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Like all other retroviruses, human immunodeficiency virus type 1 (HIV-1) and HIV-2 integrate a DNA copy of their RNA genome into the host cell's chromosomes. Integration of the viral cDNA is catalyzed by the integrase (IN) protein (3). Sequence analysis has shown that many features of the primary structure of IN are highly conserved among retroviruses and retrotransposons. The amino-terminal domain contains an array of histidine and cysteine residues that form a zinc finger motif (5, 6, 18). The central domain contains the catalytic core, which is defined by three acidic residues with stereotyped spacing. This D,D-35-E motif is a universal feature of integrase proteins and is essential for catalytic activity (9, 10, 22, 25, 36). The carboxy-terminal domain is the least conserved region of IN. It binds to the viral DNA ends and also exhibits nonspecific DNA binding properties (19, 24, 30, 31, 37). The sequence conservation of integrase suggests strong structure-function relationships. IN function has been studied extensively in vitro by using purified enzyme and short oligonucleotide substrate that mimics the ends of the viral DNA molecule (8, 21, 32). Such analysis has shown that IN proteins can utilize different retroviral DNA substrates in the in vitro integration reaction. The HIV-2 IN (IN²) has been shown to efficiently support 3' processing and strand transfer of HIV-1 substrate in vitro (37). Similarly, the feline immunodeficiency virus (FIV) IN can cleave and integrate HIV and Moloney murine leukemia virus (MoMLV) DNA ends (38). HIV-1 IN is active on FIV substrate but is barely active on MoMLV substrate (35, 38). While the analysis of the integration reaction in vitro has provided a great deal of information on IN function and has helped to elucidate the molecular mechanism of viral DNA integration, the conditions used to study IN in vitro do not fully duplicate those in vivo.

In the context of a replicating virus, HIV-1 IN is expressed and assembled into virions as the C-terminal component of a larger 160-kDa Gag-Pol polyprotein (Pr160^Gag-Pol). After proteolytic processing of Pr160^Gag-Pol and entry of the virus core into the host cell, IN exists as a 32-kDa protein together with other viral and cellular proteins that make up the viral nucleoprotein complex (34). Several in vivo studies have suggested that the IN protein may be involved in other step of the virus life cycle. Host cell proteins that bind to IN or the HIV-1 preintegration complex (PIC) and promote integration have been identified (13, 20). Through analysis in nondividing cells, mutations in the C terminus of IN have been shown to disrupt the movement of the viral PIC into the nucleus (15), suggesting a role of IN in the nuclear import machinery pathway (15, 16). In other studies, some IN mutations, including those in highly conserved amino acids residues, were found to have no apparent effect on IN activity in vitro while dramatically reducing the formation of the provirus in infected cells (11, 12, 26, 29). These results were initially explained in part by changes in the structure of the Pr160^Gag-Pol precursor protein, resulting in aberrant virus assembly and maturation. By incorporating IN protein into virions in trans, independently of Pr160^Gag-Pol, we recently demonstrated that the mature IN protein itself promotes the initiation of viral DNA synthesis (40). These results indicate that IN may play important roles in the virus life cycle at several different levels.

To examine whether IN² could function in place of HIV-1 IN during virus infection, two experimental approaches were undertaken. In the first, we replaced the HIV-1 IN coding region with that of the IN², generating a virus that contained an HIV-1-HIV-2 chimeric pol gene (Fig. 1). In the second approach, the IN² protein was incorporated into HIV-1 virions in trans by expression as a fusion partner of Vpr (Vpr-IN). This strategy is based on our previously findings and those of others, which have shown that Vpr can be used as a vehicle to deliver enzymatically active IN into HIV-1 virions in trans, independently of Pr160^Gag-Pol (14, 28, 40, 41).

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Analysis of IN2 protein function when expressed in cis with the HIV-1 genome. (A) Insertion of IN2 into the pol gene of HIV-1. The HIV-1 SG3wt DNA (41) was cut with the BamHI and SalI endonucleases to remove IN, vif, and the 5' half of vpr from the DNA genome. A fragment of pSG3wt DNA encompassing 16 bp of 3' IN sequence, vif and the 5' half of vpr was amplified by PCR, wherein the sense primer contained a MluI restriction site. In a separate reaction, an IN-containing DNA fragment was PCR amplified from the HIV-2 ST clone, wherein the sense and antisense primers contained BamHI and MluI restriction sites, respectively. The two PCR-amplified DNA fragments were ligated into the BamHI-SalI-cut pSG3wt DNA, generating the chimeric virus pSG3IN2. Sequence analysis confirmed an open chimeric pol reading frame (PR-RT-IN2). The 3' 18 nucleotides of the HIV-1 IN are retained in the chimeric protein. (B) Infectivity of SG3IN2 virions. 293T cells were transfected by calcium phosphate DNA precipitation methods with pSG3IN2 DNA. Forty-eight hours later, the culture supernatants were collected, filtered through 0.45-µm-pore-size filters, and analyzed for HIV-1 p24 antigen by enzyme-linked immunosorbent assay (Coulter Inc.). Next, 25, 5, and 1 ng of each virus (p24 antigen equivalents) was used to infect monolayer cultures of P4 indicator cells (7). Two days later, the cells were stained, and infection-positive cells were counted as described earlier (41). The virus infectivity results represent infectious units per 25-ng equivalent of p24 antigen. These results were highly reproducible in three independent experiments. The data shown are from a single representative experiment.

Characterization of HIV-1 that encodes an HIV-1-HIV-2 chimeric Pol protein. An IN-containing DNA fragment was PCR amplified from the HIV-2 ST proviral clone (23) and ligated into the BamHI and MluI restriction sites of the HIV-1 pSG3^wt molecular clone, generating pSG3^IN2 (Fig. 1A). To determine whether the SG3^IN2 virus was infectious, 293T cell monolayers were transfected with pSG3^IN2 DNA. For controls, HIV-1 wild-type (SG3^wt) and IN-minus (pSG3^S-IN; see references 28 and 41) viruses were also transfected. The supernatants of the transfected cultures were collected 72 h later and divided into three aliquots. One aliquot was ultracentrifuged to pellet virus for immunoblot analysis, the second aliquot was analyzed for reverse transcriptase (RT) activity and p24 antigen concentration (Coulter Inc.), and the third was used to infect CD4-LTR--galactosidase indicator cells (P4) (7). No differences between the wild-type and SG3^IN2 virions with respect to RT activity (relative to p24 antigen concentration) and proteolytic processing of the Gag and Pol proteins were detected (data not shown). However, the SG3^IN2 virions exhibited a marked decrease in infectivity (Fig. 1B). Compared with the IN-minus SG3^IN2 virions, the SG3^IN2 virions were reproducibly more infectious.

Incorporation of IN² protein into HIV-1 virions by expression in trans. To avoid the possible dominant-negative effects of the chimeric Gag-Pol precursor protein, we fused the HIV-1 vpr gene with the HIV-2 IN gene (IN²) and placed the vpr-IN² gene fusion into the pLR2P expression plasmid (pLR2P-vprIN²) for complementing HIV-1 IN mutant viruses in trans. The pLR2P-vprIN² plasmid was cotransfected into 293T cells with the pSG3^S-IN IN-minus clone. As controls, pSG3^S-IN was cotransfected with the Vpr HIV-1 IN expression vector (pLR2P-vprIN; references 28, 40, 41) and the pLR2P vector. Immunoblot analysis of the progeny virions detected two species of anti-HIV-1 IN-reactive proteins (Fig. 2A), a 47-kDa protein, which is consistent with the combined masses of IN (32 kDa) and Vpr (15 kDa), and a 32-kDa protein. The detection of the 32-kDa protein in SG3^S-IN virions complemented with Vpr-IN indicated proteolytic processing of the fusion protein and liberation of IN. The Vpr-IN² fusion protein was not detected with the HIV-1 anti-IN antiserum. However, using antiserum (F0784) obtained from an HIV-2-infected individual as a probe, the 47- and 32-kDa proteins were detected in both SG3^wt and SG3^S-IN virions that were complemented with either the Vpr-IN or Vpr-IN² fusion proteins (Fig. 2B). Using anti-Vpr antibody, the 47-kDa Vpr-IN and Vpr-IN² fusion proteins and their respective Vpr cleavage products were detected (Fig. 2C). While SG3 contains an open vpr reading frame, the virally encoded Vpr protein was not detected under the conditions used. Anti-Gag antibody detected approximately similar amounts of viral Gag proteins (Fig. 2D). These results confirmed that the IN² protein can be incorporated into HIV-1 virions when expressed in trans as a fusion partner of Vpr, and subsequently liberated during proteolytic maturation of the virus particle.

View larger version (36K): [in this window] [in a new window]   FIG. 2.   Incorporation of the Vpr-IN fusion protein into IN-minus virions. (A) pSG3S-IN and pSG3wt were separately cotransfected into 293T cells with pLR2P-vprIN2, pLR2P-vpr-IN, and pLR2P (vector alone), respectively. As described earlier (41), the extracellular progeny virions were concentrated from the supernatants of each culture by ultracentrifugation, lysed, and analyzed by immunoblot analysis using anti HIV-1 IN peptide antibody (A), human anti-HIV-2 antiserum (B), anti-Vpr (C), and anti-Gag (D) antibodies as probes.

The IN² protein restores the infectivity of HIV-1 IN-minus virus. To analyze whether the IN² protein was functional, SG3^S-IN virions complemented with the trans-IN² protein were analyzed on P4 indicator cells. Our results indicated that virus infectivity was rescued to a level of 16% compared with virions complemented with the homologous HIV-1 trans-IN protein (Table 1). This result represents an increase of approximately 100-fold over noncomplemented SG3^S-IN virions. Immunoblot analysis performed on the viral stocks that were used for infection confirmed that the trans-IN- and trans-IN²-complemented virions contained approximately equal amounts of the respective fusion protein (data not shown). These results suggested that the IN² protein can function in place of the HIV-1 IN in vivo, albeit with significantly reduced efficiency.

IN² supports integration of the HIV-1 provirus. To directly examine whether the heterologous trans-IN² protein could complement the defect in integration of IN-minus HIV-1, a single-cycle integration assay was used. This assay utilized VSV-G-pseudotyped virus, which contains the hygromycin resistance gene within the HIV-1 genome in place of env. In cells infected with pseudotyped virus, where viral DNA is synthesized, integrated, and expressed, the hygromycin resistance gene allows for the outgrowth of cells in the presence of selection medium. VSV-G-pseudotyped, hygromycin-resistant, IN-minus virus (hy-SG3^S-IN) was complemented by DNA cotransfection with Vpr-IN² and Vpr-IN, respectively. Transfection-derived viral stocks were filtered through 0.45-µm-pore-size filters, and divided into two aliquot sets. One aliquot set was subjected to ultracentrifugation to pellet virions and was examined by immunoblot analysis. Similar levels (relative to CA protein) of virion associated Vpr-IN and Vpr-IN² were detected (data not shown). The second aliquot set was normalized for p24 antigen concentration and was used to infect HeLa CD4 cells in hygromycin selection medium as described earlier (40). Complementation with the IN² protein produced 22% of the number of resistant colonies produced by complementation with the homologous trans-IN protein (Table 2). Complementation of wild-type virions (hy-SG3^wt) with either Vpr-IN or Vpr-IN² resulted in a slight reduction (approximately twofold) in CFU (data not shown). These result are consistent with the infectivity results (Table 1).

IN² protein does not efficiently promote HIV-1 DNA synthesis. Several reports have shown that viruses containing certain mutations in IN, including IN deletion mutants, are defective in the synthesis of their viral DNA. We reported that this is predominately due to a role that the IN protein plays in the initiation step of RT. Our published data demonstrated that IN-minus virions (SG3^S-IN) synthesize reduced levels of viral cDNA (5 to 10% of that of wild-type virus) and that this defect can be overcome by providing IN in trans (40). Therefore, the above results (Tables 1 and 2) would suggest that the defect in the infectivity of IN²-complemented SG3^S-IN virus was largely due to a block in viral DNA synthesis after infection. To test this directly, we analyzed the synthesis of the nascent viral cDNA in infected cells. SG3^S-IN virions containing Vpr-IN² were derived by DNA cotransfection and used to infect HeLa-CD4 cells. Eighteen hours later, the cell monolayers were trypsinized, washed extensively, and divided into two aliquot. One aliquot set was lysed and analyzed for intracellular p24 antigen concentration as described earlier (28, 39). DNA was extracted from the other aliquot set, normalized for intercellular p24 antigen concentration, and analyzed for early (R-U5) and late (R-Gag) viral DNA products as described earlier (40). Serial fivefold dilutions of the SG3^wt DNA were prepared and analyzed in parallel as a reference to assess the relative amounts of DNA synthesized by the mutant viruses. The IN-minus SG3^S-IN virus (lane 7) produced significantly less R-U5 DNA compared with wild-type SG3^wt virions (Fig. 3A). When complemented with the trans-IN² protein the SG3^S-IN virions produced 18% of the wild-type levels of R-U5 DNA (lane 5). This represents an increase in DNA synthesis of approximately four- to fivefold. Complementation of SG3^S-IN with the homologous trans-IN proteins generated 86% the levels of R-U5 DNA compared with SG3^wt. For control, an equivalent (p24 antigen) amount of the SG3^D116A mutant virus was analyzed and found to contain near wild-type levels (89%) of R-U5 DNA. No R-U5 DNA was detected in cells infected with the RT-IN-minus SG3^S-RT virions (40), confirming that the R-U5 DNA detected by PCR was the product of reverse transcription. The absence of this DNA product in cells infected with SG3^S-RT also indicates that the band migrating slightly slower than the R-Gag band is not a product of viral DNA contamination. Nearly identical results were obtained using the R-Gag primer pair to detect late products of viral DNA synthesis (Fig. 3B). These results show that the IN² protein can support the synthesis of HIV-1 DNA, but with significantly reduced efficiency compared with HIV-1 IN. They are also in strong agreement with our results on virus infectivity (Table 1) and together suggest that the factor limiting infectivity was primarily the defect in the synthesis of viral cDNA.

View larger version (66K): [in this window] [in a new window]   FIG. 3.   Reverse transcription of Vpr-IN2-complemented viruses. pSG3S-IN was cotransfected into 293T cells by calcium phosphate DNA transfection methods with pLR2P-vpr-IN2, pLR2P-vprIN, and pLR2P, respectively. As controls, pSG3wt, pSG3S-RT, and pSG3D116A were also transfected. Forty-eight hours later, culture supernatants were filtered through 0.45-µm-pore-size filters and analyzed by HIV-1 p24 antigen enzyme-linked immunosorbent assay (ELISA) (Coulter Inc.). The virus-containing culture supernatants were normalized to 500 ng of p24 antigen (CA), treated with RNase-free DNase H (20 U/ml for 2 h) (Promega Corporation) and placed on cultures of HeLa-CD4 cells at 37°C. After 4 h, the cell monolayers were washed, trypsinized, resuspended in fetal bovine serum, and divided into two aliquot sets. One aliquot set (which contained one-tenth of the total number of cells) was lysed in phosphate-buffered saline containing 1% Triton X-100 and analyzed by p24 antigen ELISA to quantify intracellular CA protein. The other aliquot set was placed back in culture medium at 37°C for an additional 14 h. The cells were then washed and total DNA was extracted by organic methods. Next, 250-pg equivalents (p24 antigen) of each DNA extract was analyzed by PCR methods for early (R-U5) (A) and late (R-gag) (B) viral DNA products of RT. The amplified products were resolved on 1.5% agarose gels and stained with ethidium bromide. To assess the relative amount of each of the amplified DNA products, four serial fivefold dilutions of the wild-type (SG3wt) DNA were analyzed in parallel. The undiluted 250-pg sample was arbitrarily set to 100. As a control for the efficiency of DpnI cleavage of potential carryover plasmid DNA, 6,250 copies of pSG3wt DNA were analyzed after digestion with DpnI as described previously (17, 40). The ethidium bromide staining intensity of each amplified DNA product was measured with a Lynx 5000 molecular biology workstation (Applied Imaging) as described previously (27). The data shown is from a representative experiment that was repeated three times, each time with independent transfection-derived virus preparations.

Complementation of SG3^IN2 virus with trans-IN. To better understand the defect in the infectivity of the SG3^IN2 virus (Fig. 1), pSG3^IN2 was cotransfected with the HIV-1 trans-IN protein (Vpr-IN) and analyzed for infectivity. Figure 4 shows a 27-fold increase in infectivity. This represented an increase that was near that of wild-type virus when complemented with Vpr-IN. This result suggested that the expression of a chimeric Gag-Pol precursor protein did not cause a severe defect in the assembly or maturation of the SG3^IN2 virions. Rather, these data may support our earlier findings for a role of an RT-IN intermediate in the formation of infectious HIV-1 particles (41).

View larger version (42K): [in this window] [in a new window]   FIG. 4.   Complementation of SG3IN2 virus with homologous trans-IN protein. pSG3IN2 and pSG3wt were transfected alone and separately cotransfected into 293T cells with pLR2P-vprIN. The culture supernatants were collected 48 h later, filtered through 0.45-µm-pore-size filters, and analyzed for HIV-1 p24 antigen by enzyme-linked immunosorbent assay (Coulter Inc.). Next, 25, 5, and 1 ng of each virus (p24 antigen equivalents) was used to infect monolayer cultures of P4 indicator cells. Two days later, the cells were stained and infection-positive cells were counted. The virus infectivity results represent infectious units per 25-ng equivalent of p24 antigen. The data shown are the means from three independent experiments.