable from those D116N in the active site of the IN enzyme (38) was modified to of the parental D116A mutant (not shown). We noted that contain oriT in the viral nef region. N/N.oriT replication was http://jvi.asm.org/ COS-1 cells infected with D116A.oriT supported high gene compared to WT and N/N replication in Jurkat and Jurkat expression levels regardless of whether the virus was produced TagC15 cells. in 293T cells, which express Tag protein, or HeLa cells, which Results of preliminary experiments revealed that Jurkat do not express Tag (data not shown). We also noted that cells TagC15 cells failed to support detectable levels of WT HIV- infected with the WT or the D116A or D116A.oriT mutant 1NL4-3 replication under conditions that yielded efficient virus displayed doubling times and percent viabilities indistin- growth in parental Jurkat cells (data not shown). This guishable from those of mock-infected COS-1 cells (data not prompted a comparison of CD4 levels on the Jurkat and Jurkat shown). TagC15 cell surface, which revealed the majority of Jurkat Ϫ on October 4, 2019 by guest Although the results in Fig. 1B revealed that oriP did not TagC15 cells as CD4 (data not shown). Parental Jurkat cells noticeably affect D116A gene expression in CV1/EBNA cells, were therefore transfected with a Tag expression vector, and we next assayed D116A.oriP in a second EBNA-1-expressing single-cell subclones were screened for cell surface CD4 ex- cell line, Raji T4/R5, that was derived from B-lymphoblast Raji pression by FACS and Tag protein content by Western blot- cells (44). In repeat experiments, 8 to 11% of the WT virus- ting. The 1D Jurkat cell subclone, which was approximately infected Raji T4/R5 cells were GFP positive at 5 dpi (Fig. 1D). 97% CD4 positive, expressed Tag at a level similar to that of The D116A mutant was weakly active in these cells, as only 0.2 Jurkat TagC15 cells (data not shown). to 0.3% were GFP positive at 2 to 5 dpi in repeated experi- 1D Jurkat cells infected with HIV-1NL4-3 supported peak ments (Fig. 1D and data not shown). Since approximately 1% virus replication at 5 to 6 dpi (Fig. 3). In contrast, N/N failed to of the D116A.oriP-infected cells were positive at 3 to 5 dpi replicate in 1D Jurkat cells over 2 months of observation (Fig. (Fig. 1D), oriP stimulated the level of IN mutant GFP expres- 3 and data not shown). Thus, 1D Jurkat cells, like parental sion three- to fourfold in Raji T4/R5 cells. However, since oriP Jurkat cells (38), were nonpermissive for class I IN mutant viral did not enhance D116A expression in CV1/EBNA1 cells (Fig. replication. In contrast, N/N.oriT replicated to high levels, 1B) and D116A.oriP expression was only 10 to 15% of the WT peaking at approximately 10 dpi, proving that oriT drove N/N level in Raji T4/R5 cells (Fig. 1D) under conditions in which mutant replication in 1D Jurkat cells (Fig. 3). N/N.oriT repli- D116A.oriT expressed more GFP than the WT in COS-1 cells cation also required Tag protein, as parental Jurkat cells in- (Fig. 1C), we focused our attention on SV40-based vectors for fected with as much as 5 ϫ 107 RT cpm of N/N.oriT (fivefold the remainder of this study. more virus than in Fig. 3) failed to support detectable HIV-1 D116A.oriT expression was next assayed in nonpermissive growth (data not shown). We concluded that class I IN mutant ϩ human T cells. Infections were conducted with CD4 Jurkat T HIV-1 can replicate in nonpermissive Jurkat T cells if oriT is cells (51), which were nonpermissive for class I IN mutant present in the viral genome and Tag is provided in the cell. We replication (38), and their Tag-expressing derivative Jurkat noted an apparent requirement for a class I IN in this TagC15 cells (39). The results closely paralleled the results system, as 1D Jurkat cells infected with 107 RT cpm of the obtained with CV-1 and COS-1 cells: whereas D116A and pleiotropic class II IN C-terminal deletion mutant 1-212 (33, D116A.oriT supported similar low levels of GFP expression in 38) carrying oriT failed to support detectable levels of HIV-1 Jurkat cells (Fig. 2A), D116A.oriT was significantly more ac- replication (not shown). tive than D116A in Jurkat TagC15 cells (Fig. 2B). The To investigate the mechanism of N/N.oriT replication, virus D116A.oriT infection also subsided much faster than the WT harvested from 1D Jurkat cells was passed onto fresh 1D Ju- infection in Jurkat TagC15 cells (Fig. 2B). rkat and Jurkat cells. Whereas parental Jurkat cells infected 662 LU ET AL. J. VIROL.

FIG. 5. N/N.Tag-oriT replication in Jurkat cells requires functional Tag protein. (A) Jurkat cells infected with WT (ᮀ), N/N.Tag-oriT (‚), N/N.U19-oriT (E), or mock-treated (ϩ) supernatant were analyzed for Downloaded from RT activity at the indicated times. (B and C) Jurkat cells infected with the indicated levels of virus derived from peak days of WT and N/N.Tag-oriT replication in panel A. Arrows in panels A and C indi- cate cultures lysed by Hirt extraction. Similar replication curves were FIG. 4. Spreading N/N.oriT replication in 1D Jurkat cells. (A) 1D ϫ 6 6 ‚ obtained following a second set of primary and secondary infections of Jurkat cells (2 10 ) infected with 10 RT cpm of WT ( ), N/N.oriT Jurkat cells. pssg, passage. (Œ), or mock-treated supernatant (ϩ) were assayed for RT activity at the indicated times. (B) RT assays of cell supernatants following co- culture of 2 ϫ 105 cells from panel A with 1.8 ϫ 106 Jurkat TagC15 cells. (C and D) RT assays following coculture of 2 ϫ 105 cells from the Ϫ 6 CD4 Jurkat TagC15 cells failed to yield detectable HIV-1 experiment whose results are shown in panel A with 1.8 ϫ 10 Jurkat ϩ and 1D Jurkat cells, respectively. levels under conditions in which CD4 Jurkat and 1D Jurkat http://jvi.asm.org/ cells supported efficient WT virus expression (Fig. 4B to D). If N/N.oriT replicated in 1D Jurkat cells in the absence of viral spread (Fig. 3 and 4A), then each coculture condition would, in with 106 RT cpm did not support detectable virus growth, 1D theory, yield equivalent levels of N/N.oriT in cell supernatants. Jurkat cells infected with either 106 or 105 RT cpm supported However, akin to the WT virus, high levels of N/N.oriT were efficient HIV-1 replication (data not shown). Thus, infectious only detected after coculture with cells that supported replica- ϩ ϩ 1D Jurkat cell-derived N/N.oriT maintained its starting cell- tion of cell-free virus, namely, CD4 Tag 1D Jurkat cells type-dependent phenotype upon passage. 1D Jurkat cells were (Fig. 4). On the basis of this, we concluded that 1D Jurkat cells on October 4, 2019 by guest lysed by Hirt extraction at the peak of the second round of support the spreading replication of IN mutant N/N.oriT virus. infection, and the resulting Hirt supernatant was amplified by N/N.Tag-oriT replication in Jurkat cells requires functional PCR with pol-specific primers. Sequencing revealed that six Tag protein. In the previous section, N/N.oriT replication re- independent molecular clones each retained the D64N and quired trans expression of Tag protein in 1D Jurkat cells. We D116N codon changes in IN. Thus, N/N.oriT replicated in 1D next tested whether expression of Tag in cis from the incoming Jurkat cells without reverting to an integration-competent phe- viral genome would support N/N.oriT replication in parental Ϫ notype or genotype. Tag Jurkat cells. For this, the gene for Tag was inserted into Spreading N/N.oriT replication in 1D Jurkat cells. The re- the viral nef position upstream of oriT. A second virus express- sults of the previous section showed that the supernatant of ing the U19 Tag mutant, which is unable to replicate oriT- N/N.oriT-infected 1D Jurkat cells contained high levels of RT containing DNA substrates (26, 41), was also made. We noted activity (Fig. 3). Although this was interpreted as evidence for that incorporating Tag increased the normal size of the HIV-1 spreading HIV-1 replication, we considered the alternative RNA genome from 9.1 to 11.3 kb. 6 model in which 1D Jurkat cells might yield high levels of Jurkat cells infected with 10 RT cpm of WT HIV-1NL4-3 supernatant N/N.oriT in the absence of viral spread. Since cells supported peak virus replication at 8 dpi (Fig. 5A), and in infected with SV40 can produce very high levels of uninte- agreement with our previous report (38), N/N failed to detect- grated DNA (12), N/N.oriT DNA replication could, in theory, ably replicate under these conditions (data not shown). In yield high levels of viral gene expression in the absence of virus contrast, N/N.Tag-oriT replication was detected in Jurkat cells. spread. We therefore devised the following coculture experi- In repeated experiments, N/N.Tag-oriT grew with a delay of ment to investigate whether virus spread was required for high about 4 days compared with the WT and yielded about four- levels of N/N.oriT expression in 1D Jurkat cells. fold less virus (Fig. 5A). Importantly, N/N.U19-oriT failed to At 40 h postinfection (hpi), 1D Jurkat cells infected with replicate under these conditions, proving that the DNA repli- either WT or N/N.oriT virus were extensively washed and cation function of Tag was necessary for N/N.Tag-oriT virus cocultured at a ratio of 1:10 with three different types of un- replication. ϩ Ϫ Ϫ ϩ infected cells: Tag CD4 Jurkat TagC15 cells, Tag CD4 To investigate the mechanism of N/N.Tag-oriT replication, ϩ ϩ parental Jurkat cells, and Tag CD4 1D Jurkat cells (Fig. 4). viruses harvested from cells infected as described in the legend This experimental strategy investigated the CD4 and Tag re- to Fig. 5A were passed onto fresh Jurkat cells at two different quirements for efficient WT versus N/N.oriT gene expression multiplicities of infection (Fig. 5B and C). Infections initiated after coculture. As expected, high levels of WT expression with 106 RT cpm yielded viral replication profiles similar to required virus spread: coculturing of WT-infected cells with first-round profiles (compare Fig. 5B and A), and infections VOL. 78, 2004 HIV-1 IN MUTANT REPLICATION IN NONPERMISSIVE CELLS 663

fractions was assayed by Southern blotting and inverse PCR, respectively, as previously described (38). WT and N/N.oriT each reached their replication peak at 3 dpi in this experiment. Consistent with results of previous studies (2, 15, 23, 29, 52), cells infected with class I IN mutant N/N displayed a transient increase in the level of unintegrated DNA: whereas cells in- fected with N/N contained more 1-LTR and 2-LTR circles than did WT-infected cells at 1 dpi, the N/N circles decreased 2.5-fold to below the WT levels by 3 dpi (Fig. 7A). In stark contrast, the level of N/N.oriT circles greatly increased during FIG. 6. Functional analysis of N/N.Tag-oriT replication. (A) Aga- the course of the experiment. Whereas the level of N/N.oriT rose gel stained with ethidium bromide. Lanes: 1, IN-containing pol fragment amplified from plasmid pN/N.Tag-oriT; 2, PCR of Hirt su- circles was similar to that of the WT at 1 dpi, cells infected with pernatant from approximately 5 ϫ 104 second-round N/N.Tag-oriT- N/N.oriT contained approximately 10-fold more 1-LTR and

infected cells (Fig. 5C, arrow); 3, same as lane 2 except that lysate was 2-LTR circles than did cells infected with the WT by 3 dpi (Fig. Downloaded from treated with DpnI to degrade residual plasmid DNA prior to PCR; 4, 7A). Tag amplified from pN/N.Tag-oriT; 5 and 6, minus and plus DpnI Genomic DNA isolated at 3 dpi was next analyzed for evi- treatment of first-round Hirt supernatant (Fig. 5A, arrow), respec- tively; 7 and 8, minus and plus DpnI treatment of second-round lysates dence of HIV-1 integration. Inverse PCR detects the fre- (Fig. 5C, arrow), respectively. (B) Ethidium bromide-stained poly- quency and distribution of genomic HindIII sites flanking a acrylamide gel. Lanes: 1, oriT amplified from pN/N.Tag-oriT; 2 and 3, population of integrated proviruses (30, 38; Fig. 7C). In addi- minus and plus DpnI treatment of first-round lysates, respectively; 4 tion to these variably sized products indicative of provirus and 5, minus and plus DpnI treatment of second-round lysates. Whereas migration positions of PCR products are marked to the right distribution (labeled X in Fig. 7C), all forms of WT HIV-1NL4-3 of each gel, sizes of molecular mass standards in kilobases are marked yield an internal 1,102-bp inverse PCR product (Fig. 7C and to the left. reference 38). 2-LTR circles, which are recovered at a low level http://jvi.asm.org/ in genomic DNA preparations, yield a novel internal fragment of 261 bp (Fig. 7C). Because of the presence of a HindIII site initiated with 10-fold less virus did not significantly alter this in oriT, we note that the internal 1,102-bp WT inverse PCR result (Fig. 5C). At 16 dpi, first (Fig. 5A, arrow)- and second product was, instead, 358 bp for N/N.oriT (Fig. 7C). (Fig. 5C, arrow)-round N/N.oriT-Tag-infected cells were lysed As predicted, genomic DNA recovered from WT-infected by Hirt extraction and Hirt supernatants were subjected to cells yielded a heterogeneous ladder of inverse PCR products PCR with pol-, Tag-, and oriT-specific primers. Whereas the in addition to the 1,102- and 261-bp products (Fig. 7B, lane 3). overall size of each PCR product was monitored by gel elec- Although genomic DNA from N/N.oriT-infected cells revealed on October 4, 2019 by guest trophoresis (Fig. 6), the pol and oriT products were addition- both internal products, a ladder representing the normal fre- ally analyzed by DNA sequencing. Although Tag and oriT quency and distribution of provirus integration was not de- sequences were both maintained following two rounds of tected (Fig. 7B, lane 4). We did note the presence of a heter- N/N.Tag-oriT replication (Fig. 6A and B), an internal deletion ogeneous background smear that extended upward from the in oriT arose during virus passage (Fig. 6B and data not 261-bp 2-LTR circle product (lane 4). Owing to the difficulty of shown). Substituting this deletion-containing oriT for the full- quantifying this signal, we next used RQ-PCR to get a better length element in N/N.Tag-oriT revealed that each origin sup- handle on the extent of SV40-based IN mutant integration. ported a similar profile of N/N.Tag-oriT replication in Jurkat HIV-1 DNA quantitation by RQ-PCR. RQ-PCR was used to cells (not shown). Since the sequence of the second-round quantify levels of WT and IN mutant DNAs in acutely infected (Fig. 5C) pol PCR product (Fig. 6A, lane 3) revealed mainte- cells. PCR primers and TaqMan probes were chosen to detect nance of both substitutions in the IN active site, we concluded the following HIV-1 species: (i) LRT products, which repre- that N/N.Tag-oriT replicated through two rounds in nonper- sent the total level of intracellular HIV-1 DNA after the sec- missive Jurkat cells without acquiring an integration-compe- ond template switch of reverse transcription, (ii) 2-LTR circles, tent genotype. which represent a fraction of the total unintegrated DNA pop- Southern blotting and inverse PCR analyses of unintegrated ulation, and (iii) Alu RQ-PCR, which represents the fraction and integrated HIV-1 DNA. Our results demonstrate that Tag of integrated provirus DNA. Infections for RQ-PCR assays can drive the replication of oriT-containing class I IN mutant were conducted with single-round env deletion-containing vi- HIV-1 in nonpermissive Jurkat T cells. We next investigated ruses to restrict cDNA synthesis and integration to single in- the mechanism of SV40-dependent IN mutant replication, fectious cycles. Viral RQ-PCR signals were normalized to considering two alternative models. Since SV40 replicates from ERV-3, an -like element present at two amplified copies of unintegrated DNA (12), it seemed plausi- copies per normal diploid human cell (55). ble that SV40-based HIV-1 IN mutants might likewise repli- In repeated experiments, Jurkat cells infected with the WT cate from amplified copies of unintegrated DNA. Alterna- and N/N viruses supported peak LRT values at 6 to 12 hpi (Fig. tively, this predicted increase in unintegrated DNA might yield 8A and B). As expected from the results in Fig. 7A and nu- a parallel increase in the level of illegitimate DNA recombi- merous previous reports (2, 15, 23, 29, 33, 52), N/N converted nation, which might also contribute to mutant viral replication four to fivefold more of its total DNA into 2-LTR circles than (38). To begin to distinguish between these two possibilities, did the WT in repeated experiments (Fig. 8A and B and Table nuclei purified from WT-, N/N-, and N/N.oriT-infected 1D 1). Whereas the WT converted between 15% (Fig. 8A) and Jurkat cells were lysed and DNA in the supernatant and pellet 69% (Table 1) of its total DNA into integrated proviruses in 664 LU ET AL. J. VIROL. Downloaded from

FIG. 8. Real-time profiles of HIV-1 LRT, 2-LTR, and Alu RQ-

PCR DNAs in WT-, N/N-, and N/N.Tag-oriT-infected cells. (A) Total http://jvi.asm.org/ cellular DNA was isolated from WT-infected Jurkat cells at the indi- cated times. Levels of LRT (ࡗ), 2-LTR (ᮀ), and Alu (Œ) RQ-PCR products were normalized to ERV-3. (B and C) RQ-PCR values for N/N- and N/N.Tag-oriT-infected cells, respectively, were determined as described for panel A. Error bars indicate variation between dupli- cate RQ-PCR assays. AU, arbitrary units.

repeated experiments, the level of N/N provirus formation was below the detection limit of the RQ-PCR assay (Ͻ0.2% of the on October 4, 2019 by guest total N/N DNA integrated). We noted the requirement for the VSV-G Env glycoprotein in this system (6), as Jurkat cells

infected with single-round HIV-1NL4-3 carrying WT IN and the HIV-1 Env glycoprotein failed to yield a detectable Alu RQ- PCR signal (data not shown). Consistent with this observation, we previously demonstrated that Jurkat cells infected with VSV-G pseudotypes supported approximately 30-fold more FIG. 7. N/N.oriT replication in the absence of normal provirus cDNA synthesis than did cells infected via the HIV-1 Env (34). ϫ 8 ϫ 8 formation. (A) 1D Jurkat cells (1.6 10 ) infected with 8.0 10 RT N/N.Tag-oriT yielded LRT levels similar to those of the WT cpm of WT (W), N/N (N), N/N.oriT (T), or mock-treated (m) super- natant for4hat37°C were lysed at the indicated times, and uninte- and N/N viruses at early times postinfection (Fig. 8C and data grated nuclear DNA was detected by Southern blotting. Whereas the not shown). However, by 72 hpi, N/N.Tag-oriT LRT and first four lanes were exposed to X-ray film overnight, the remaining 2-LTR circles far exceeded the peak values attained with the lanes were exposed to film for 3 h. Linear DNA was not detected in this WT and N/N viruses (Fig. 8). In repeat experiments, between analysis, likely because of its loss during alkaline lysis (3). (B) Inverse PCR. Lanes: 1, genomic DNA isolated from mock-infected cells; 2, 1 57% (Fig. 8C) and 75% (Table 1) of the N/N.Tag-oriT DNA ng of plasmid pNL4-3 (1) mixed with 10 ␮g of mock-infected genomic was present as 2-LTR circles. Unlike those infected with N/N, DNA prior to digestion with HindIII; 3 and 4, genomic DNA isolated Jurkat cells infected with N/N.Tag-oriT yielded detectable Alu at 3 dpi with the indicated viruses; 5, mock-infected DNA mixed with 4 RQ-PCR signals. In repeated experiments, between 0.8 and Hirt supernatant from approximately 10 N/N.oriT-infected 1D Jurkat 2.0% of the total N/N.Tag-oriT became integrated (Table 1). cells prior to HindIII digestion; 6, mock-infected DNA plus 1 ng of pN/N.oriT. Whereas the gel in lane 3 was exposed to film overnight, However, since N/N.Tag-oriT-infected cells contained approx- the gel in lanes 1, 2, and 4 to 6 was exposed to film for 5 h. The imately 50-fold more HIV-1 DNA than WT-infected cells migration positions of the 1,102-, 358-, and 261-bp internal PCR prod- (compare Fig. 8C and A), N/N.Tag-oriT yielded two- to four- ucts are indicated on the right; migration positions of molecular mass fold more integrated proviruses than the WT in repeated ex- standards (sizes in base pairs are shown) are indicated on the left. (C) Inverse PCR strategy. Solid lines, HIV-1 DNA; open boxes, HIV-1 periments. LTRs; vertical arrows, relevant HindIII sites; dashed lines, cellular To investigate which DNA population(s) promoted gene DNA; black box, oriT; P, location of PCR primers (not drawn to scale); X, variably sized products indicative of provirus distribution. Whereas the HindIII sites at positions 8,131 and 9,606 in WT HIV-1NL4-3 yielded a 1,102-bp internal PCR product (38), oriT introduced an 358 bp. The WT and N/N.oriT viruses each yielded a 2-LTR-specific additional HindIII site, thereby reducing the size of the WT product to 261-bp product (38). VOL. 78, 2004 HIV-1 IN MUTANT REPLICATION IN NONPERMISSIVE CELLS 665 Downloaded from http://jvi.asm.org/ on October 4, 2019 by guest FIG. 9. WT and N/N.Tag-oriT viruses support similar kinetics of gene expression in single-round infections. (A) RT activity (■) was plotted against the WT Alu RQ-PCR (छ) signal from Fig. 8A. (B) Comparison of WT RT (■) and LRT (E) profiles. (C) RT activity (■) plotted against the N/N.Tag-oriT RQ-PCR Alu signal (छ) from Fig. 8C. (D) N/N.Tag-oriT RT (■) and LRT (E) profiles. Although N/N.Tag-oriT and the WT supported similar kinetic profiles of gene expression, we noted that the level of N/N.Tag-oriT expression was approximately 50% of that of the WT. We infer that this reduced level of mutant gene expression was in large part responsible for the delay in N/N.Tag-oriT replication observed in spreading infection assays (Fig. 5). AU, arbitrary units. expression from N/N.Tag-oriT in these single-round infection tween the peaks of WT integration and gene expression (Fig. assays, the WT and N/N.Tag-oriT gene expression profiles 9A). In contrast, the N/N.Tag-oriT Alu RQ-PCR signal, which were compared to their RQ-PCR profiles. Although these vi- was undetectable until 36 hpi, did not peak until 72 hpi (Fig. ruses carried env deletions, efficient gene expression was pre- 9C). Since the peak of N/N.Tag-oriT integration was delayed dicted to yield viruslike particles containing readily detectable approximately 60 h from that of the WT under conditions in RT activity in cell supernatants (46). The WT and N/N.Tag- which the two viruses displayed similar gene expression pro- oriT RT activities each peaked at 60 hpi under these conditions files, we speculate that the large pool of unintegrated N/N.Tag- (Fig. 9). Since the WT Alu RQ-PCR signal peaked at 12 hpi oriT DNA, which reached its peak slightly after the RT activity (Fig. 8A and 9A), there was an approximately 48-h delay be- peak (Fig. 9D), likely contributed to IN mutant gene expres- sion under these assay conditions. N/N.Tag-oriT replication in MDM. Our experiments thus TABLE 1. 2-LTR and Alu RQ-PCR levels in WT and IN far focused on IN mutant replication in nonpermissive Jurkat a mutant infections T cells. Since primary cells such as MDM were also nonper- Expt 1b Expt 2 missive for class I IN mutant replication (38), we next investi- Virus gated N/N.Tag-oriT replication in MDM. Macrophage-tropic 2-LTR Alu 2-LTR Alu Env AD8 (16) was introduced into WT and IN mutant molec- WT 0.9 (0.2)c 15.3 (8.5) 2.3 (1.0) 68.7 (13.8) ular clones to facilitate this analysis. Ͻ Ͻ N/N 4.6 (2.3) 0.2 10.0 (8.6) 0.1 As expected (38), MDM supported efficient WT HIV-1 N/N.Tag-oriT 57.0 (7.2) 0.8 (0.1) 74.9 (15.4) 2.0 (0.5) NL4- 3(AD8) replication under conditions in which class I IN mutant a Percentage of peak LRT, calculated by using peak 2-LTR and Alu RQ-PCR N/N replication was undetectable (Fig. 10). And, as was ob- values. b Data from Fig. 8. served with nonpermissive Jurkat T cells, N/N.Tag-oriT repli- c Values in parentheses indicate variations between duplicate RQ-PCR assays. cated to detectable levels in MDM. Whereas the cells in Fig. 10 666 LU ET AL. J. VIROL.

integrated proviruses are expressed and replicated as if they were host , unintegrated HIV-1 DNA is lost from cells via a proteosome-dependent process (7, 45) and/or following di- vision (7, 43). In this study, we investigated whether incorpo- rating a functional might overcome the block to class I IN mutant viral replication in nonpermissive cell types. SV40-based amplification of class I IN mutant gene expres- sion. Our initial approach was to insert oriP or oriT into a class FIG. 10. Efficient N/N.Tag-oriT replication in MDM requires Tag I IN mutant viral expression vector and assay infectivity in cells DNA replication and transforming activities. (A) MDM infected with macrophage-tropic WT (ᮀ), N/N (छ), N/N.Tag-oriT (E), N/N.GKH- expressing either the EBNA-1 or the Tag protein (Fig. 1). This oriT (‚), N/N.D402H-oriT (Œ), N/N.U19-oriT (■), or mock-treated experimental design is reminiscent of a previous report supernatant (ϫ) were assayed for RT activity at the indicated times. wherein the polyomavirus origin of replication conferred ex- Similar to N/N.GKH-oriT, neither N/N.E107K-oriT nor N/N.C105G/ trachromosomal DNA replication on a murine retroviral vec- Downloaded from E107K-oriT supported detectable levels of HV-1 replication in MDM tor in cells expressing polyomavirus large T (4). Apart from the (not shown). (B) Growth curves in panel A replotted to highlight the various IN mutant viruses. different viruses, the main difference between that study and ours was that the murine vector carried functional IN whereas functionally inactive IN was studied here. supported N/N.Tag-oriT replication at about 10% of the WT Since oriP did not substantially alter the pattern of IN mu- level, cells derived from different blood donors supported 4 to tant gene expression in two different cell types under condi- 24% of the WT replication in repeated experiments (data not tions in which oriT greatly stimulated expression (Fig. 1), our shown). Since N/N.U19-oriT failed to support a detectable results indicate that simply maintaining the copy number of level of HIV-1 replication (Fig. 10B), the DNA replication unintegrated DNA does not yield a functional HIV-1 http://jvi.asm.org/ function of Tag was essential for N/N.Tag-oriT virus replica- and instead copy number amplification was required for effi- tion in MDM. cient expression from unintegrated DNA templates. This con- Tag is an oncoprotein that transforms cells through its ability clusion is consistent with the notion that unintegrated DNAs to bind and sequester the cellular tumor suppressor retinoblas- formed by retroviral infection are significantly poorer tran- toma binding (pRB) and proteins (reviewed in references scriptional templates than are integrated proviruses. 12 and 49). Whereas the Tag sequence 103LFCSE (residues Mechanism of SV40-based HIV-1 IN mutant replication in conserved in other viral oncoproteins in bold) mediates bind- nonpermissive cells. Our results demonstrate that the com- bined action of oriT in cis and Tag protein in trans conferred a ing to pRB, p53 interacts with a larger C-terminal region of on October 4, 2019 by guest Tag (12, 49). Since IN mutant viral replication assays were spreading replication phenotype on class I IN mutant N/N in until now conducted with oncogenic Tag protein, we next in- nonpermissive 1D Jurkat T cells (Fig. 3 and 4). Interestingly, vestigated the replication capacity of td variants of Tag. the pleiotropic C-terminal IN deletion mutant 1-212 (38) failed Whereas Tag changes C105G and E107K each disrupt pRB to function under these conditions. Since these so-called class binding (13, 56), D402H interferes with p53 binding (31, 35). II mutants accumulate significantly less viral DNA in cell nu- The following Tag mutants were tested: single mutants E107K clei than either WT HIV-1 or class I IN mutants (33), we and D402H, double mutant C105G/E107K, and triple mutant speculate that prenuclear defects in HIV-1 cDNA metabolism C105G/E107K/D402H (GKH). Jurkat cells infected with each precluded class II mutant function under these novel infection td variant supported HIV-1 replication at a level that was conditions. Furthermore, by incorporating the gene for Tag similar to that of parental N/N.Tag-oriT (data not shown). into the oriT-containing class I IN mutant, we developed Although N/N.D402H-oriT reproducibly replicated to a higher 11.3-kb SV40–HIV-1 chimeras that replicated in nonpermis- level than did either N/N or N/N.U19-oriT, none of the td sive Jurkat T cells (Fig. 5) and primary MDM (Fig. 10). Since variants replicated to the same level as parental N/N.Tag-oriT the replication-defective Tag mutant U19 (26, 41) failed to in MDM (Fig. 10B and data not shown). function in these assays, the DNA replication function of Tag was essential for class I IN mutant viral replication under these infection conditions. DISCUSSION We performed a variety of experiments to address the mech- IN-mediated integration of retroviral cDNA into a cell chro- anism of SV40-based IN mutant replication. Results of South- mosome is required for productive HIV-1 replication in most ern blotting (Fig. 7A) and RQ-PCR (Fig. 8) assays indicated instances (38). Although IN-defective HIV-1 expressed rela- that cells infected with class I IN mutant-SV40 replicons con- tively high levels of the (9, 47), Tat (2, 15, 52), and Nef tained 10- to 50-fold more unintegrated DNA than their WT- (53) proteins in a variety of cell types, only a limited number of infected counterparts. Although these increased levels of un- highly permissive T-cell lines supported HIV-1 replication in integrated DNA were anticipated (4), an important issue was the absence of IN function (38). These observations suggested to determine levels of IN mutant DNA recombination and that integrated proviruses were more efficient transcription assess to what extent integration played a role in IN mutant templates than unintegrated HIV-1 DNA. Although the rea- gene expression and virus replication. son(s) behind differential gene expression from integrated ver- Both inactivating changes in the IN active site were main- sus unintegrated DNA was unclear, one possibility was the tained following N/N.oriT replication in 1D Jurkat cells and relative instability of unintegrated DNA templates. Whereas N/N.Tag-oriT replication in Jurkat cells. Consistent with these VOL. 78, 2004 HIV-1 IN MUTANT REPLICATION IN NONPERMISSIVE CELLS 667 observations, genomic DNA prepared from N/N.oriT-infected of efficient td Tag function in an established cell line (13). In 1D Jurkat cells failed to reveal the normal pattern of HIV-1 contrast, macrophage-tropic derivatives of these viruses failed integration by inverse PCR (Fig. 7B). However, since this to yield substantial levels of gene expression in infected MDM DNA revealed a heterogeneous smear of inverse PCR prod- (Fig. 10). On this basis, we speculate that binding of both pRB ucts, we used an RQ-PCR assay to quantify the extent of and p53 by Tag was required for maximum N/N.Tag-oriT gene N/N.Tag-oriT integration in Jurkat T cells. expression in MDM. By extension, SV40 gene therapy vectors Results of Alu RQ-PCR assays revealed 0.8 to 2.0% carrying td Tag may have limited applications in primary hu- N/N.Tag-oriT integration under conditions in which the WT man cells, because sequestering pRB and p53 is likely impor- accomplished 15 to 69% provirus formation (Fig. 8 and Table tant to drive cells into for efficient expression from 1). Since N/N.Tag-oriT-infected Jurkat cells contained approx- DNA replication-based vectors. Although this may limit the imately 50-fold more total HIV-1 DNA than WT-infected utility of chimeric SV40-HIV gene delivery vectors in primary cells, N/N.Tag-oriT yielded two- to fourfold more proviruses human cells, such constructs may be useful in suicidal gene than the WT in repeat experiments (Fig. 8 and data not therapy approaches in which destruction of cycling tumori-

shown). Since the peak of N/N.Tag-oriT integration was de- genic cells is the desired result (reviewed in reference 54). Downloaded from layed approximately 60 h from the peak of WT provirus for- mation under conditions in which both viruses yielded similar ACKNOWLEDGMENTS kinetics of gene expression, it seems likely that the large pool We thank J. DeCaprio and M. D. Miller for plasmid DNAs, J. of unintegrated DNA contributed to N/N.Tag-oriT expression DeCaprio for monoclonal antibody PAb101, R. Bram and G. Crabtree (Fig. 9). However, this analysis did not rule out the possibility for Jurkat TagC15 cells, H. Choe for Raji T4/R5 cells, and J. DeCaprio that integrated proviruses also contributed to N/N.Tag-oriT for valuable discussion. Plasmid p83-10 was obtained from Ronald Desrosiers through the AIDS Research and Reference Reagent Pro- gene expression. gram. Similar to our previous approach (38), we attempted to This work was supported by NIH grants AI45313 (A.E.) and determine precise frequencies of IN mutant DNA recombina- AI28691 (Dana-Farber Cancer Institute Center for AIDS Research), http://jvi.asm.org/ tion by comparing the number of D116A.Tag-oriT puromycin- the G. Harold and Leila Y. Mathers Foundation, the Friends 10, the resistant colonies to those of the WT following stable trans- Bristol-Myers Squibb Foundation, the International AIDS Vaccine Initiative, and the Japanese Foundation for AIDS Prevention (N.N.). duction. Despite numerous attempts, however, we repeatedly failed to recover any pac-expressing colonies from Jurkat cells REFERENCES infected with the VSV-G-pseudotyped D116A.Tag-oriT or 1. Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, and D116A.GKH-oriT expression vector. These results indicate M. A. Martin. 1986. 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ERRATUM Simian Virus 40-Based Replication of Catalytically Inactive Human Immunodeficiency Virus Type 1 Integrase Mutants in Nonpermissive T Cells and Monocyte-Derived Macrophages Richard Lu, Noriko Nakajima, Wolfgang Hofmann, Monsef Benkirane, Kuan-Teh Jeang, Joseph Sodroski, and Alan Engelman Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, and Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115, and Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

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