Complementation of integrase function in HIV-1 virions

Article15 August 1997free access Complementation of integrase function in HIV-1 virions Thomas M. Fletcher III Thomas M. Fletcher III Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Marcelo A. Soares Marcelo A. Soares Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Silvia McPhearson Silvia McPhearson Department of Microbiology, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Huxiong Hui Huxiong Hui Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author MaryAnn Wiskerchen MaryAnn Wiskerchen Promega, Inc., Madison, WI, 53711 USA Search for more papers by this author Mark A. Muesing Mark A. Muesing Aaron Diamond AIDS Research Center, New York, NY, 10016 USA Search for more papers by this author George M. Shaw George M. Shaw Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Department of Microbiology, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Howard Hughes Medical Institute, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Andrew D. Leavitt Andrew D. Leavitt Departments of Laboratory Medicine and Internal Medicine, University of California at San Francisco, San Francisco, CA, 94143-0100 USA Search for more papers by this author Jef D. Boeke Jef D. Boeke Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Beatrice H. Hahn Corresponding Author Beatrice H. Hahn Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Department of Microbiology, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Thomas M. Fletcher III Thomas M. Fletcher III Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Marcelo A. Soares Marcelo A. Soares Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Silvia McPhearson Silvia McPhearson Department of Microbiology, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Huxiong Hui Huxiong Hui Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author MaryAnn Wiskerchen MaryAnn Wiskerchen Promega, Inc., Madison, WI, 53711 USA Search for more papers by this author Mark A. Muesing Mark A. Muesing Aaron Diamond AIDS Research Center, New York, NY, 10016 USA Search for more papers by this author George M. Shaw George M. Shaw Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Department of Microbiology, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Howard Hughes Medical Institute, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Andrew D. Leavitt Andrew D. Leavitt Departments of Laboratory Medicine and Internal Medicine, University of California at San Francisco, San Francisco, CA, 94143-0100 USA Search for more papers by this author Jef D. Boeke Jef D. Boeke Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Beatrice H. Hahn Corresponding Author Beatrice H. Hahn Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Department of Microbiology, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA Search for more papers by this author Author Information Thomas M. Fletcher1, Marcelo A. Soares1, Silvia McPhearson2, Huxiong Hui1, MaryAnn Wiskerchen3, Mark A. Muesing4, George M. Shaw1,2,5, Andrew D. Leavitt6, Jef D. Boeke7 and Beatrice H. Hahn 1,2 1Department of Medicine, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA 2Department of Microbiology, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA 3Promega, Inc., Madison, WI, 53711 USA 4Aaron Diamond AIDS Research Center, New York, NY, 10016 USA 5Howard Hughes Medical Institute, University of Alabama at Birmingham, 701 S.19th Street, LHRB 613, Birmingham, AL, 35294 USA 6Departments of Laboratory Medicine and Internal Medicine, University of California at San Francisco, San Francisco, CA, 94143-0100 USA 7Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:5123-5138https://doi.org/10.1093/emboj/16.16.5123 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Proviral integration is essential for HIV-1 replication and represents an important potential target for antiviral drug design. Although much is known about the integration process from studies of purified integrase (IN) protein and synthetic target DNA, provirus formation in virally infected cells remains incompletely understood since reconstituted in vitro assays do not fully reproduce in vivo integration events. We have developed a novel experimental system in which IN-mutant HIV-1 molecular clones are complemented in trans by Vpr–IN fusion proteins, thereby enabling the study of IN function in replicating viruses. Using this approach we found that (i) Vpr-linked IN is efficiently packaged into virions independent of the Gag–Pol polyprotein, (ii) fusion proteins containing a natural RT/IN processing site are cleaved by the viral protease and (iii) only the cleaved IN protein complements IN-defective HIV-1 efficiently. Vpr-mediated packaging restored IN function to a wide variety of IN-deficient HIV-1 strains including zinc finger, catalytic core and C-terminal domain mutants as well as viruses from which IN was completely deleted. Furthermore, trans complemented IN protein mediated a bona fide integration reaction, as demonstrated by the precise processing of proviral ends (5′-TG…CA-3′) and the generation of an HIV-1-specific (5 bp) duplication of adjoining host sequences. Intragenic complementation between IN mutants defective in different protein domains was also observed, thereby providing the first evidence for IN multimerization in vivo. Introduction Efficient replication of retroviruses (including HIV-1) requires the insertion of a DNA copy of the viral genome into the chromosome of infected host cells (Goff, 1992; Kulkosky and Skalka, 1994; Farnet and Bushman, 1996). This integration process generates the proviral template for subsequent viral gene expression and is mediated by the viral integrase (IN) protein. IN is one of three enzymes encoded by the viral pol gene, and is expressed and incorporated into virions as part of a Gag–Pol polyprotein. After budding and release of virus particles from infected cells, this polyprotein is cleaved by the viral protease into individual components, a process essential for virus replication. Upon infection of new target cells, IN remains associated with a large nucleoprotein (preintegration) complex which contains the newly synthesized viral DNA as well as Gag and Pol proteins (Bowerman et al., 1989) and, in the case of HIV/SIV, the accessory proteins Vpr and Vpx (Heinzinger et al., 1994; Fletcher et al., 1996) as well as at least one cellular protein (Farnet and Bushman, 1997). The preintegration complex migrates to the nucleus where proviral integration takes place. Most of the mechanistic details of the integration reaction are derived from in vitro studies of purified IN protein acting on short oligonucleotides that mimic viral ends and also serve as target DNA (Bushman and Craigie, 1991; Engelman et al., 1991; Kulkosky and Skalka, 1994). From these and other analyses it has become apparent that integration proceeds as a two step process. In the cytoplasm, IN mediates an endonucleolytic reaction that generally removes two nucleotides from the 3′ ends of the newly synthesized (blunt ended) linear viral DNA in a 3′ processing reaction. After transport to the nucleus, the recessed 3′ ends of the viral DNA are joined to host chromosomal DNA in a concerted strand transfer reaction. The two ends of viral DNA join the target DNA in a staggered fashion, which results in the duplication of host cell sequences immediately flanking the inserted provirus. The length of this duplication is virus-specific and, in the case of HIV-1, comprises a 5 bp direct repeat (Muesing et al., 1985; Bushman et al., 1990; Vink et al., 1990). Purified IN protein can also catalyze the reverse strand transfer reaction, termed ‘disintegration’, when supplied with a synthetic gapped intermediate substrate (Chow et al., 1992). Phylogenetic comparisons, mutational analyses, partial protease cleavage and structural studies have all shown that the HIV-1 IN protein (like that of other retroviruses) consists of functionally distinct subdomains (see Kulkosky and Skalka, 1994; Plasterk, 1995; Farnet and Bushman, 1996). The N-terminal region (located between residues 1 and 50) contains a highly conserved ‘HHCC’ motif, which resembles the zinc finger domains of some transcription factors (Burke et al., 1992; McEuen et al., 1992; Bushman et al., 1993). The exact contribution of this zinc finger domain to IN catalytic activity remains unclear, because mutational analyses have produced varying results, ranging from minor effects to complete abrogation of function (Drelich et al., 1992; Engelman and Craigie, 1992; Schauer and Billich, 1992; Bushman et al., 1993; Leavitt et al., 1993; Vincent et al., 1993; Vink et al., 1993). Nevertheless, there is evidence suggesting that the HHCC domain plays a role in the formation of stable complexes between integrase and viral DNA (Hazuda et al., 1994; Ellison and Brown, 1994; Ellison et al., 1995). The central region (located between residues 50 and 212) contains a triad of three invariant acidic residues (Asp64, Asp116 and Glu152), commonly called the D,D-35-E domain, which are evolutionarily highly conserved among retroviral IN proteins as well as various eukaryotic and prokaryotic transposases (Kulkosky et al., 1992; Doak et al., 1994; Rice and Mizuuchi, 1995). Replacement of any of these acidic residues results in the loss of all enzymatic activities including the disintegration reaction, indicating that this domain constitutes the catalytic core of the enzyme (Engelman and Craigie, 1992; van Gent et al., 1992; Leavitt et al., 1993). Finally, a less conserved C-terminal domain is also required for 3′ processing and forward reactions; although its boundaries are not clearly defined, most investigators place it between residues 212 and 288. This domain contains extensive positively charged surfaces and is believed to have non-specific DNA binding activity (Vink et al., 1993; Woerner and Marcus-Sekura, 1993; Engelman et al., 1994). The precise function of the C-terminus of IN remains unknown. Functional subdomains of integrase have also been defined by in vitro complementation studies (Engelman et al., 1993; van Gent et al., 1993) which demonstrated that certain combinations of enzymatically inactive IN mutants efficiently catalyze 3′ processing and strand transfer reactions when assayed as mixed multimers. For example, zinc finger and catalytic domain mutants complemented each other in trans, i.e. they could be supplied on two different monomers, while the C-terminal region of integrase could function both in trans and in cis relative to the catalytic core. By contrast, no complementation was observed between proteins with mutations in the same functional domain (e.g. different active site mutations). IN can thus form functional multimers in vitro and domains critical for integration can be supplied by different subunits in an oligomeric complex (Engelman et al., 1993; van Gent et al., 1993). Based on these in vitro experiments, it has been proposed that provirus formation in vivo (i.e. in virally infected cells) is also mediated by active IN multimers (Plasterk, 1995). However, direct evidence for this is lacking, since in vitro integration assays generally examine only ‘half reactions’, i.e. insertion of a single viral DNA end into a single strand of target DNA, and thus do not fully reproduce the integration events that occur in vivo. To further define the functions of integrase during in vivo integration, several groups of investigators have begun to analyze IN mutants in the context of infectious molecular clones of HIV-1. Interestingly, these studies have provided evidence for additional roles of IN in HIV-1 replication (Engelman et al., 1995; Masuda et al., 1995; Wiskerchen and Muesing, 1995; Cannon et al., 1996; Leavitt et al., 1996; Taddeo et al., 1996). For example, mutations in the C-terminal domain of integrase, which have little to no effect on in vitro IN enzymatic activity (Leavitt et al., 1993), abrogate proviral integration when introduced into an infectious molecular clone (Cannon et al., 1996; Leavitt et al., 1996). Careful analysis of particle morphology, protein composition, viral DNA synthesis and nuclear import revealed no differences between the C-terminal integrase mutants and wild-type HIV-1, suggesting an altered interaction of integrase with the target cell DNA (Cannon et al., 1996; Leavitt et al., 1996). Another interesting phenotype resulted from analyses of viral constructs with substitutions in the conserved His or Cys residues of the N-terminal HHCC domain (Masuda et al., 1995; Leavitt et al., 1996). Upon infection of new cells, these HHCC mutants were severely impaired in their ability to synthesize viral DNA, although they contained a fully functional reverse transcriptase enzyme and wild-type levels of packaged viral RNA (Masuda et al., 1995; Leavitt et al., 1996). These data thus indicated an effect of integrase on reverse transcription, possibly through alteration of the preintegration complex. Given the complexity of IN activities in vivo, we wished to develop a trans-complementation system that would allow us to probe integrase function in the context of replicating virions. To mediate virion incorporation in the absence of genomic expression, we fused IN to Vpr, an HIV-1 accessory protein which is present in virions in equimolar quantities to the viral Gag proteins (Lu et al., 1993; Paxton et al., 1993), represents a known component of the viral preintegration complex (Heinzinger et al., 1994) and has previously been shown to have the capacity to target heterologous fusion proteins to the HIV-1 particle (Fletcher et al., 1995; Wu et al., 1995). Coexpressing Vpr–IN fusion constructs with IN-mutant HIV-1 molecular clones, we found that IN can be efficiently packaged by this novel route and that trans complemented IN protein can restore provirus formation to IN-defective virions. We also found that proteolytic cleavage of IN from its Vpr fusion partner is required for efficient complementation. Finally, we demonstrated that intragenic complementation between IN mutants defective in different protein domains is possible, thus providing the first evidence for IN multimerization in vivo. Results Integrase is efficiently packaged into HIV-1 virions as a Vpr fusion protein We have previously shown that virion-associated accessory proteins of HIV (i.e. Vpr, Vpx and Vif) can be utilized to target foreign proteins to the HIV particle (Fletcher et al., 1995; Wu et al., 1995). To investigate whether this same strategy could be used to complement functionally impaired virion components, e.g. a defective IN protein, we prepared Vpr-integrase gene fusions and control constructs (Figure 1A, left panel) and tested their ability to express proteins with virion targeting capabilities. R–IN was generated by ligating the 3′ end of vpr in-frame to the 5′ end of integrase, while R–PC–IN was engineered to contain an additional 45 bp of pol sequences upstream of IN conserving the natural RT/IN protease cleavage site (PC). Control constructs contained either vpr alone (R) or vpr fused to PC sequences (R–PC). These gene fusions were cloned into an HIV-2 LTR/rev responsive element (RRE) regulated vector (pLR2P) known to mediate high level expression (Wu et al., 1995) and cotransfected with HIV-1 molecular clones containing either wild-type (R7–3) or mutant (H12A) integrase coding regions (Figure 1A, right panel). Figure 1.Efficient packaging of Vpr–IN fusion proteins into HIV-1 virions. (A) Schematic representation of Vpr–integrase fusion constructs (R–IN; R–PC–IN), control constructs (R; R–PC), and HIV-1 molecular clones containing wild-type (R7-3) or mutant (H12A) integrase genes (IN domains and their boundaries are indicated). PC comprises 45 bp of pol sequence immediately upstream of the natural RT/IN cleavage site (also see Figure 3). R7-3 and H12A are isogenic, except for a single amino acid substitution in the zinc finger domain (highlighted). (B) Western blots of transfection-derived virion preparations (200 ng of p24 per lane) probed with anti-IN and anti-Vpr antibodies. R7-3 and H12A molecular clones were transfected alone (left lanes of each panel) or in combination with R, R–PC, R–IN and R–PC–IN constructs. Bands corresponding to the various fusion proteins as well as wild-type integrase (IN) are indicated. An additional IN-reactive protein of 38 kDa in R–PC–IN complemented virions likely represents a cleavage product processed at a non-natural site (non-specific processing is known to occur in the context of Vpr fusion proteins; Wu et al., 1995). The same protein is also apparent in R–PC–IN containing virion preparations shown in Figure 3B (middle panel) and Figure 5A (right upper panel). Neither the R7-3 nor the H12A molecular clones encode a functional Vpr protein (the 22 kDa band present in all virion preparations probed with the anti-Vpr antiserum is an a non-specific reaction product). Download figure Download PowerPoint To assess packaging of the Vpr fusion proteins, transfection-derived virions were pelleted through 20% sucrose and their protein profiles were examined by Western blot analysis. As shown in Figure 1B, R–IN and R–PC–IN fusion proteins were readily detectable in R7-3 as well as H12A derived virions. As expected, R–PC–IN was slightly larger than R–IN. However, the intensity of the R–PC–IN band was diminished relative to that of R–IN, while the intensity of the corresponding wild-type integrase band (IN) was increased, suggesting partial cleavage by the viral protease. This was confirmed by blots probed with anti-Vpr antibodies which showed that only virions containing the R–PC–IN (but not the R–IN) fusion protein exhibited a 13 kDa Vpr-reactive protein. Since both R7-3 and H12A encode a prematurely truncated (and thus unstable) Vpr protein, which is undetectable on immunoblots (Wiskerchen and Muesing, 1995), this small Vpr-reactive protein most likely represents a protease cleavage product. Moreover, comigration with the R–PC translation product (which contains 15 amino acids of PC sequence in addition to Vpr), rather than the slightly smaller R translation product (which resembles the native Vpr protein), suggests that protease processing had occurred at the intended (i.e. natural) site. These results indicate that Vpr–integrase fusion proteins are efficiently packaged into HIV-1 virions and accessible to processing by the viral protease at the RT/IN cleavage site. Vpr-mediated packaging of integrase restores the biological activity of a zinc finger mutant HIV-1 molecular clone To investigate whether Vpr-mediated packaging supplied a functional integrase protein, we cotransfected an IN-defective HIV-1 molecular clone (H12A) with R–PC–IN and R–IN, and tested the resulting virions for biological activity in the MAGI cell assay (wild-type R7-3 HIV-1 was analyzed in parallel for control). MAGI (HeLa-CD4-LTR-β-gal) cells contain a β-galactosidase gene (β-gal) stably integrated under the control of an HIV-1 LTR (Kimpton and Emerman, 1992). Since the β-gal gene also encodes a nuclear localization signal (NLS), infection with wild-type HIV-1 results in the formation of blue nuclei. Viruses with defective integrase genes, including H12A, score negative in this assay, because the induction of blue nuclei requires tat gene expression from an integrated provirus to activate the LTR-β-gal construct. The MAGI cell assay has thus been widely used to characterize the biological activity of IN-mutant molecular clones of HIV-1, except for catalytic triad mutants which are believed to express Tat from unintegrated viral DNA and generate blue nuclei even in the absence of viral integration (Engelman et al., 1995; Wiskerchen and Muesing, 1995). Transfection-derived virion preparations were normalized for p24 content and used to infect MAGI cells. As expected, wild-type HIV-1 (R7-3) yielded large numbers of blue nuclei when transfected alone (∼1×104 per 10 ng of p24) or in combination with R–IN, R–PC–IN, R and R–PC constructs (0.3–0.5×104 per 10 ng of p24; Figure 2A depicts results for cotransfection with R–PC–IN). By contrast, virions derived from the H12A molecular clone produced no blue nuclei (Figure 2B), consistent with previous reports of a severe DNA synthesis defect associated with mutations of the HHCC domain (Engelman et al., 1995; Masuda et al., 1995; Wiskerchen and Muesing, 1995; Leavitt et al., 1996). There were also no blue nuclei detectable in cultures infected with virions derived from H12A/R–IN cotransfections (Figure 2C), despite efficient packaging of the R–IN fusion protein (see Figure 1B). However, virions derived from H12A/R–PC–IN cotransfections yielded considerable numbers of blue nuclei (Figure 2D). Since equivalent amounts of virions (based on p24 content) were used for all MAGI cell infections, the biological activity of wild-type and complemented IN-mutant HIV-1 could be compared. Counting several different fields from two independent experiments, we estimated that R–PC–IN restored the IN defect of H12A-derived virions to ∼20% of wild-type activity. Importantly, complementation efficiency appeared to depend on the amount of R–PC–IN fusion protein packaged into virions. As shown in Figure 2E, maximal numbers of blue nuclei were generated when H12A and R–PC–IN constructs were cotransfected in ratios (wt/wt) of 1:4–1:8 (a ratio of 1:5 was used for all subsequent experiments). These results thus indicate that the R–PC–IN fusion protein is functionally active and can restore IN function to a zinc finger mutant HIV-1 molecular clone. Figure 2.Vpr-mediated packaging of integrase restores IN function to an IN-defective (H12A) HIV-1 molecular clone. (A)–(D) Analysis of fusion protein containing wild-type (R7-3) and IN-mutant (H12A) HIV-1 virions in the MAGI cell assay. Blue nuclei indicate single cell infections with viruses containing a functional IN protein (see text for details of the experiment). (E) Determination of optimal cotransfection ratios for efficient Vpr-mediated IN complementation. Maximal numbers of blue nuclei were generated when H12A and R–PC–IN were cotransfected in ratios (wt/wt) of 4:1 to 8:1. Download figure Download PowerPoint Proteolytic cleavage of integrase is required for efficient in vivo complementation The fact that R–IN-complemented H12A virions failed to produce blue nuclei in the MAGI cell assay indicated that virion incorporation of the fusion protein alone was not sufficient for restoration of integrase function. To investigate directly whether cleavage of the fusion protein was required, we mutated the RT/IN cleavage site in R–PC–IN (Figure 3A) by substituting a single nucleotide (CTA→ATA) in the codon immediately 5′ of the N-terminus of IN (P1 position of the cleavage site), thus generating R–PCM–IN. Based on previous analyses of HIV-1 protease processing sites (Pettit et al., 1991), we expected the resulting amino acid substitution (Leu to Ile) to abrogate, or at least greatly diminish, protease processing. Sequence analysis of the entire R–PCM–IN construct confirmed the C to A substitution and excluded inadvertent PCR-induced mutations. Figure 3.Proteolytic cleavage of IN from its Vpr fusion partner is required for efficient in vivo complementation. (A) Schematic representation of wild-type (PC) and mutated (PCM) RT/IN protease cleavage sites. The single Leu to Ile substitution is highlighted. Two amino acids generated by the introduction of a BamHI restriction site used for fusion gene construction are denoted by asterisks (*). (B) Protein profiles of H12A-derived virions containing R, R–PC, R–IN, R–PC–IN and R–PCM–IN fusion proteins. The absence of an R–PC band in virions containing the R–PCM–IN fusion protein indicates that proteolytic cleavage at the RT/IN site did not occur (the blot is overexposed to rule out partial cleavage). (C) R–PCM–IN-containing H12A virions lack biological activity in the MAGI cell assay. Download figure Download PowerPoint Figure 3B depicts the protein profiles of sucrose pelleted virions derived from cotransfections of H12A with R, R–PC, R–IN and R–PC–IN as well as the newly generated R–PCM–IN construct. The results show that the introduced amino acid substitution indeed inhibited (or at least greatly reduced) protease cleavage of the R–PCM–IN fusion protein. No Vpr-reactive cleavage product was detectable on blots probed with an anti-Vpr antibody (upper panel), and there was no decrease in the intensity of the full length R–PCM–IN fusion protein relative to R–IN (middle panel). Analysis of the same virion preparations in the MAGI cell assay (Figure 3C) documented that cleavage was essential for trans-complementation. H12A cotransfected with R–PC–IN yielded the expected number of blue nuclei (see Figure 2). However, the same clone cotransfected with R–PCM–IN produced virtually no blue nuclei (there were only one or two per plate). These data thus indicate that cleavage of IN from its Vpr fusion partner is required for complementation of an HIV-1 molecular clone defective in its HHCC domain. Defects in all three functional domains of IN can be complemented by packaging integrase in trans To examine whether R–PC–IN could complement a broader spectrum of integrase mutations and to prove that this complementation indeed resulted in bona fide provirus formation, we characterized additional HIV-1 molecular clones with point or deletion mutations in their IN coding region (Figure 4A). These included a catalytic core mutant (D116A), a combined central region/C-terminus mutant which lacked 68 amino acid residues between positions 181 and 249 and contained two amino acid substitutions (E85A/E87A) in the central domain (M2) and a mutant in which integrase expression was abrogated due to stop codons at the RT/IN junction (ΔIN). These clones were selected because they contained mutations in all three functional domains of integrase and were known to be integration defective (Wiskerchen and Muesing, 1995). They were also available as proviral constructs containing a selectable marker cassette (SV40 gpt) in place of their env coding region (Figure 4B), allowing characterization in a single round integration assay (Wiskerchen and Muesing, 1995). Figure 4.Generation of an expanded set of IN-mutant molecular clones. (A) Schematic representation of wild-type (R7-3 gpt) and IN-mutant HIV-1 molecular clones with defects in three functional domains (highlighted). Amino acid substitutions and deletions are indicated. ΔIN contains two in-frame stop codons at the RT/IN junction. (B) Experimental outline of the single round integration assay (see text for details). Download figure Download PowerPoint Viral stocks were prepared by transfecting the various gpt constructs with and without R–PC–IN. All constructs were also pseudotyped with MuLV env to provide a functional envelope glycoprotein. Virus stocks were pelleted through a sucrose cushion, normalized for p24 content, and examined by Western blot analysis. This was done to ensure efficient packaging and cleavage of the R–PC–IN fusion protein, and to confirm the authenticity of the proviral gpt constructs used for transfection (Figure 5A). For example, ΔIN gpt-derived virions exhibited no reactivity with anti-IN antibodies, confirming the inability of the ΔIN construct to express integrase. After transfection of ΔIN gpt with R–PC–IN, however, wild-type IN (32 kDa) and R–PC (13 kDa) bands were apparent, indicating efficient packaging and cleavage of the fusion protein. Virus stocks were then used to infect susceptible target cells and infected cell clones were selected as described (Landau et al., 1991). Because the gpt constructs are defective in their env gene (compare Figure 4B), all progeny virions produced after the first round of infection are replication incompetent. Moreover, each mycophenolic acid