Title: Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells
Abstract: Article15 March 2001free access Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells Douglas Braaten Douglas Braaten Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY, 10032 USA Search for more papers by this author Jeremy Luban Corresponding Author Jeremy Luban Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY, 10032 USA Department of Medicine, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY, 10032 USA Search for more papers by this author Douglas Braaten Douglas Braaten Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY, 10032 USA Search for more papers by this author Jeremy Luban Corresponding Author Jeremy Luban Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY, 10032 USA Department of Medicine, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY, 10032 USA Search for more papers by this author Author Information Douglas Braaten1 and Jeremy Luban 1,2 1Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY, 10032 USA 2Department of Medicine, Columbia University College of Physicians and Surgeons, 701 W. 168th Street, New York, NY, 10032 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1300-1309https://doi.org/10.1093/emboj/20.6.1300 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds most members of the cyclophilin family of peptidyl-prolyl isomerases. Of 15 known human cyclophilins, cyclophilin A (CypA) has been the focus of investigation because it was detected in HIV-1 virions. To determine whether CypA promotes HIV-1 replication, we deleted the gene encoding CypA (PPIA) in human CD4+ T cells by homologous recombination. HIV-1 replication in PPIA−/− cells was decreased and not inhibited further by cyclosporin or gag mutations that disrupt Gag's interaction with cyclophilins, indicating that no other cyclophilin family members promote HIV-1 replication. The defective replication phenotype was specific for wild-type HIV-1 since HIV-2/SIV isolates, as well as HIV-1 bearing a gag mutation that confers cyclosporin resistance, replicated the same in PPIA+/+ and PPIA−/− cells. Stable re-expression of CypA in PPIA−/− cells restored HIV-1 replication to an extent that correlated with steady-state levels of CypA. Finally, virions from PPIA−/− cells possessed no obvious biochemical abnormalities but were less infectious than virions from wild-type cells. These data formally demonstrate that CypA regulates the infectivity of HIV-1 virions. Introduction Human immunodeficiency virus type 1 (HIV-1) gag encodes proteins that play roles in practically every step of the virus life cycle (Freed, 1998). The Gag polyprotein orchestrates the formation and release of enveloped virions from infected cells. Concurrent with budding of nascent virions, the Gag polyprotein is cleaved by the viral protease to produce, among other products, matrix, which lines the virion envelope; capsid, which forms the virion core; and nucleocapsid, which coats viral genomic RNA. To initiate infection, the mature virion binds to cell surface receptors on a susceptible target cell and fuses its membrane with the cell's plasma membrane. A viral ribonucleoprotein complex then enters the target cell cytoplasm where gag-encoded proteins participate in reverse transcription, nuclear transport and establishment of the provirus. Since viruses are obligate intracellular parasites, host– cell factors might be required for any of the HIV-1 gag-encoded functions mentioned above. Attempts to identify these putative factors have revealed a number of Gag-interacting proteins, including actin (Rey et al., 1996; Liu et al., 1999; Wilk et al., 1999; Ott et al., 2000), ubiquitin (Ott et al., 1998), calmodulin (Radding et al., 2000), the motor protein KIF-4 (Tang et al., 1999), the nuclear transporter karyopherin-α (Gallay et al., 1996; Agostini et al., 2000), translation elongation factor 1-α (Cimarelli and Luban, 1999), translation initiation factor 2 (Wilson et al., 1999), the HO3 histidyl-tRNA synthetase (Lama and Trono, 1998) and a human member of the trithorax/polycomb group of proteins (Peytavi et al., 1999). Convincing evidence that any of these factors are required for HIV-1 replication has remained elusive. In contrast, it is instructive to consider studies that exploited CD4-negative or chemokine receptor-negative cells to demonstrate that these cell surface proteins are required for viral entry (Maddon et al., 1986; Feng et al., 1996). To date, cells that do not express the Gag-binding factors have yet to be identified or generated. In addition to the factors mentioned above, members of the large family of proteins known as the cyclophilins (Table I) were found to bind to HIV-1 Gag in one of the first reported two-hybrid screens for a cDNA encoding an interacting protein (Luban et al., 1993). Cyclophilins were discovered originally because of their high affinity for cyclosporin (Handschumacher et al., 1984), an immunosuppressive drug used to prevent allograft rejection. The clinical effect of the drug is not thought to result from inhibition of a cyclophilin function. Instead, immunosuppression results when the cyclophilin–cyclosporin complex binds and inhibits calcineurin (Friedman and Weissman, 1991; Liu et al., 1991b), a calcium-dependent, serine-threonine phosphatase required for transcriptional activation of many cytokine genes in stimulated T cells. Considering the immunosuppression associated with HIV-1 infection, cyclophilins seemed intriguing Gag-binding partners. Gag and cyclosporin were later found to compete for the same binding site on cyclophilin (Gamble et al., 1996; Braaten et al., 1997; Dorfman et al., 1997), but the Gag–cyclophilin complex does not interact with calcineurin (Luban et al., 1993). Table 1. The known human cyclophilins Protein name Protein size Subcellular localization Binds to HIV-1 Gaga HUGO gene name GenBank accession Nos CypA 18 kDa cytoplasm yes PPIA X52851 Y00052 CypB, SCYLP 18–20 kDa ER and secretory pathway yes PPIB M60857 M63573 M60457 CypC 18 kDa secretory pathway yes PPIC S71018 CypF, Cyp3 18–22 kDa mitochondrial inner membrane ND PPIF M80254 PPIL1, CypM 18 kDa cytoplasm ND PPIL1 AF090992 USA-CyP, Cyp20 20 kDa nucleus; human U4/U6 snRNP-associated protein ND USA-CYP AF016371 CypE, Cyp33A 40 kDa RNA-binding nuclear protein ND PPIE AF042385 AF104013 Cyp33B shorter isoform of 33A RNA-binding nuclear protein ND PPIE AF042386 Cyp40 40 kDa cytoplasm and nucleus; associated with steroid receptors yes PPID L11667 D63861 Serologically defined colon cancer 50 kDa nucleus ND SDCCAG10 AF039692 antigen 10 Cyp60 60 kDa nucleus; interacts with the proteinase inhibitor elgin C yes PPIL2 U37219 KIAA0073, Hal539-CyP 60 kDa unknown ND KIAA0073 D38552 CARS-CyP, Clk-associating RS cyclophilin 89 kDa associated with the nuclear matrix and splicing factors yes CYP U40763 X99717 NK-TRCyP 89 kDa associated with the nuclear matrix and splicing factors yes NKTR L04288 RAN-BP2, NUP-358 358 kDa associated with the nuclear pore, cytoplasmic face no RAN-BP2 L41840 D42063 aHIV-1 Gag binding to GST fusions with CypA, B or C as previously described (Franke et al., 1994); identical methods were used to determine binding to GST fusions with the cyclophilin domains from Cyp40, 60, CARS, NK-TR and RAN-BP2/NUP-358. ND, not determined. Cyclophilins are defined by a conserved sequence of ∼150 amino acids that form an eight-stranded β-barrel with a hydrophobic pocket that serves as the binding site for cyclosporin and HIV-1 Gag (Ke et al., 1991; Mikol et al., 1993; Gamble et al., 1996). Some cyclophilins, such as CypA, consist of just this core domain. In other cases, the cyclophilin domain is embedded within a more complex protein. Proteins containing a cyclophilin domain have been implicated in a number of cellular processes, including protein secretion, mitochondrial function, RNA processing and transcriptional regulation (Colgan et al., 2000), but the exact biochemical function in cells of the core cyclophilin domain is unknown. One function is, presumably, the maintenance of proper protein conformation, since cyclophilins catalyze the cis–trans interconversion of peptide bonds N-terminal to proline, an activity that has been shown to stimulate the rate of refolding of model proteins in vitro (Fischer et al., 1989; Takahashi et al., 1989). It has been suspected, therefore, that cyclophilins regulate the conformation of HIV-1 Gag (Luban et al., 1993). Following the discovery of the Gag–cyclophilin interaction, CypA in particular was found to be a constituent of the HIV-1 virion (Franke et al., 1994; Thali et al., 1994). Additional studies have attempted to provide evidence of a functional role for CypA, either early in infection of susceptible cells (Steinkasserer et al., 1995; Braaten et al., 1996b; Sherry et al., 1998; Saphire et al., 1999) or in the assembly of HIV-1 virions (Agresta and Carter, 1997; Streblow et al., 1998; Bristow et al., 1999). These studies relied largely on the use of mutations in gag or competitive inhibitors such as cyclosporin to block the Gag–cyclo philin interaction; however, neither of these experimental conditions abrogates the interaction completely and both potentially can cause pleiotropic effects. A third confounding issue is the number and abundance of cyclophilins in mammalian cells; at present there are 15 known human cyclophilins (Table I) and nearly all that have been tested are capable of binding HIV-1 Gag (Luban et al., 1993; Franke et al., 1994). Thus, it has not been possible to determine conclusively which cyclophilin family members, if any, promote HIV-1 replication. As a means to address these issues, we produced PPIA−/− Jurkat T-cell lines by homologous recombination. These cell lines have enabled us formally to demonstrate that CypA is required for wild-type replication kinetics of HIV-1 and, more specifically, for the infectivity of HIV-1 virions. We also present data indicating that, with regard to replication of HIV-1, none of the 14 other known cyclophilins substitutes functionally for CypA in PPIA−/− Jurkat cells. Results Gene targeting of PPIA by homologous recombination A cell line lacking PPIA expression would be an ideal reagent with which to study the functional role of CypA for HIV-1. Initially, we screened 10 cell lines and 40 primary tumors by northern blotting for the absence of PPIA expression; none was found (data not shown). We therefore set out to produce a PPIA−/− cell line. Production of PPIA−/− cells proceeded first by obtaining genomic clones of the human PPIA locus, which was complicated by the fact that multiple reverse transcribed PPIA pseudogenes are present in the genome (Haendler and Hofer, 1990). Two PCR primer sets, designed to amplify distinct regions of the functional PPIA genomic locus, were thus used to screen a human foreskin fibroblast P1 phage library. Three P1 clones were obtained, one of which was used for subsequent cloning and for fluorescence in situ hybridization (FISH) on metaphase spreads of two human CD4+ T-cell lines to determine the PPIA copy number (Braaten et al., 1996d). The Jurkat T-cell line was determined to be diploid for PPIA, and the H9 T-cell line tetraploid (data not shown). To produce PPIA−/− Jurkat cells, then, two consecutive rounds of gene targeting would be required. The promoter and all but the last exon of both PPIA alleles (∼5 kb of contiguous genomic DNA) in Jurkat cells was deleted by homologous recombination using two targeting plasmids encoding different selectable markers (Figure 1A). Five PPIA+/− and two PPIA−/− clones were produced at a targeting efficiency of ∼1 in 350 drug-resistant cultures. The recombinant clones were screened by Southern blot hybridization with a PPIA locus-specific probe (Figure 1B) and by western blotting total cell lysates probed with a polyclonal antibody raised against CypA (Figure 1C). These experiments demonstrated that homologous recombination had occurred specifically at the functional PPIA gene and that CypA-null cell lines had been produced. Figure 1.Gene targeting of the PPIA locus by homologous recombination. (A) Strategy for deleting all but the fifth exon of both copies of PPIA in Jurkat T cells by two consecutive rounds of gene targeting. Linearized targeting plasmids are shown above and below a schematic of the genomic locus. ‘Neo’, neomycin (G418) resistance gene; ‘Hygro’, hygromycin resistance gene; S, SacI; X, XbaI. (B) Southern blot hybridization of PPIA+/+, PPIA+/− and PPIA−/− cell lines; total cellular DNAs were digested with both SacI and XbaI and probed with the 5′ probe (shown in A). (C) Western blot of total cell lysates from PPIA+/+, PPIA+/− and PPIA−/− cell lines, probed with polyclonal anti-actin and anti-CypA antibodies. Download figure Download PowerPoint Initially, the growth rate of PPIA+/− and PPIA−/− cell lines was compared with that of PPIA+/+ cells by measuring incorporation of [3H]thymidine; no differences were observed (data not shown). The cell lines were also compared for cell surface expression of CD4 and CXCR4, which are required for both attachment and entry of HIV-1 into Jurkat T cells; again, no significant differences were observed between the cell lines in these assays (data not shown). Decreased replication of HIV-1 in PPIA−/− Jurkat T cells We next compared the PPIA+/+, PPIA+/− and PPIA−/− cell lines for replication of HIV-1NL4-3 (Figure 2A). In PPIA+/+ and PPIA+/− cells, replication of HIV-1NL4-3 was the same, with the onset of exponential kinetics by day 3 and the peak of virus production at day 9; since the relative amount of CypA was similar in the two cell lines (Figure 1C), we had expected the kinetic profiles to be the same. In contrast to PPIA+/+ and PPIA+/− cells, in PPIA−/− cells the onset of exponential growth of HIV-1NL4-3 was significantly delayed, by between 4 and 5 days (Figure 2A). Figure 2.HIV-1 replication is decreased in PPIA−/− Jurkat T cells. (A) Replication of HIV-1NL4-3 in PPIA+/+ (filled circles), PPIA+/− (shaded triangles) and PPIA−/− (open squares) cell lines, demonstrating delayed kinetics in PPIA−/− cells. (B) Replication of HIV-1Eli in PPIA+/+ (filled circles) and PPIA−/− (open squares) cell lines, demonstrating an even greater defect in replication than HIV-1NL4-3. Download figure Download PowerPoint To determine whether decreased HIV-1 replication in PPIA−/− cells was peculiar to the laboratory-adapted viral strain HIV-1NL4-3, we tested HIV-1Eli, an isolate that was cloned directly from peripheral blood mononuclear cells of an infected individual (Peden et al., 1991). Replication of HIV-1Eli in PPIA−/− cells was, if anything, even more decreased than that of HIV-1NL4-3 (Figure 2B). The observation that reverse transcriptase activity eventually accumulated to wild-type level in the medium of PPIA−/− cells infected with HIV-1NL4-3 could be explained by the appearance of a viral clone bearing a gain-of-function mutation resulting in replication that is independent of CypA. Such ‘CypA-independent’ mutants of HIV-1 have in fact been selected by serial passage of HIV-1 in the presence of a competitive inhibitor of the Gag–cyclophilin interaction (Aberham et al., 1996; Braaten et al., 1996a). To test this possibility, samples of virus taken from the peak of infection of PPIA+/+ and PPIA−/− cells (days 9 and 14, respectively) with HIV-1NL4-3 were used to infect fresh PPIA+/+ and PPIA−/− cells; the replication kinetics in the re-infection were the same as in the initial experiment (data not shown). In addition, RT–PCR sequencing of the region of gag that encodes residues required for binding to (Gamble et al., 1996) and which determines the functional dependence on (Aberham et al., 1996; Braaten et al., 1996a) CypA was performed on virion RNA isolated from the peak of infection. No mutations were observed (data not shown). Thus, neither re-initiated infections nor the results of RT–PCR sequencing indicated that virus had been produced with a gain-of-function mutation. Replication of HIV-1NL4-3/A224E and HIV-2/SIVSM is not defective in PPIA−/− cells A possible explanation for the decreased replication of HIV-1 in the PPIA−/− Jurkat clones is that the cells are non-permissive for replication of any virus, irrespective of the viruses' functional reliance on CypA. To rule out this possibility, we tested PPIA−/− cells for replication of HIV-1NL4-3/A224E, one of the CypA-independent HIV-1 mutants that was selected by serial passage of virus in the presence of an analog of cyclosporin (Aberham et al., 1996; Braaten et al., 1996a). HIV-1NL4-3/A224E replicated very similarly in both PPIA−/− and PPIA+/+ cells (Figure 3A). We also tested PPIA−/− cells for replication of HIV-277618, HIV-2GB122, HIV-27312A and SIVSMpbj1.9 (Dewhurst et al., 1990; Owen et al., 1998), viruses related to HIV-1 but that have no known functional interaction with cyclophilins (Franke et al., 1994; Thali et al., 1994; Braaten et al., 1996c). All of the HIV-2/SIVSM viruses replicated the same in PPIA−/− and PPIA+/+ cells (Figure 3B and C; data not shown). These data demonstrating that the PPIA−/− Jurkat clones are fully permissive for the replication of viruses closely related to wild-type HIV-1 are consistent with the interpretation that the reduced replication seen for HIV-1 is a specific consequence of the engineered mutation. Figure 3.The decreased replication phenotype in PPIA−/− Jurkat T cells is specific to wild-type HIV-1. Replication of HIV-1NL4-3/A224E (A), HIV-2GB122 (B), HIV-277618 (C) or HIV-1NL4-3/P222A (D) is nearly the same in PPIA+/+ (filled circles) and PPIA−/− cells (open squares), indicating that replication of these viruses is independent of CypA. Download figure Download PowerPoint Replication of HIV-1 in PPIA−/− cells is not inhibited further by conditions that abrogate the Gag–cyclophilin interaction Given our finding that CypA regulates but is not essential for replication of HIV-1NL4-3, and given that many cyclophilin family members bind to Gag (Table I) (Luban et al., 1993; Franke et al., 1994), it was possible that cyclophilins other than CypA might promote HIV-1 replication in the PPIA−/− Jurkat cells. Cyclosporin, an immunosuppressive compound with high affinity for the hydrophobic pocket of most cyclophilins, competitively inhibits HIV-1 Gag binding to cyclophilin family members (Luban et al., 1993; Franke et al., 1994; Rosenwirth et al., 1994; Thali et al., 1994; Steinkasserer et al., 1995; Braaten et al., 1996b, 1997; Franke and Luban, 1996), thereby effectively inhibiting HIV-1 replication. Thus, to determine whether HIV-1 replication in PPIA−/− cells is dependent upon other cyclophilin family members, we compared PPIA−/− and PPIA+/+ cells for replication of HIV-1NL4-3 in the presence of methyl-Ile4-cyclosporin, an even more effective inhibitor of HIV-1 replication than the parent compound cyclosporin (Rosenwirth et al., 1994; Thali et al., 1994; Franke and Luban, 1996). In PPIA+/+ cells, 2.5 μM methyl-Ile4-cyclosporin inhibited replication of HIV-1NL4-3 between 100- and 1000-fold compared with no drug (Figure 4A); under the same conditions, replication of HIV-2 was unaffected by the drug (Figure 4B), consistent with what has been published previously (Thali et al., 1994). In contrast, in PPIA−/− cells, 2.5 μM methyl-Ile4-cyclosporin inhibited replication of HIV-1NL4-3 <10-fold compared with no drug (Figure 4C) and, as in PPIA+/+ cells, replication of HIV-2 was unaffected by the drug (Figure 4D). Importantly, the magnitude and kinetics of HIV-1 replication in the presence of 2.5 μM methyl-Ile4-cyclosporin were very similar in PPIA+/+ and PPIA−/− cells (compare Figure 4A and C). Figure 4.HIV-1 replication in PPIA−/− cells is insensitive to a competitive inhibitor of the Gag–cyclophilin interaction. Effect of methyl-Ile4-cyclosporin on replication of HIV-1NL4-3 (A) and HIV-277618 (B) in PPIA+/+ cells. Effect of methyl-Ile4-cyclosporin on replication of HIV-1NL4-3 (C) and HIV-277618 (D) in PPIA−/− cells. No drug (filled symbols), 1.25 μM (shaded symbols) and 2.5 μM (open symbols) methyl-Ile4-cyclosporin. Download figure Download PowerPoint In addition to abrogation by competitive inhibitor, interaction between HIV-1 Gag and cyclophilin family members is significantly diminished by particular gag mutations, e.g. G221A and P222A (Franke et al., 1994; Braaten et al., 1996b, 1997). Viruses bearing these mutations, HIV-1NL4-3/G221A and HIV-1NL4-3/P222A, would thus be predicted to replicate poorly, but any replication capacity that they exhibited would be cyclophilin independent. If these viruses are independent of cyclophilins, their replication should be the same in PPIA+/+ and PPIA−/− cells. These predictions were confirmed, as replication of HIV-1NL4-3/G221A and HIV-1NL4-3/P222A in PPIA+/+ cells was similar to that of wild-type HIV-1NL4-3 in PPIA−/− cells, but abrogated no further in PPIA−/− cells (Figure 3D and data not shown). Taken together, the results with methyl-Ile4-cyclosporin and with the gag mutant viruses are consistent with the conclusion that no cyclophilins other than CypA promote HIV-1 replication. Rescue of HIV-1 replication kinetics in PPIA−/− cells by re-introduction of CypA If the absence of CypA expression is the sole cause of the defective replication of HIV-1 in PPIA−/− Jurkat cells, re-expression of CypA in the cells should restore the replication kinetics to wild type. To test this, PPIA−/− cells were transfected with a CypA expression plasmid, and cell lines expressing CypA were derived by limit-dilution cloning; western blot analysis confirmed the presence of CypA in individual clones (Figure 5A). The clones were screened for replication of HIV-2 to eliminate those with general defects unrelated to CypA; three clones not defective in this assay were then tested for replication of HIV-1. Compared with the parent PPIA−/− cells, clones re-expressing CypA demonstrated faster replication kinetics of HIV-1NL4-3 (Figure 5B); similar results were obtained with HIV-1Eli as well as with another set of clones re-expressing CypA that had been generated with the second of the two PPIA−/− cell lines (data not shown). Importantly, the degree to which the replication kinetics was restored to wild type correlated with the level of CypA expression in the individual clones. Figure 5.HIV-1 replication kinetics depend on the level of CypA expression. (A) Western blot of total cell lysates showing the relative level of CypA in the cell lines. (B) Replication of HIV-1NL4-3 in PPIA+/+ cells (filled circles), in three cell lines derived from PPIA−/− cells stably re-expressing CypA (shaded symbols) and in PPIA−/− cells (open squares). Higher CypA expression levels correlate with more rapid replication kinetics. Download figure Download PowerPoint HIV-1 virions produced by PPIA−/− cells are less infectious than virions from PPIA+/+ cells Previous work has demonstrated that conditions which block CypA binding to Gag, such as treating infected cells with cyclosporin or creating specific point mutations in gag, render virions defective at an early step of the viral life cycle before the start of reverse transcription (Braaten et al., 1996b). These studies found no defects in the RNA or protein content, or in endogenous reverse transcriptase activity, of CypA-deficient virions (Braaten et al., 1996b; Grattinger et al., 1999). We performed metabolic labeling and pulse–chase analysis of HIV-1 proteins produced by PPIA+/+ and PPIA−/− cells and observed no significant differences between the cell lines in terms of Gag translation efficiency, protein stability or polyprotein processing (data not shown). In addition, we analyzed, by western blotting, protein constituents of virions (e.g. capsid, matrix, Nef and gp160/120) produced by PPIA−/− cells and, except for the absence of CypA, detected no differences from virions produced by PPIA+/+ cells. We next measured the relative infectivity of virions produced by PPIA−/− cells in a single round of infection. Wild-type and ‘CypA-null’ virions were harvested from cultures of PPIA+/+ cells and two PPIA−/− clones after 6 and 10 days, respectively, of infection with wild-type HIV-1NL4-3. Similarly, a stock of HIV-1NL4-3/G221A was produced from PPIA+/+ cells; this virus bears one of the gag mutations that significantly reduces binding to and virion incorporation of CypA. All virions produced from the PPIA+/+ and PPIA−/− infections were purified and normalized by exogenous reverse transcriptase assay. GHOST cells (Morner et al., 1999), a CD4+/CCR5+/CXCR4+ human osteosarcoma cell line containing an LTR-gfp reporter gene, were infected with the virus stocks and then processed either for the presence of reverse transcriptase products by real-time PCR (12 h post-infection) or for expression of green fluorescent protein (GFP) by flow cytometry (48 h post-infection). Both full-length viral cDNA (indicative of completion of reverse transcription) and 2-LTR circles (indicative of nuclear translocation of the pre-integration complex) were measured by real-time PCR. As a percentage of product measured for wild-type HIV-1NL4-3 produced in PPIA+/+ cells, both full-length cDNA and 2-LTR circles for wild-type HIV-1NL4-3 produced in the two PPIA−/− cell lines were significantly reduced (Figure 6A). The apparent difference in magnitude seen in virus produced by the two PPIA−/− clones appears not to be a fixed property of the cell lines, as experimental variation in this range was observed. As expected from previous work (Braaten et al., 1996b), full-length cDNA and 2-LTR circles were reduced for HIV-1NL4-3/G221A as well (Figure 6A). Figure 6.Virions produced from PPIA−/− cells are less infectious than those produced from PPIA+/+ cells. Virus stocks (wild-type HIV-1NL4-3 and HIV-1NL4-3/G221A) were produced in either PPIA+/+ or PPIA−/− cells (as indicated) and used to infect equal numbers of GHOST cells. (A) Low molecular weight DNA was isolated 12 h post-infection. Real-time PCR was performed to quantitate full-length viral cDNA and 2-LTR circles. The relative levels of products (percentage of wild-type signal) are shown. (B) The percentage of GFP+ cells (of total cells) was determined by flow cytometry 48 h post-infection. Download figure Download PowerPoint The GHOST cells were also screened by flow cytometry for expression of GFP, which, in these cells, is indicative of HIV-1 integration. Relative to the 40% GFP-positive cells for wild-type HIV-1NL4-3 produced in PPIA+/+ cells (Figure 6B), the number of GFP-positive cells for wild-type HIV-1NL4-3 produced in the two PPIA−/− cell lines was between 4- and 10-fold lower (the same difference as that for 2-LTR circles), similar to the percentage of GFP-positive cells for HIV-1NL4-3/G221A produced in PPIA+/+ cells. These results indicate that virions produced by PPIA−/− cells are defective at some early step of the virus life cycle before, or concurrent with, reverse transcription. Discussion We have produced PPIA−/− cell lines by homologous recombination in the widely used Jurkat T-cell line. As reagents for studying the biology of HIV-1, these cells have enabled us to address several issues concerning the role of cyclophilins in the replication of HIV-1. CypA is required for wild-type HIV-1 replication kinetics Since we were unable to identify CypA-null conditions with which to study HIV-1 replication, demonstration of the functional relevance of CypA necessitated the generation of PPIA−/− cells by homologous recombination in a human T-cell line. Our targeting efficiency was low, but within the range previously reported for somatic cell targeting with non-isogenic DNA (Sedivy et al., 1999). Once the PPIA−/− cells were generated, HIV-1 replication was then rescued with a CypA expression plasmid; this ‘rescue experiment’, in which CypA was formally proven to be a determinant of HIV-1 replication kinetics, is analagous in outline to studies proving that CD4 and chemokine receptors are essential viral entry factors (Maddon et al., 1986; Feng et al., 1996). Other than the host N-myristoyltransferase, a protein that catalyzes a co-translational modification required for Gag polyprotein targeting to the inner face of the plasma membrane (Göttlinger et al., 1989; Bryant and Ratner, 1990), CypA is perhaps the first Gag-associated host factor that has been clearly demonstrated to play a significant role in HIV-1 replication. Replication of primary isolate HIV-1Eli was found to be even more decreased in PPIA−/− cells than was replication of the laboratory-adapted strain HIV-1NL4-3. This suggests the intriguing possibility that primary isolates of HIV-1 as a group are more functionally dependent on CypA than are laboratory-adapted strains, which in turn may be indicative of a greater importance of CypA for HIV-1 replication kinetics in vivo than in vitro. This would not be unprecedented since a number of HIV-1 factors appear more important in vivo than in tissue culture. As but one example, vpr is s