The V86M mutation in HIV-1 capsid confers resistance to TRIM5α by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import
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HIV-1 is inhibited early after entry into cells expressing some simian orthologues of the tripartite motif protein family member TRIM5α. Mutants of the human orthologue (TRIM5αhu) can also provide protection against HIV-1. The host protein cyclophilin A (CypA) binds incoming HIV-1 capsid (CA) proteins and enhances early stages of HIV-1 replication by unknown mechanisms. On the other hand, the CA-CypA interaction is known to increase HIV-1 susceptibility to restriction by TRIM5α. Previously, the mutation V86M in the CypA-binding loop of HIV-1 CA was found to be selected upon serial passaging of HIV-1 in cells expressing Rhesus macaque TRIM5α (TRIM5αrh). The objectives of this study were (i) to analyze whether V86M CA allows HIV-1 to escape mutants of TRIM5αhu, and (ii) to characterize the role of CypA in the resistance to TRIM5α conferred by V86M.
We find that in single-cycle HIV-1 vector transduction experiments, V86M confers partial resistance against R332G-R335G TRIM5αhu and other TRIM5αhu variable 1 region mutants previously isolated in mutagenic screens. However, V86M HIV-1 does not seem to be resistant to R332G-R335G TRIM5αhu in a spreading infection context. Strikingly, restriction of V86M HIV-1 vectors by TRIM5αhu mutants is mostly insensitive to the presence of CypA in infected cells. NMR experiments reveal that V86M alters CypA interactions with, and isomerisation of CA. On the other hand, V86M does not affect the CypA-mediated enhancement of HIV-1 replication in permissive human cells. Finally, qPCR experiments show that V86M increases HIV-1 transport to the nucleus of cells expressing restrictive TRIM5α.
Our study shows that V86M de-couples the two functions associated with CA-CypA binding, i.e. the enhancement of restriction by TRIM5α and the enhancement of HIV-1 replication in permissive human cells. V86M enhances the early stages of HIV-1 replication in restrictive cells by improving nuclear import. In summary, our data suggest that HIV-1 escapes restriction by TRIM5α through the selective disruption of CypA-dependent, TRIM5α-mediated inhibition of nuclear import. However, V86M does not seem to relieve restriction of a spreading HIV-1 infection by TRIM5αhu mutants, underscoring context-specific restriction mechanisms.
KeywordsNuclear Transport TE671 Cell V86M Mutation CRFK Cell CypA Knockdown
TRIM5α was isolated as a retroviral restriction factor in 2004  and acts within the post-entry, pre-integration window [2, 3]. The viral molecular target of TRIM5α is the correctly matured N-terminal domain of capsid (CA) proteins forming the outer surface of the retroviral core [2, 4, 5, 6, 7, 8]. A direct interaction between the two proteins, each present as high molecular weight multimers, occurs shortly after entry and is required for downstream inhibition of viral replication [8, 9, 10, 11, 12]. The mechanism of TRIM5α-mediated restriction can be broken down to discrete events, some of them inter-dependent: (i) virus entrapment into TRIM5α cytoplasmic bodies , (ii) decreased stability of the virus core [8, 14, 15, 16], (iii) targeting to a proteasome-dependent degradation pathway [17, 18, 19, 20], and (iv) inhibition of nuclear transport [17, 21, 22].
CypA, a host peptidyl-prolyl cis/trans isomerase that is ubiquitously expressed in tissues, is known to play roles in both HIV-1 infection of human cells and in HIV-1 restriction by TRIM5α in monkey cells. In dividing permissive human cells, CypA enhances HIV-1 infectivity by regulating the disassembly of its core [23, 24, 25] independently of TRIM5α [26, 27]. On the other hand, restriction of HIV-1 by simian TRIM5α orthologues is enhanced by CypA, and inhibition of CypA expression or of its activity partially rescues infectivity in restrictive conditions [21, 26, 28, 29, 30]. CypA binds to an exposed proline-rich loop on the viral CA [31, 32] and catalyzes the isomerisation of the peptide bond G89-P90 [33, 34]. The mutation V86M in the CypA-binding loop of HIV-1 CA has been identified as conferring partial resistance to TRIM5αrh. The mechanism of HIV-1 resistance to TRIM5αrh conferred by V86M CA was not addressed in this study. However, it was established that this mutation in the CypA-binding loop did not disrupt CA-CypA interactions in vitro or in cell cultures .
We and others have proposed that point mutations in the variable region 1 (v1) of TRIM5αhu could confer HIV-1 restriction capability [36, 37, 38, 39, 40, 41]. These mutations were discovered by mapping of HIV-1 restriction determinants in non-human TRIM5α orthologues [36, 39, 40, 41] or through the use of random mutagenesis-based screens [38, 42]. Such antiviral genes are promising candidates for gene therapy applications, owing to a few key characteristics: (i) They block replication early after virus entry and before integration can occur; (ii) They are human-like and thus probably nonimmunogenic; (iii) They inhibit HIV-1 by a well-established, natural mechanism with little side effect expected. However, it is currently unknown whether HIV-1 can acquire resistance to TRIM5αhu mutants. Here we investigate the extent and mechanism of resistance of the HIV-1 CA mutant V86M to R332G-R335G TRIM5αhu and other mutants of the v1 domain. Our data show that this mutation affects physical and functional interactions with CypA in order to decrease HIV-1 sensitivity to TRIM5α while retaining replication-enhancement functions also conferred by CypA binding.
CA-V86M HIV-1 is partially resistant to restriction by TRIM5αhumutant R332G-R335G
Restriction of V86M CA HIV-1 by R332G-R335G TRIM5αhuis independent of cyclophilin A
CsA concentration-dependent assays
Altogether, data in Figures 2 and 3 show that restriction of V86M HIV-1 replication by R332G-R335G TRIM5αhu is poorly sensitive to the presence of CypA compared to restriction of the WT control. However, replication of V86M HIV-1 in permissive human cells is as much sensitive as WT HIV-1 to the presence of CypA.
The sensitivity of HIV-1 restriction by TRIM5αhumutants to the V86M mutation correlates with its sensitivity to CsA treatment
Restriction of V86M CA HIV-1 by R332G-R335G TRIM5αhuin cat cells is insensitive to CsA
V86M CA interactions with CypA studied by NMR
Upon addition of CypA, symmetrical exchange peaks were observed for WT and V86M indicating that both capsids are a substrate for CypA and that increased cis-trans exchange occurs during the ZZ-exchange experiment. However, in the V86M mutant, at least two cis exchange peaks were observed compared to a single peak in the WT. Furthermore, the intensities and line shapes of these additional peaks were directly affected by addition of CypA and dependent upon the mixing time of the experiment (Figure 6B). This supports the hypothesis that the V86M mutation results in distinct populations of conformers for the G89-P90 bond.
Next, we determined the catalysed isomerisation rate of the G89-P90 bond in both WT and V86M capsids in the presence of CypA (Figure 6B). For WT capsid we observed a similar rate as previously published, of ~5 s-1. For the V86M capsid, the exchange rate for the major G89 peak was ~20 s-1. This suggests that the V86M mutation has both altered the conformations adopted by the CypA-binding loop and increased the rate at which it is isomerised. The presence of an additional G89 cis peak in V86M in the absence of CypA suggests that the mutant capsid may also undergo faster uncatalysed isomerisation, however we were not able to quantify this under the conditions tested.
Effects of V86M on early stages of viral replication
Effect of CypA and V86M on a spreading HIV-1 infection
HIV-1 resistance to treatment is a hallmark of pharmacological interventions against this virus. Invariably, mutations appear in the coding sequences of the proteins targeted by antiretroviral drugs, including protease, reverse transcriptase and integrase . It is expectable that genetic interventions will similarly lead to the occurrence of viral resistance. Indeed, restriction of HIV-1 by CPSF6-358, a truncated form of the RNA processing factor, cleavage and polyadenylation specific factor 6 (CPSF6), is counteracted by the mutation N74D in CA . This mutation, isolated by serial passages of HIV-1 in cells expressing CPSF6-358, abrogates direct binding of CA to CPSF6-358 . Several groups, including ours, have produced mutants of TRIM5αhu to be used as antiviral transgenes [36, 38, 39, 40, 41]. In order to predict potential HIV-1 escape from inhibition mediated by these TRIM5αhu variants, it is important to isolate those escape mutants in vitro, and to understand by which mechanism they decrease sensitivity to restriction. Our initial efforts had not led us to isolate R332G-R335G TRIM5αhu-resistant HIV-1 by serial passaging. We then decided to test whether V86M, an HIV-1 CA mutant isolated from cells expressing TRIM5αrh by the Sodroski group and shown to confer some level of protection against this orthologue, would also make HIV-1 resistant to TRIM5αhu mutants.
Our data show that V86M indeed confers some level of protection against various mutants of TRIM5αhu, at least in the context of single-cycle infections with HIV-1 vectors. We saw no protection in the context of HIV-1 spreading infections, although we tested only one HIV-1 strain (NL4-3) in one cell line (Sup-T1). Sodroski and collaborators  have isolated the V86M mutant in HeLa-CD4 cells, which we have not tested here. They observed modest levels (2-fold) of protection against TRIM5αrh in these HeLa-derived cells when infectivity was measured in single-cycle assays, and they saw an even more modest effect in canine Cf2Th cells . Altogether, V86M can confer HIV-1 protection against restriction by various TRIM5α proteins in specific replication settings. However, even in situations in which protection takes place, restriction still occurs, albeit weakened. On the basis of our data, we do not expect V86M to be highly significant in an in vivo context, although this is of course rather hazardous to predict.
In order to investigate the mechanism of CA-V86M HIV-1 resistance to R332G-R335G TRIM5αhu, we analyzed the role of CypA in the restriction. Altogether, our results show that restriction of V86M CA HIV-1 is largely insensitive to the presence of functional CypA, while the same virus is still inhibited in human cells devoid of CypA. In other words, HIV-1 is able to subtly alter its interactions with CypA in order to downregulate a mechanism of restriction while preserving other benefits associated with this interaction. Accordingly, our NMR data showed that the molecular interactions between CA and CypA were altered by the V86M mutation, as was the isomerisation reaction catalysed by CypA.
Finally, our qPCR data correlate modifications in CypA-CA interactions with effects on nuclear transport in restrictive conditions. Interestingly, experiments pre-dating the discovery of TRIM5α restriction had demonstrated that CsA treatment of Old World monkey cells increased nuclear transport of HIV-1 in these cells . More recently, Lin and Emerman similarly observed that CsA treatment of the sMAGI Rhesus macaque cell line increases HIV-1 nuclear transport more than it does enhance reverse transcription . Other recently published data also link nuclear transport and CA-CypA interactions in permissive human cells. Specifically, CypA knockdown and CsA treatment reduce HIV-1 dependency toward nucleopore components Nup153 and Nup358 for its nuclear transport [56, 57]. It should be informative to analyze whether V86M modifies the interactions between HIV-1 CA and these nucleoporins.
The V86M mutation in HIV-1 CA can confer partial resistance against restriction of HIV-1 replication by mutants of human TRIM5α. V86M abrogates CypA-dependent restriction mechanisms, resulting in an increase of HIV-1 DNA nuclear transport in restrictive conditions. However, this mutation might not confer significant resistance to the restriction of a spreading HIV-1 infection.
pMIP-TRIM5αhu and pMIP-TRIM5αrh express C-terminal FLAG-tagged versions of the corresponding proteins and have been extensively described before [28, 38, 44, 58, 59]. pMIP-TRIM5αhu with the mutation R332G-R335G has been described , and additional mutants have been described as well . pSRBl-CypA expresses a short hairpin RNA (shRNA) targeting the human CypA mRNA. Shortly, it is the previously described pSRP-CypA plasmid  in which the puromycin resistance cassette has been changed to one conferring resistance to blasticidin. The control plasmid, pSRBl-Luc, encodes a shRNA targeting luciferase . pNL-GFP, pMD-G and pCL-Eco have all been described elsewhere [21, 28, 58, 61, 62, 63]. The fully replication-competent HIV-1 clone pNL4-3  was used for propagation experiments.
Human rhabdomyosarcoma TE671 cells, human embryonic kidney 293 T cells and feline renal CRFK cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics at 37°C. Human T lymphoblast Sup-T1 cells and monocytic cells THP-1 were maintained in RPMI-1640 supplemented with 10% fetal bovine serum and antibiotics at 37°C. All cell culture reagents were from Hyclone (Thermo Scientific, Logan, UT, USA). Human PBMCs were isolated from diluted whole blood of a healthy donor by Ficoll density gradient centrifugation as previously described . CD4+ T cells were then isolated using a negative isolation kit (Dynabeads Untouched Human CD4 T Cells, Invitrogen) according to manufacturer specifications. The isolated cells were cultivated in RPMI-1640 supplemented with 10% fetal bovine serum and antibiotics. Activation and proliferation of CD4+ T cells were performed by supplementing the culture medium with 60 U/ml IL-2 (Peprotech, Canada) and incubating the cells with anti-CD3/CD28 coated beads (Dynabeads Human T-Activator CD3/CD28, Invitrogen) according to the manufacturer instructions. Infections of these cells were performed in the absence of IL-2 and activating beads. THP-1 monocytic cells (7.5 x 105) were differentiated into M0 macrophages by incubation in serum-free medium supplemented with 50 ng/ml PMA and 10 ng/ml GM-CSF for 48 h. Polarization into pro-inflammatory M1 macrophages was induced by addition of 100 ng/ml LPS and 25 U/ml IFN-γ. Anti-inflammatory M2 macrophages were induced by addition of 20 ng/ml IL-4 and 20 ng/ml IL-10 (all reagents were from Peprotech). In all cases, non-adherent cells were washed away before infection.
Retroviral vectors production
HIV-1 and MLV-based vectors were produced through transient transfection of 293 T cells and collected as previously described [38, 44]. To produce HIV-1NL-GFP, cells were co-transfected with pNL-GFP and pMD-G. To produce the MLV-based vectors SRBl and MIP, cells were transfected with the relevant pMIP or pSRBl plasmid and co-transfected with pCL-Eco and pMD-G. To produce HIV-1 VLPs, cells were co-transfected with pΔR8.9 and pMD-G. When necessary, amounts of viruses were normalized using the nonradioactive EnzChek RT Assay Kit (Invitrogen, Burlington, ON), or based on their titer in permissive human cells.
pSRBl-based retroviral vectors encoding an shRNA targeting either CypA or Luc mRNAs were produced in 293 T cells as described [28, 38]. TE671 and Sup-T1 cells were plated at 250,000 cells per well in 12-well plates and exposed to 1 mL of either SRBL-CypA or -Luc vector preparation. Two days post-transduction, cells were placed in medium containing 10 μg/ml of blasticidin (Sigma-Aldrich). Selection was allowed to proceed for 1 week. Efficiency of the knockdown was verified by assaying CypA expression levels in the transduced cells by western blotting, using antibodies directed against CypA (rabit polyclonal IgG; Sigma-Aldrich, StLouis, MI) and actin (mouse monoclonal; Chemicon International).
Cells were plated in 24-well plates at 50,000 cells/well (TE671 and CRFK), 100,000 cells/well (Sup-T1), 200,000 cells/well (primary CD4 T cells) or 750,000 cells/well (differentiated THP-1) and infected with HIV-1 vectors (either HIV-1TRIP-CMV-GFP or HIV-1NL-GFP) with or without CsA treatment (2 or 5 μM). Drugs were added 15 min prior to infections. In CsA dose-dependent experiments, virus doses were adjusted for each virus-cell combination so that approximately 1% of the cells would be infected in the absence of CsA. Two days post-infection, cells were trypsinised when necessary, fixed in 1 to 2% formaldehyde in a PBS solution. The % of GFP-positive cells were then determined by analyzing 10,000 to 20,000 cells with a FC500 MPL cytometer (Beckman Coulter) using the CXP software (Beckman Coulter). For spreading infections, quantification of RT activity in the supernatants of infected cells was done exactly as before .
Quantitative real-time PCR of HIV-1 DNA
Cells were plated in 12-well plates at 3x105 cells/well and infected with HIV-1NL-GFP WT or V86M vectors. Viruses were passed through 0.45 μm filters and pretreated for 1 hour at 37°C with 20U/ml DNAse I (New England Biolabs) to prevent contamination by carry-over plasmid DNA. In addition, control infections were performed in presence of 80 μM nevirapine to ascertain the absence of such contaminating plasmid DNA. Total cellular DNA was prepared after 6 hours of infection (late RT products) or 6 hours of infection followed by 18 hours of incubation in virus-free medium (2LTR-circles) and DNA preparation was done using the DNeasy Blood and Tissue Kit (Qiagen, California).
The primer sets to detect each DNA species were as follows: GFP forward, GACGACGGCAACTACAAGAC; GFP reverse, TCGTCCATGCCGAGAGTGAT; 2LTR-circles forward (MH535), AACTAGGGAACCCACTGCTTAAG ; 2LTR-circles reverse (MH536), TCCACAGATCAAGGATATCTTGTC; GAPDH forward, GTCAGTGGTGGACCTGACCT; GAPDH reverse, TGAGCTTGACAAAGTGGTCG. In each experiment, a standard curve of the amplicon being measured was run in duplicate ranging from 30 to 3 × 105 copies plus a no-template control. Reactions contained 1× SensiFAST SYBR Lo-Rox kit (Bioline, UK), 300nM forward and reverse primers, and 5 μl template DNA (100-300 ng) in 20 μl-sized reactions. After initial incubation step of 3 min at 95°C, 40 cycles of amplification were carried out at 10s at 95°C, 10s at 62°C (GFP) or 65°C (2LTR, GAPDH) and 10s (GFP) or 15 s (2LTR, GAPDH) at 72°C on a MX3000P qPCR system (Agilent Technologies, California). Results were analyzed with the MxPro software (Agilent Technologies).
Statistical data were calculated using GraphPad Prism version 5.03. Student’s unpaired t-tests were used for tests of difference between means.
2D 1H-15 N Heteronuclear (ZZ) Exchange Spectroscopy
Uniformly 15 N-labeled CAN domain of HIV1 (15 N HIV1caN) was expressed in DE3 bacteria in K-MOPS buffer supplemented with 20 mM 15NH4Cl as a sole source of nitrogen, 4 mM pH 8.0 KPOi, 0.1 o/oo sodium ampicillin salt, vitamins and 0.4% glucose. Bacterial culture was grown until 0.500-0.700 OD600 at 37°C, induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), and the protein was expressed at 20°C overnight followed by purification as previously described  except that the final step of purification, i.e. gel filtration, was run in 50 mM potassium phosphate, pH 6.5, 100 mM NaCl and 1 mM DTT. Uniformly 15 N-labeled CAN domain of HIV1 V86M (15 N HIV1caN V86M) was expressed and purified as per 15 N HIV1caN. Non-isotopically labeled N-terminally his6-tagged human CypA was expressed in DE3 bacteria in 2xTY medium supplemented with 0.6% glucose and 0.1 o/oo sodium ampicillin salt, induced at 0.6-0.8 OD600 at 37°C with 1 mM IPTG, expressed at 20°C and purified using Ni-NTA beads (Qiagen) and gel-filtration chromatography in 75 mM Tris pH8.0, 50 mM NaCl and 1 mM DTT buffer. For all NMR experiments, proteins were dialysed against the same 50 mM KPOi, pH 6.5 buffer and supplemented with 1 mM DTT. The capsids were used at 12-fold excess concentration over CypA as previously described [33, 68]: 430 μM CAN/35 μM Cyp A or 430 μM CAN only. All NMR samples contained 5% D2O.
2D 1H-15N heteronuclear (ZZ) exchange experiment is described in detail in  and was previously applied for a similar model [33, 68]. The experiments were performed on a Bruker 800 MHz spectrometer at 298 K using in-house written pulse program with the following mixing times in a randomised order: 3 ms, 25 ms, 47 ms, 69 ms, 75 ms, 97 ms, 147 ms, 169 ms, 197 ms, 247 ms, 297 ms, 397 ms, 547 ms and 796 ms. The first time-point was acquired twice to assess the error. G89 assignment for 15 N HIV1caN was assumed from ppm chemical shifts referred to in [33, 68]. It was also assumed that chemical shifts for G89 in 15 N HIV1caN V86M did not change dramatically. The data was processed in TopSpin 2.0 (Bruker, Karlsruhe) and after analysis in Sparky (T. D. Goddard and D. G. Kneller, University of California, San Francisco).
This work was funded by the Canadian Institutes for Health Research and the Canada Research Chairs program (LB). LCJ and KB were supported by the Medical Research Council [U105181010] and the Darwin Trust of Edinburgh, respectively. MV received a FRQS Master’s Training Award.
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