Cyclophilin A interacts with diverse lentiviral capsids
© Lin and Emerman. 2006
Received: 27 July 2006
Accepted: 12 October 2006
Published: 12 October 2006
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© Lin and Emerman. 2006
Received: 27 July 2006
Accepted: 12 October 2006
Published: 12 October 2006
The capsid (CA) protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA). This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay.
We find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA.
These results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection.
Cyclophilin A (CypA) is a highly conserved peptidyl prolyl isomerase (PPIA) that is incorporated into HIV-1 virions and plays a yet undefined role in the early stages of viral replication in some cell types [1–3]. CypA interacts with HIV-1 by virtue of a direct binding between residues in a loop between the fourth and fifth alpha-helices of the capsid (CA) protein of HIV-1 and the active site of CypA [4–6]. Cyclosporin A (CsA), an immunosuppressive drug, binds to the same region of binding groove of CypA and disrupts the CypA/CA interaction which leads to an attenuation of wild type HIV-1 infectivity by 2–5 fold in T cells [7–9]. Although CypA can bind to viral CA in the producer cell during viral assembly, it is CypA in the newly infected target cells that is important for infectivity rather than the CypA that is present in the producer cell [8–11]
Tripartite motif 5 isoform alpha (Trim5α) proteins also bind to retroviral CA early after viral entry, and can have a negative effect on the viral lifecycle by accelerating the viral core uncoating or CA degradation [12–15]. Trim5α contains a C-terminal B30.2 domain that recognizes retroviral CA and restricts viral replication in a species-specific manner [12, 16]. The resistance of cells from the owl monkey (Aotus trivirgatus) to HIV-1 infection is due to the presence of a natural fusion protein in this species, called TrimCyp, in which the B30.2 recognition domain of Trim5α was replaced by the CypA gene [17, 18]. CypA is important for the activities of some Trim5α alleles [19, 20].
The CypA/CA interaction was initially identified by two-hybrid analysis in yeast [1, 5]. In those studies, it was reported that only HIV-1 and the closely related SIVcpz encoded Gag proteins (which are cleaved by viral protease to generate CA during maturation) that could interact with CypA, while the Gag proteins from HIV-2, SIVmac, SIVs from African green monkeys-Sabaeus (SIVagmSAB), feline immunodeficiency viruses (FIV), and Mason-Pfizer monkey viruses (MPMV) failed to interact with CypA in this assay . Furthermore, incorporation of CypA into virions was detected in HIV-1 and SIVcpz, but not in HIV-2, SIVmac, SIVagm-Grivet (SIVagmGRI), and murine leukemia viruses (MLV) . However, here we show that the CypA/CA interaction is not unique to HIV-1/SIVcpz. By both genetic and biochemical experiments we show that FIV can bind CypA and its replication is affected by this interaction to the same extent as HIV-1. Moreover, we have identified an amino acid that is essential for FIV CA interaction with CypA that is in a nearly identical context to an amino acid necessary for the interaction of HIV-1 CA with CypA. Finally, we show that SIVagm-Tantalus (SIVagmTAN) is restricted by TrimCyp, strongly suggesting that this viral CA also interacts with CypA. While these studies were in progress, two other groups also provided evidence that FIV and SIVagm are both susceptible to the TrimCyp restriction in a CsA sensitive manner [21, 22]. Together, these results demonstrate that lentiviral interactions with CypA are more conserved than had been previously assumed and suggest that CypA/CA interactions play an evolutionarily conserved role in the life cycle of many lentiviruses.
We began this study by looking at the pattern of restriction effect of Trim5α isolated from diverse primate species with a panel of different retroviruses. Remarkably, we found that HIV-1 and FIV had an identical pattern of restriction in that both were strongly restricted by rhesus Trim5α, had slight sensitivity to human and Tamarin Trim5α, and were resistant to Titi Trim5α (data not shown). Similar data was also reported by another group . Because Trim5α restriction, like the CypA interaction, is dictated by CA, this led us to examine if FIV is also similar to HIV-1 in its interaction with CypA.
These constructs were then co-transfected to 293T cells with VSV-G and GFP reporter plasmids to generate VSV-G pseudotyped FIVs that were used to infect CRFK or CRFK-TrimCyp cells. The infectivity of each virus was normalized to give an amount of virus that resulted in about the same number of infected CRFK cells. Similar to wild-type FIV, the mutations P84A, P85A, P88A, P92A, and R89A are also restricted by TrimCyp (Fig. 3B). Moreover, in each case, the restriction by TrimCyp is abrogated by the addition of CsA. These data indicate that mutating each of these residues did not destroy a functional interaction between CypA and the viral CA. However, the P90A mutant is not sensitive to the TrimCyp restriction and the infectivity is not affected by the addition of CsA. This indicates that amino acid P90 is critical for a functional interaction between FIV CA and CypA (Fig. 3B).
In addition to the TrimCyp assay, we also applied the GST-CypA pull down assay to test the interaction of CypA and FIV mutants. The FIV wild type and mutant virions were incubated with the GST-CypA and the interactions were analyzed by Western Blotting (Fig. 3C). Wild-type FIV and other mutants are sensitive to the TrimCyp restriction and are pulled down by the GST-CypA. On the other hand, the P90A mutant, which is not sensitive to the TrimCyp restriction, is also not pulled down by GST-CypA (Fig. 3C). These data demonstrate that amino acid P90 is a critical target for CypA binding to FIV CA.
The CypA/CA interactions had originally been described only for HIV-1 and SIVcpz . Here, we show that this phenotype is more widespread among lentiviruses because both FIV and SIVagmTAN also interact with CypA. Moreover, we identified an amino acid in FIV CA that is critical for CypA binding that is in a similar position in CA of HIV-1. Finally, we show that the CypA/CA interaction is functionally significant for FIV replication.
It is not clear why a functional assay (Fig. 1) and a direct binding assay (Fig. 2) detected the CypA/CA interaction in FIV, whereas previous reports that looked for interactions with a yeast-two hybrid assay or by expression of recombinant Gag and CypA in E. Coli did not . It is possible that the formation of mature viral core may be more important for CypA recognition of FIV, or that subtle folding problems with FIV Gag was expressed in yeast and E. Coli might have prevented binding. Nonetheless, the two assays used here (TrimCyp restriction and GST-CypA pull down) were conducted in mammalian cells and thus more closely mimic the structure of CA found in natural targets cells.
The finding that FIV and SIVagmTAN also bind host CypA demonstrates that the CypA/CA interaction is a more wide spread phenotype among lentiviruses than just the HIV-1/SIVcpz lineage. At this point it is not possible to determine if this property arose independently in three separate lentivirus lineages, or if an ancestral retrovirus was able to bind CypA and then other lentiviruses (for example, SIVmac) each lost this ability. Although the latter possibility is more parsimonious, it will be important to test a much broader range of lentiviruses for CypA binding. It is also possible that the ability of a lentivirus to bind CypA is evolutionarily dynamic and changes upon adaptation to new hosts.
The exact role of CypA/CA interaction in HIV-1 life cycle is still not clear. However, several groups have suggested that CypA/CA interaction mediates the lentiviral susceptibility to Trim5α restriction [17–19, 26, 27]. The discovery of TrimCyp links the CypA/CA interaction with the post-entry restriction mechanism [17, 18]. The potent restriction against HIV-1 infection requires the recognition of HIV-1 CA by the C-terminal CypA domain of TrimCyp. In addition, the CypA/CA interaction in HIV-1 was shown to correlate with viral sensitivity to rhesus and African green monkey Trim5α restriction [19, 26, 27]. Blocking CypA/CA interaction by CsA or down-regulating endogenous CypA by RNAi rescues HIV-1 replication from rhesus and African green monkey Trim5α restriction, suggesting the rhesus and African green monkey Trim5α restrict HIV-1 via the CypA-binding pathway. Human Trim5α, on the other hand, weakly restricts HIV-1 via a pathway that is independent of CypA binding [26–28]. It is not clear whether the Trim5α restriction is mediated by direct binding to CypA or requires an unidentified adaptor protein. Another explanation is that CypA/CA interaction changes the CA conformation which provides access for rhesus and African green monkey but not human Trim5α. We report here that SIVagmTAN interacts with CypA, but it has a different recognition pattern by different Trim5α proteins when compared to HIV-1 . SIVagmTAN is resistant to the African green monkey Trim5α but susceptible to rhesus Trim5α restriction, whereas HIV-1 is restricted by rhesus and African green monkey Trim5α via the CypA-binding pathway. The SIVagmTAN susceptibility to rhesus Trim5α is probably determined by the patch on the B30.2 domain and the CypA/CA has little to do with the restriction .
CypA binding is necessary for Trim5α restriction of HIV-1 in rhesus cells, but not for that in human cells. It has been hypothesized that CypA binding protects HIV from an unknown restriction factor in humans. If so, then this hypothesis would have to be extended to account for the fact that diverse lentiviruses also bind CypA. Our report here will provide a parallel line to investigate the role that CypA plays in lentiviral life cycle.
293T and CRFK (Crandall Feline Kidney) cells were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum (FBS). Jurkat T cells were grown in RPMI with 10% FBS. 293T cells were used for generation of vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviruses and for production of GST-CypA fusion proteins. CRFK cells expressing TrimCyp protein from different species were generated as described previously .
2.5 × 105 cells/ml of 293T cells were plated in 2 ml/well in a 6-well plate 16 hours prior to transfection. The VSV-G pseudotyped HIV-1 WT-GFP, HIV-1 G89V-GFP mutant, HIV-1 WT-Luc, SIVmac-Luc, and SIVagmTAN-Luc were generated as described previously [30, 31]. Plasimds pFGinSin and pFP93  used to generate FIV vectors and virions were gifts of Eric Poeschla (The Mayo Clinic College of Medicine, Rochester). For generating VSV-G pseudotyped FIV WT-GFP, 0.75 μg of enhanced green fluorescent protein transfer vector pFGinSin was co-transfected with 0.4 μg of pL-VSV-G, 0.1 μg of pCMV-tat, and 0.75 μg of pFP93 to 293T cells by FuGene 6 transient transfection. For generating the VSV-G pseudotyped mutant FIVs, the same method was applied but replacing pFP93 with pFP93 proviral DNA with mutations of P84A, P85A, P88A, P90A, P92A, and R89A. The mutations were generated with the QuickChange Site-directed mutagenesis kit by following the instruction of manufacturer's protocol (Stratagene). Culture media were collected on 48 hours, 72 hours, and 96 hours after transfection, and were passed through a 0.2 μm filter (Nalgene) for harvesting the viruses. FIV P84A-GFP and FIV P90A-GFP were ultra-centrifuged in an SW28 rotor at 23000 rpm for 1.5 hours for concentrating viruses 10-fold and 100-fold, respectively. All viruses were aliquoted in 1.5 ml micro-tubes and frozen at -80°C until use. The FIV viral titer was tested by infecting CRFK cells with different volumes of the original viral stocks; the amount of virus causing 20% GFP+ (20% infected cells) was defined as 0.2 CRFK infectious equivalents.
CRFK or CRFK cells expressing TrimCyp proteins were plated 16 hours before infection at a density of 8 × 104 cells/ml on a 12-well (1 ml/well) or a 24-well (0.5 ml/well) plate. Jurkat T cells for the CypA-dependency experiment were prepared before infection at a density of 2.5 × 105 cells/ml on a 12-well (1 ml/well) plate. The VSV-G pseudotyped viruses were added to target cells in the presence of 20 μg/ml DEAE/Dextran, and spinoculated at 1200 relative centrifugal force (rcf) at room temperature for 2 hours . The infected cells were placed at 37°C incubator for 48 hours until the analysis of the infectivity tested with flow cytometry (for viruses expressing GFP) or luminometer (for viruses expressing luciferase). The HIV-1 WT-GFP, HIV-1 G89V-GFP, and all FIVs (WT and mutants) infected cells was harvested and fixed with 1% paraformaldehyde for 1 hour at 4°C. The fixed cells were washed with PBS and re-suspended with 300 μl of PBS supplemented with 5% calf serum, and subjected to FACScan (Becton Dickinson) for the analysis of the infectivity. The HIV-1 WT-Luc, SIVmac-Luc, and SIVagmTAN-Luc infected cells were washed with PBS and lyzed with 80 μl of the cell culture lysis buffer (Promega), and 10 μl of the lysates were used for the measurement of luciferase activity (infectivity) with the Luciferase assay system (Promega) and luminometer.
The mRNA from 293T cells were extracted with RNeasy kit (Qiagen). The human CypA was cloned from the human mRNA by using the OneStep RT-PCR (Qiagen) with primer sets 5'-GGATCCACGGTTCAGGTGGTTCTGGAGGTTCAGGAGTCAACCCCACCGTGTTC-3' (forward) and 5'-CTCGAGTTATTCGAGTTGTCCACAGTCAGCAATGGT-3' (reverse). The cyclophilin cDNA was used to replace huTrim5α in the plasmid pEF/GST-huTrim5α  (a kind gift of Jeremy Luban, Columbia University, New York). To generate GST-CypA protein in human cells, 1 μg of pEF/GST-CypA or pEF/GST were transiently transfected to 293T cells with FuGene 6 (Roche). 72 hours after transfection, one 6-well plate of cells were harvested and lysed with 1.6 ml of GST buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% NP40, 0.1% SDS, and supplemented with complete protease inhibitor). The lysates were centrifuged at 13200 rpm for 1 minute, and the supernatants were harvested. To balance the glutathione-sepharose beads (BD Biosciences) for optimal binding, 200 μl of beads were centrifuged at 5000 rpm for 5 minutes and washed with 800 μl of GST buffer for 3 times. The beads were re-suspended with 200 μl of GST buffer and incubated with 800 μl of GST or GST-CypA supernatants at 4°C on a slowly rotated shaker for 1 hour. After incubation, beads were centrifuged at 5000 rpm for 5 minutes and washed three times with 800 μl of GST buffer. Viruses for the pull down assay (HIV-1 WT, FIV WT, and FIV mutants) were prepared by concentrating the same amount of virions (about 1 ml) into 200 μl of PBS. The concentrated virions then were incubated with the GST or GST-CypA bound beads at 4°C on a slowly rotated shaker for 1 hour. Beads were centrifuged and washed with 800 μl of GST buffer for 3 times. The beads then were re-suspended with 60 μl of GST buffer and frozen at -80°C until the Western blot analysis.
Lysates from the GST-pull down assay were mixed with 20 μl of sample buffer (2% SDS, 1% 2-mercaptomethanol, 1% glycerol, 65 mM Tris-hydrochloride ). 30 μl of the mixture were loaded on SDS-10% polyacrylamide gels. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes. The membranes were blocked for 30 minutes at room temperature with 5% fat-free milk in PBS, and then incubated with a 1:1000 dilution of a mouse anti-FIV capsid monoclonal antibody (clone 2C1, from NIH AIDS Research and Reference Reagent program) overnight at 4°C. The membranes were washed 10 minutes for 3 times in PBS containing 0.1% Tween 20 and then incubated with a 1:10000 dilution of a horseradish peroxidase-conjugated anti-mouse monoclonal antibody (Santa Cruz Biotechnology) for 30 minutes at room temperature. The membranes were washed 3 times for 30 min, and the bound antibody was detected with the ECL detection system (Amersham).
Lentiviral Gag sequences were from GenBank. The accession numbers are: HIV-1 (M19921), HIV-2 (X05291), SIVcpz (AF115393), SIVmac (AY588946), SIVsm (AF334679), SIVagmTAN (U58991), and FIV (M25381). The CA sequences from these lentiviruses were aligned by the ClustalX software [35, 36]. The computer modeling of N-terminal CA structure from SIVmac and SIVagmTAN was performed with the SWISS-MODEL server [37, 38, 39] by using the CA crystal structure of HIV-1 which bound to human CypA (PDB:1AK4) . The predicted CA structures were viewed and displayed by DeepView (Swiss Pdb-Viewer).
We thank two rotation students, Olivier Humbert and Jon Reed, for initial experiments in this project to test the sensitivity of FIV to Trim5α and to TrimCyp, the FHCRC Flow Cytometry lab, Jeremy Luban and the NIH AIDS Reagent Repository for the TrimCyp construct, Eric Poeschla for the FIV GFP vector and packaging construct, and Eric Poeschila, Semih Tareen, Masahiro Yamashita, and Yegor Voronin for comments on the manuscript.
This work was supported by NIH grant R37 AI30937 to M.E.
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