Vol. 65, No. 1

JOURNAL OF VIROLOGY, Jan. 1991, p. 396404

0022-538X/91/010396-09$02.00/0 Copyright C) 1991, American Society for Microbiology

Adeno-Associated Virus Rep Protein Inhibits Human Immunodeficiency Virus Type 1 Production in Human Cells BETH ANN ANTONI,1 ARNOLD B. RABSON,2t IRVING L. MILLER,' JAMES P. TREMPE, t NOR CHEJANOVSKY,'§ AND BARRIE J. CARTER'* Laboratory of Molecular and Cellular Biology, Room 304, Building 8, National Institute of Diabetes and Digestive and Kidney Diseases,' and Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases,2 Bethesda, Maryland 20892 Received 9 July 1990/Accepted 4 October 1990

The adeno-associated virus (AAV) rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep4O) required for AAV DNA replication and AAV gene regulation. In addition, the Rep proteins may have pleiotropic regulatory effects in heterologous systems, and in particular Rep78 may mediate a negative regulatory effect. We analyzed the effects of the AAV rep gene on human immunodeficiency virus type 1 (HIV-1) gene expression. The rep gene proteins of AAV type 2 (AAV2) inhibited the trans-activating ability of HIV-1. Constructs containing the AAV2 rep gene (pHIVrep) or a CAT gene (pBennCAT) expressed from the 5' HIV-1 long terminal repeat were inducible for Rep78 and Rep68 or CAT expression, respectively, when cotransfected with a plasmid containing the HIV-1 tat gene (pARtat). When equivalent amounts of pHIVrep and pBennCAT were cotransfected with increasing amounts of pARtat, expression of CAT activity was decreased. The pHIVrep construct was more inhibitory than plasmids expressing rep from the wild-type AAV2 p5 transcription promoter. rep expression from pHIVrep almost completely inhibited the replication of an HIV-1 proviral clone as measured by reverse transcriptase activity and p24 protein levels. Inhibition of HIV-1 production by Rep protein was also seen at the transcriptional level in that all HIV-1 transcripts were decreased when pHIVrep was present. The inhibitory effects of pHIVrep appear to be mediated primarily by Rep78 and perhaps Rep68. These results suggest that a trans-acting protein from a heterologous virus might be used to inhibit HIV-1 growth.

(17) generally by enhancing transcription directed by the HIV long terminal repeat (HIV LTR). In this study, we analyzed the effect on HIV gene expression of a trans-acting gene, rep, from the human parvovirus, adeno-associated virus (AAV). AAV exhibits inhibitory effects in several systems such as inhibition of tumor induction by oncogenic adenoviruses in hamsters (14, 33, 41), decrease in tumorigenicity of cells transformed by viral or cellular oncogenes (26, 32, 34, 47), inhibition of growth of the helper virus (36), and inhibition of viral or carcinogen-induced gene amplification (4, 25). Some of these inhibitory events do not require expression of an AAV gene product (14), but others may be mediated by the AAV rep gene which has several trans-acting negative regulatory properties (5, 35, 54). The rep gene, comprising the left half of the AAV genome, is expressed from overlapping mRNA species transcribed from two promoters, p5 and P19 (for a review, see reference 7). At least four overlapping rep proteins, Rep78 and Rep68 coded by P5 transcripts and Rep52 and Rep4O coded by P19 transcripts, have been identified (44, 56). As well as being required for AAV DNA replication (27, 52), the AAV rep gene mediates multiple positive and negative regulatory effects (35, 54, 55). Rep shows strong negative autoregulation of its own synthesis (5, 9) but may activate expression from AAV promoters depending in part on the presence of helper virus (35, 54, 55). Rep may negatively or positively regulate expression of reporter genes from AAV type 2 (AAV2) promoters (5, 43, 54, 55) or from some heterologous promoters (5, 34, 42, 55). For a reporter gene (chloramphenicol acetyltransferase [CAT]) expressed from the AAV P40 promoter, rep mediated both a positive activation of the chimeric p40-CAT mRNA and a negative posttranscriptional

AIDS, a chronic and lethal disease caused by the human immunodeficiency virus (HIV), is characterized by a profound deficiency in T-cell-mediated cellular immune responses due to a drastic reduction in CD4 T lymphocytes (15, 19). Development of AIDS generally leads to death, and thus identification of effective therapies against this virus is essential. Generation of effective therapies against HIV infection has been extremely difficult to date because of the apparent inability of the immune system to mount an effective response to HIV compounded by the ability of HIV to evade normal immune responses. A complete understanding of the molecular biology of HIV growth is not yet available, but current evidence suggests several points in the virus life cycle at which a therapeutic agent might intervene (38, 45). The life cycle of HIV is controlled by a complex system of trans-acting gene regulation mediated by several HIV genes (11, 12, 57), one dominant factor being tat. Expression of tat is critical both for replication (13, 17) and for high-level gene expression of HIV (12, 46, 48, 50, 51). The TAR element at the 5' end of all HIV mRNAs is the target for the Tat protein, and Tat markedly activates transcription and perhaps translation of its own gene and all other HIV genes (2, 11-13). A variety of trans-acting genes of heterologous viruses (adenoviruses, herpesviruses, papovaviruses, papillomaviruses) have been reported to activate HIV gene expression * Corresponding author. t Present address: New Jersey Center for Advanced Biotechnology and Medicine, Piscataway, NJ 08855-0759. t Present address: Department of Biochemistry, Medical College of Ohio, Toledo, OH 43614. § Present address: Institute for Plant Protection, The Volcani Center, Entomology Department, Bet Dagan, Israel.

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INHIBITION OF HIV BY AAV Rep PROTEIN

regulation of CAT activity (55). Recently, Rep78 was reported to inhibit transformation of mouse cells by bovine papillomavirus (26) and to block herpes simplex virusinduced gene amplification (25). In this study we addressed two questions. First, could the AAV rep gene be expressed from an HIV promoter so that it was inducible by HIV? Second, would the high level of induced Rep proteins in turn be inhibitory for HIV growth? This would then demonstrate a system in which HIV generated its own trans-acting inhibitor. We have used DNA transfection assays in several human cell lines to analyze the effect of the AAV rep gene on HIV gene expression and growth. We report here that an AAV rep gene expressed from its homologous AAV promoter gave a modest inhibition of Tat-mediated CAT reporter gene expression from an HIV LTR. However, a chimeric gene comprising the rep reading frame expressed from an HIV LTR produced a higher basal level of Rep78 that was further inducible by Tat. This chimeric HIV rep gene was highly inhibitory for Tat-mediated reporter gene expression from the HIV LTR and also prevented production of infectious HIV from a proviral clone. These results suggest a possible approach to therapeutic intervention in the treatment of HIV-mediated disease by use of an interfering heterologous trans-acting regulatory protein whose expression is induced by HIV. MATERIALS AND METHODS

Cell lines. All human cell lines were grown in monolayers at 37°C in 5% CO2 in Dulbecco minimal essential medium supplemented with fetal bovine serum (10% [vol/vol]) and the antibiotics penicillin and streptomycin. Human 293 cells are human embryonic kidney cells transformed by adenovirus type 5 (22) obtained originally from N. Jones (Purdue University, Lafayette, Ind.). HeLaJW cells, a subclone of the human cervical carcinoma cell line HeLa, were obtained at passage 35 from J. Janik (National Institutes of Health). SW480 cells are human colon carcinoma cells obtained from the American Type Culture Collection at passage number 100. Plasmids. Figure 1 summarizes the structure of the AAV2 genome and the relevant features of several plasmids including pAV2, pJDT269, pHIVrep, and pHIVrepam. pAV2 and pJDT269 have been previously described (37, 54). The plasmid pHIVrep contains the AAV2 rep gene expressed from an HIV LTR and was constructed in several steps. First, the CAT gene was excised from pBennCAT (20) by digestion with HindIII and BamHl and replaced by a 1,968-bp AvaI fragment from pAV2 (AAV nucleotides 263 to 2231) which was inserted into this site by blunt-end ligation to produce pBennHIVrep. This plasmid was then digested with SalI to remove the smaller fragment and replaced with the smaller Sall fragment from pJDT95, a capsid deletion mutant of pAV2 (44). This process added the correct 3' end of the rep gene open reading frame and the AAV polyadenylation site but retained the deletion of most of the capsid gene. pHIVrepam is identical to pHIVrep, except that AAV2 nucleotide 1033 was mutated from C to A by using oligonucleotide-directed mutagenesis to produce an amber codon at that position in the rep gene sequence (8). This mutation eliminates expression of Rep proteins (8). pHIVLTR was derived simply by deletion of the CATcontaining HindIII-BamHI fragment from pBennCAT. pNL43 contains an infectious HIV proviral genome (1),

397

and pARtat (pHlVtat) contains the first exon of the Tat coding sequence expressed from the HIV LTR region of pNL43 (20). pSVtat contains the first exon of the Tat coding sequence but expressed from a simian virus 40 early region promoter (31). pNL432 contains the same infectious HIV plasmid genome as pNL43 and was derived from pNL43 by deletion of flanking cellular sequences. Thus, pNL432 contains only 1.5 kb of flanking cell sequence, whereas pNL43 contains 9 to 12 kb of flanking sequence on either side. Two pHIVCAT plasmids expressing CAT from an HIV LTR promoter were used. In pBennCAT, (20), the HIV LTR is that shown in Fig. 1 and was obtained from an HIV provirus pB2 inserted into pUCCAT (6). The HIV LTR-CAT region of pBennCAT was transferred from the pBR322based plasmid and inserted into a pUC18 vector (18). pBennCAT and pUCCAT are functionally equivalent and, in this text, are collectively referred to as pHIVCAT. pAliP, pUC18, pUC19, or pGEM4Z (Promega Corp., Madison, Wis.) were used as control plasmid DNAs. Plasmids were constructed and grown and the DNA was purified by using standard techniques as generally described by Maniatis et al. (40). Transfection of DNA into cells. Monolayers of human 293, SW480, or HeLa cells were transfected with DNA by use of the calcium phosphate precipitation procedure (23). Monolayers of cells plated at approximately 106 cells per 10-cm dish or per 25-cm2 T-flask were transfected with equivalent amounts of DNA adjusted by adding appropriate amounts of the control plasmid DNA pAllP, pUC18, or pGEM4Z. In some experiments on SW480 cells, DNA was introduced into cells by lipofection with the Lipofecton reagent (Life Technologies Inc., Gaithersburg, Md.) as described by the supplier. In calcium phosphate transfections on HeLa cells, the cells were glycerol shocked 4 h after transfection. Assay of CAT activity. CAT activity was measured by acetylation of [14C]chloramphenicol in reactions containing 1 to 40 ,ul of cell extract (21). All extracts were assayed in the linear range, i.e., not more than 50% acetylation of the substrate. Results are expressed in arbitrary units of CAT activity, where 1 unit results in acetylation of 1% of the substrate in 1 h at 37°C. Analysis of RNA. RNA was extracted from the cytoplasm of infected cells by using the Nonidet P-40 lysis method (16) with the kit from 5 Prime-3 Prime Inc. or by using the guanidinium-acid phenol method (10) with the kit from Stratagene as described by the suppliers. RNA was analyzed by electrophoresis on 1.4% agarose gels containing formaldehyde, blotting to nitrocellulose paper followed by hybridization with 32P-labeled AAV2 DNA, 32P-labeled chicken actin DNA (obtained from Oncor, Gaithersburg, Md.), or 32P-labeled HIV type 1 (HIV-1) LTR DNA sequences (a 507-bp Bgll fragment spanning from 9.0 to 9.5 kb in the HIV genome encompassing the U3 LTR), and autoradiography. Immunoblotting analysis of proteins. Proteins were extracted from cells and analyzed by polyacrylamide gel electrophoresis and immunoblotting (Western blotting) by using a rabbit antibody to AAV2 Rep protein raised against a synthetic oligopeptide (S18K) as described previously (44) followed by incubation with 125I-labeled goat anti-rabbit immunoglobulin G and autoradiography. Analysis of HIV production. The production of infectious HIV-1 was determined by assaying portions of cell culture medium for the p249aB protein by use of the antigen capture assay kit (Dupont NEN Research Products) and for viral reverse transcriptase by use of a [32P]TTP-based assay (59).

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RESULTS Structure of plasmids containing the rep gene. In pAV2, the AAV rep gene is contained in its normal AAV2 background with Rep78 and Rep68 or Rep52 and Rep4O expressed from the p5 or plg transcription units, respectively (Fig. 1). In pHIVrep, the P5 promoter is replaced by an HIV LTR sequence which extends to nucleotide +80 relative to the HIV RNA start site and therefore includes the Tat target sequence TAR (Fig. 1). Thus it was anticipated that, in pHIVrep, expression of Rep78 and Rep68 but not Rep52 and Rep4O should be inducible by Tat. Although pHIVrep retains the normal AAV P5 RNA start site (but not the ps TATA box), transcription begins mainly at the HIV RNA start site (la). Inhibition of Tat-activated gene expression by Rep. We first tested if rep could inhibit Tat-activated gene expression from an HIV promoter (Fig. 2). Human 293 cells were transfected with pHlVcat which expresses CAT from an HIV LTR and with increasing amounts of pARtat which expresses Tat from an HIV LTR. As expected in the presence of the control plasmid DNA pAllP, there was a linear dose response for induction of CAT by Tat. When pAV2 was used instead of control plasmid, there was a modest inhibition of

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INHIBITION OF HIV BY AAV Rep PROTEIN

VOL. 65, 1991

399

TABLE 1. Effect of Rep on expression from HIV CAT in the presence of Tata Cell line

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Tat-induced and basal CAT activity. This inhibition was due to Rep protein expression because the rep mutant pJDT269 had no inhibitory effect. When pHIVrep was used, there was a very strong inhibition of both the Tat-induced level of CAT expression and also the basal (uninduced) level of CAT expression. In additional experiments, we examined the effect of rep on the expression of CAT from pHIVCAT in human 293 cells, SW480 cells, and HeLaJW cells as a function of the gene dosage of rep. In these experiments (Table 1), we used a 5- to 25-fold-higher gene dosage of tat than in the experiments of Fig. 2. Nevertheless, pHIVrep consistently showed strong inhibition in all three cell lines (Table 1). The inhibition by pHIVrep was primarily due to the expression of Rep protein because neither pHIVrepam (that does not express Rep protein) nor pHIVLTR (which does not contain any rep gene sequence) showed a similar degree of inhibition. In some cells (e.g., human 293 and SW480), the moderate inhibition by pHIVrepam or pHIVLTR probably reflects competition for factors binding to the HIV LTR or TAR region. Since similar results were seen in the several cell lines tested, the strong inhibition by pHIVrep in human 293 cells is not accounted for simply by inhibition of the adenovirus transcriptional activator ElA which is present in these cells but not in SW480 or HeLaJW cells. Results similar to those shown in Fig. 2 and Table 1 were obtained in other experiments (data not shown) when Tat was supplied from a simian virus 40 promoter by using pSVtat or when lipofection, rather than calcium phosphate precipitation, was used to introduce DNA into cells. In these preliminary experiments in which the level of tat was varied (Fig. 2 and Table 1), pHIVrep was much more inhibitory than pAV2 at all doses of tat. However, at higher gene dosages of tat, there was poor inhibition, if any, by pAV2 (as compared with the rep mutant pJDT269 control). This presumably reflected Tat induction of Rep78 (and Rep68) from pHIVrep but not from pAV2. Therefore, expression of Rep proteins and rep mRNA from pHIVrep was examined. Expression of Rep proteins from pHIVrep. In cells infected with AAV2 particles or transfected with pAV2 in the presence of helper adenovirus, Rep78 and Rep68 accumulate mainly in the cell nucleus whereas Rep52 and Rep4O are

distributed in both nucleus and cytoplasm upon cell fractionation (44). In the absence of adenovirus, the distribution of Rep proteins is similar but the level of expression is much lower (44). Expression of Rep proteins in transfected cells was examined by Western blotting. Human 293 cells transfected with pAV2 expressed only low levels of Rep proteins in the nucleus or cytoplasm, and there was no obvious inductive effect of Tat (Fig. 3C and D). The apparent increase in cytoplasmic Rep52 and Rep4O (Fig. 3C, lane 4) was not observed in other experiments. In contrast, Rep78 was readily induced by Tat at low inputs of pHIVrep (Fig. 3A, tracks 1 to 4), whereas Rep52 and Rep4O were not induced. Furthermore, the cytoplasmic levels of Rep52 and Rep4O were about equivalent for pHIVrep and pAV2, respectively, consistent with their expression from the AAV Plg promoter in each case. Therefore, induction by Tat was specific for Rep78 (and Rep68) in pHIVrep, consistent with their expression from a chimeric HIV-rep mRNA containing the TAR sequence (Fig. 1). Rep68 was not well defined because of a cross-reacting cell protein (44) and the low level of expression of Rep68 resulting from inefficient splicing of mRNA in the absence of adenovirus (55) (Fig. 3). Nevertheless, the results (Fig. 3) suggest that the inhibition of HIV LTR activity produced by pHIVrep is due to efficient expression of Rep78 and perhaps Rep68. An unexpected feature was revealed by the experiment in Fig. 3. Even in the absence of Tat (lanes 1), the basal level of Rep78 expression was much higher from pHIVrep than from pAV2. This apparently reflects the fact that rep exerts a strong negative autoregulation on expression from its homologous P5 promoter (5) but is much less strongly autoregulated from heterologous promoters (la). Finally, we note that induction of Rep78 by Tat was readily seen at the low gene dosage of pHIVrep (Fig. 3A), but at a 10-fold-higher level of pHIVrep (Fig. 3B) there was much less induction. This suggested that, as the level of Rep78 increased, there was feedback inhibition that prevented further Tat induction. This is consistent with the ability of rep to decrease Tat-activated CAT expression from the HIV LTR. In similar experiments with HeLa cells, both Rep78 and Rep68 were inducible from pHIVrep by Tat (Fig. 4). Both of these proteins were also specifically induced by Tat in

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