Virology 408 (2010) 204–212

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Adaptation of HIV-1 to cells expressing rhesus monkey TRIM5α

Beatriz Pacheco a, Andrés Finzi a, Matthew Stremlau a, Joseph Sodroski a,b,⁎a Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Pathology, Division of AIDS, Harvard Medical School, Boston, MA 02115, USAb Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA

⁎ Corresponding author. Dana-Farber Cancer InstitutBoston, MA 02115, USA. Fax: +1 671 632 4338.

E-mail address: joseph_sodroski@dfci.harvard.edu (

0042-6822/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.virol.2010.09.019

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 July 2010Returned to author for revision12 August 2010Accepted 20 September 2010Available online 16 October 2010

Keywords:RetrovirusRestriction factorTripartite motifCapsidCyclophilin-binding loopMutant

The cross-species transmission of retroviruses is limited by host restriction factors that exhibit inter-speciesdiversity. For example, the TRIM5α proteins of OldWorld monkeys block the early, post-entry steps in humanimmunodeficiency virus (HIV-1) infection. We adapted an HIV-1 isolate to replicate in cells expressingTRIM5αrh from rhesus monkeys, an Old World species. A single amino acid change in the cyclophilin-bindingloop of the HIV-1 capsid protein allowed virus replication in cells expressing TRIM5αrh. The capsid of theescape virus exhibited a reduced affinity for TRIM5αrh, but retained the ability to bind cyclophilin A efficiently.Thus, a preferred HIV-1 escape pathway involves decreased binding to TRIM5α, a capsid-destabilizing factor,and retention of binding to cyclophilin A, a capsid-stabilizing factor.

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Introduction

Human immunodeficiency virus (HIV-1), the major cause ofacquired immunodeficiency syndrome (AIDS) in humans, arose as aresult of cross-species transmission of related viruses in chimpanzeesand gorillas (Hahn et al., 2000; Keele et al., 2006; Sharp et al., 2005;Van Heuverswyn et al., 2006; Van Heuverswyn and Peeters, 2007).These simian immunodeficiency viruses (SIVs) in apes are thought tohave been acquired from African monkeys, many species of which areendemically infected by particular SIVs (Heeney et al., 2006; VanHeuverswyn and Peeters, 2007). In establishing infections in newspecies, the primate immunodeficiency viruses have encountered andadapted to a variety of host restriction factors that exhibit lineage-related differences in viral specificity. Appreciation of these restrictionfactors is relevant not only to natural cross-species transmission ofviruses, but also to attempts to establish animal models of HIV-1infection in humans.

Current animal models for HIV-1 infection are based on theinfection of macaques with SIV or simian-immunodeficiency virus(SHIV) chimeras (Ambrose et al., 2007; Li et al., 1995). In the cells ofOld World monkeys, HIV-1 encounters three main blocks: TRIM5α,APOBEC3 and BST2 (Mariani et al., 2003; McNatt et al., 2009; Stremlauet al., 2004). After HIV-1 entry, the cytoplasmic factor TRIM5αrecognizes the incoming capsid and prematurely accelerates theuncoating of the retroviral capsid, compromising virus infectivity

(Stremlau et al., 2006a). APOBEC3G and APOBEC3F are cellularcytidine deaminases that can be incorporated into newly producedvirions and block virus replication by various mechanisms, includinghypermutation of viral cDNA (Bishop et al., 2008; Lecossier et al.,2003; Mbisa et al., 2007). The HIV-1 and SIV Vif proteins can inhibitvirion incorporation of APOBEC3G/F from some species by promotingproteasome-mediated degradation (Yu et al., 2003). BST2 tethersbudding viral particles to the infected cell surface, blocking theirrelease (Neil et al., 2008; Van Damme et al., 2008). The HIV-1 Vpuprotein counters the impact of BST2 on virus release (Neil et al., 2008;Van Damme et al., 2008). Overcoming these blocks may allow thedevelopment of macaque models of infection with HIV-1-like viruses.Recently, two groups have generated HIV-1/SIV chimeras composedof about 90% HIV-1 sequences that efficiently infect macaque cells(Hatziioannou et al., 2006; Kamada et al., 2006). These monkey-tropicHIV-1 derivatives encode the SIVmac239 Vif protein and either a short7-amino acid segment of the SIV capsid corresponding to the HIV-1cyclophilin A-binding loop or the whole capsid of SIVmac.

In the present work, we adapted an HIV-1 isolate to replicate inHeLa-CD4 cells expressing TRIM5αrh.

Results

Adaptation of HIV-1 to cells expressing rhesus monkey TRIM5α

To generate an HIV-1 variant resistant to rhesus monkey TRIM5α,we employed an approach that was previously used to adapt HIV-1 toreplicate in CD4-negative, CCR5-expressing cells (Kolchinsky et al.,1999) or in cells expressing CD4 and CXCR4 from commonmarmosets

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(Pacheco et al., 2008). The parental virus, HIV-1NL4-3, was generatedby transfection of an infectious proviral molecular clone into 293Tcells, and the resulting virus was passaged on HeLa cells expressingCD4. HeLa cells naturally express CXCR4, the coreceptor utilized bythe NL4-3 envelope glycoproteins (Feng et al., 1996). This viralstock was used to infect a culture of HeLa-CD4 cells in which 50% ofthe cells expressed TRIM5αrh. Viral replication was determined bymeasuring reverse transcriptase (RT) activity in the supernatantof the cultures every 2–3 days. Viral supernatants derived from thisculture at the peak of RT activity were used to infect cultures ofHeLa-CD4 cells with 75% of cells expressing TRIM5αrh. This processwas repeated with cultures consisting of 90% and then 100% HeLa-CD4cells expressing TRIM5αrh. In this way, viruses that demonstratedthe ability to replicate in cultures of 100% HeLa-CD4-TRIM5αrh cellswere obtained.

The gag and pol sequences of the adapted viruses wereexamined by PCR amplification of the genomic DNA of infectedcells extracted on the day when the supernatant RT activity reacheda maximum. The Gag, Pro, RT and RNaseH amino acid changesobserved in the adapted HIV-1NL4-3 variants are summarized inTable 1. After the first passage of the virus in cultures of 100% HeLa-CD4-TRIM5αrh cells, all of the sequenced proviral clones exhibited amutation that converted valine 86 in the capsid protein tomethionine. This change (V86M) was also present in all theproviral clones derived after additional passages of virus in culturesof 100% HeLa-CD4-TRIM5αrh cells (see Table 1). The V86M changehas not been observed in natural HIV-1 strains (Kuiken et al., 2008),suggesting that its appearance may result from specific selectivepressure associated with these experiments. A number of clonescontained additional changes that affected HIV-1 proteins otherthan the capsid; however, most of these changes were found onlyin single clones and none were maintained between the first andthird passages of virus in the 100% HeLa-CD4-TRIM5αrh cultures.Therefore, we focused on assessing the contribution of the V86Mchange in the capsid protein to virus adaptation to TRIM5αrh.

Table 1Amino acid changes in the HIV-1NL4-3 gag and pol protein products following adaptationto TRIM5αrh-expressing cells.a

Region Predicted amino acidchange

Presence of amino acid change in1st passage

Clone1

Clone2

Clone3

Clone4

Clone5

MA R20W X XCA V86M X X X X XProtease V11I X XProtease M46I XRT G196R XRT E296K XRNase V119I X

Region Predicted amino acidchange

Presence of amino acid change in3rd passage

Clone1

Clone2

Clone3

Clone4

Clone5

MA S126R XCA V86M X X X X Xp6 F17I XRT R206G XRT A26T XRNase D48N XIntegrase G70E X

a HIV-1NL4-3 viruses adapted to cultures of 100% HeLa-CD4-TRIM5αrh cells werepassaged additional times in these cells. The gag and pol genes of the proviruses fromthe first and third passages were amplified by PCR and cloned. The differences betweenthe predicted amino acids of the proteins of the adapted virus and those of the parentalHIV-1NL4-3 are shown.

Characterization of the sensitivity of the HIV-1 V86M capsidmutant to TRIM5αrh

Valine 86 is located in the cyclophilin A-binding loop of the HIV-1capsid protein (Fig. 1A and B). Changes in the cyclophilin-binding loopof the capsid have been previously shown to reduce HIV-1 sensitivity toearly-acting restriction factors in Old World monkey and owl monkeycells (Chatterji et al., 2005; Gatanaga et al., 2006; Hatziioannou et al.,2004; Ikeda et al., 2004; Kootstra et al., 2003, 2007; Nagao et al., 2009;Owens et al., 2004). Of note, some of these changes involve the histidine87 residue, which is adjacent to valine 86. Other changes involvingglycine 89 and proline 90 in the capsid cyclophilin-binding loop disruptthe binding of cyclophilin A, which in some cases appears to potentiateTRIM5αrh restriction of HIV-1 (Berthoux et al., 2005; Stremlau et al.,2006b; Towers et al., 2003). We compared the effect of some of thesecapsid changes with that of the V86M change on HIV-1 infection ofHeLa-CD4cells expressingTRIM5αrh. The effect of the capsid changes onthe replication of theHIV-1NL4-KB9 SEMQviruswas examined; the SEMQalteration in Vif is reported to overcome partially the replication blockconferredby rhesusmonkeyAPOBEC3G(Schrofelbauer et al., 2006). Thereplication of the HIV-1NL4-KB9 SEMQ virus with the wild-type andmutant capsids in HeLa-CD4-TRIM5αrh cells is shown in Fig. 2A. HIV-1viruses containing the V86M or H87Q changes replicated efficientlyin these cells, reaching a peak of RT activity around days 15 and 19,respectively. Beginning at approximately 19 days after infection,RT activity was detected in the culture infected by the P90A mutant;the RT activity in this culture reached a peak around day 33 post-infection. Reverse transcriptase remained at background levels incultures inoculated with HIV-1NL4-KB9 SEMQ bearing the wild-typecapsids, even after 40 days in culture. In parallel, as a control, weincubated this panel of viruses with Cf2Th cells stably expressinghuman CD4 and CXCR4, but not TRIM5αrh. All the viruses replicatedefficiently in this cell line (Fig. 2B); the delay in replication of theP90Amutant is consistent with the consequences of poor cyclophilinA binding. These results demonstrate that the V86M capsid changeassociated with virus passage in TRIM5αrh-expressing cells increasesthe resistance of HIV-1 to the inhibitory effects of rhesus monkeyTRIM5α.

To examine whether the combination of the V86M or the H87Qcapsid changes with the VifSEMQ changes were sufficient to allow HIV-1 to replicate in rhesus macaque cells, we infected the 221-89lymphoid cell line (Alexander et al., 1997) and rhesus monkey PBMCswith HIV-1NL4-KB9 viruses containing these changes. Virus replicationwas not detected in this rhesus T cell line nor in rhesus macaquePBMCs (data not shown). This result suggests that additional changesin HIV-1 are needed to allow efficient replication in rhesus monkeycells. This result was not unexpected, given the existence of rhesusmonkey restriction factors such as BST2 (tetherin) that operate duringthe late phase of HIV-1 replication (Neil et al., 2008; Van Damme et al.,2008). Rhesus monkey BST2 is not effectively countered by the HIV-1Vpu protein (Jia et al., 2009; McNatt et al., 2009). Moreover, the SEMQchanges in the Vif protein may be insufficient to allow HIV-1 toovercome APOBEC3G restriction and to replicate in the cells of OldWorld monkeys (Hatcho et al., 2008; Kamada et al., 2009).

To examine the ability of V86M and other capsid variants to escapespecifically from the early restriction imposed by TRIM5αrh, we infectedcells with recombinant HIV-1 vectors containing the capsid changes.These viruses were pseudotyped with the vesicular stomatitis virus(VSV)Gglycoprotein,whichallows entry into awide rangeof vertebratecells. The recombinant viruses express either green fluorescent protein(GFP) or luciferase. The V86M change in the capsid protein allowedmore efficient single-round infection of HeLa-CD4-TRIM5αrh cells thanwas observed for the wild-type recombinant HIV-1 vector (Fig. 2C).Because the R20W change in thematrix protein appeared in two clonesduringanearly passage of thevirus inHeLa-CD4-TRIM5αrh cells,we alsotested the contribution of this change. An HIV-1 vector containing the

Fig. 1. Thecyclophilin A-binding loopof theHIV-1 capsid protein. (A) The ribbon structureof a cyclophilin A-HIV-1 p24 capsidprotein complex is shown (Gamble et al., 1996). ThecyclophilinA-binding loop is colored yellow. Thecomplex isoriented so that, in an assembled capsid, the exposed surfaceof the capsidprotein faces the viewer. (B) TheHIV-1 capsidprotein is oriented asin (A), with the side chains of the amino acid residues relevant to this study colored according to the following key: valine 86 (red), other residues in the cyclophilin A-binding loop that havebeen shown to influence HIV-1 sensitivity to rhesus monkey restriction (green) (Owens et al., 2004), and residues that directly contact cyclophilin A (magenta). (C) Three HIV-1 capsidhexamers are shownas theyare related ina crystal lattice, approximating their relationship in anassembled capsid (Pornillos et al., 2009). Thesurfaceof theassembled capsid faces theviewer,and the cyclophilin A-binding loops (yellow) andvaline 86 residues (red) are highlighted. The right panel provides a close-up of the region at the intersection of the three hexamers in the leftpanel. The local 3-fold (triangle) and2-fold (ellipses) axes of symmetry are shown.Thecyclophilin A-binding loops at thedistal endof eachhexamer spoke are locatednear thedepressions onthe surface of the assembled capsid that track along the 2- and 3-fold axes of pseudosymmetry.

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R20W change in the matrix protein in addition to the V86M change incapsid infected the HeLa-CD4-TRIM5αrh cells similarly to the viruswithonly the V86M change. All three recombinant viruses infected thecontrol HeLa-CD4 cells efficiently (Fig. 2D).We conclude that the V86Mchange in the capsid protein confers an advantage during the infectionof HeLa cells expressing TRIM5αrh.

HeLa cells express human TRIM5α, which only minimally inhibitsHIV-1 infection (Stremlau et al., 2004); the presence of human TRIM5αmight influence the potency with which TRIM5αrh restricts HIV-1infection. Therefore, we used single-round, luciferase-expressing HIV-1to examine the contribution of the V86M capsid change to the earlyphase of infection of another cell type expressing TRIM5αrh. Wecompared the infectivity of recombinant HIV-1 with the V86M change,or with different changes in the capsid previously reported to affectsensitivity to TRIM5α restriction or cyclophilin A binding (Chatterjiet al., 2005; Gatanaga et al., 2006; Hatziioannou et al., 2004; Ikeda et al.,2004; Kootstra et al., 2003, 2007; Owens et al., 2004; Towers et al.,2003). CanineCf2Th cells,whichdonot express an endogenous TRIM5αprotein (Sawyer et al., 2007),were used as target cells; in addition to thecontrol cells transduced with the empty LPCX vector, we used Cf2Th

cells expressing TRIM5αrh and Cf2Th cells expressing the owl monkeyTRIMCyp protein. The latter protein is expressed in the NewWorld owlmonkey lineage as a result of a retrotransposition that fuses the amino-terminal part of TRIM5 with the cyclophilin A protein (Nisole et al.,2004; Sayah et al., 2004). In control canine Cf2Th cells transduced withan empty LPCX vector, all of the HIV-1 capsid mutants, with theexception of H87Q, infected less efficiently than the wild-type HIV-1(Fig. 3A). An HIV-1 vector (SCA) containing the SIVmac capsid (Owenset al., 2003) also infected these control cells less efficiently than wild-type HIV-1. In Cf2Th cells expressing TRIM5αrh, the SCA virus exhibiteda higher level of infectivity than wild-type HIV-1. The V86M and H87QHIV-1 variants also infected these cells slightly more efficiently thanwild-type HIV-1 (Fig. 3B). The G89A and P90A HIV-1 capsid mutants,which are defective for cyclophilin A binding (Braaten et al., 1996;Franke et al., 1994), infected the TRIM5αrh-expressing cells poorly. Bycontrast, these HIV-1 variants, as well as SCA, infected Cf2Th cellsexpressing owl monkey TRIMCyp muchmore efficiently than the otherviruses, including wild-type HIV-1 (Fig. 3C). This was expected, asneither the SIVmac nor the G89A or P90A HIV-1 capsids bind TRIMCyp(Hatziioannou et al., 2004; Sayah et al., 2004; Towers et al., 2003). These

Fig. 2. Replication of HIV-1 capsid mutants in HeLa-CD4 cells stably expressing TRIM5αrh. (A, B) HeLa-CD4 cells expressing TRIM5αrh (A) or control Cf2Th-CD4/CXCR4 cells(B) were incubated with 30,000 RT units of the indicated HIV-1NL4-KB9 SEMQ variants for 14 h and then washed once with PBS. Every 3 or 4 days, cell supernatants wereremoved and used for RT assays. Cells were then trypsinized, diluted 1/10 in fresh medium and replated. (C, D) Recombinant, single-round HIV-1 expressing GFP wereincubated with HeLa-CD4 cells expressing TRIM5αrh (C) or control HeLa-CD4 cells transduced with the empty LPCX vector (D). GFP expression was measured 48 h later.

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results indicate that theV86MandH87Qchanges in theHIV-1 capsid arespecifically advantageous during the infection of Cf2Th-TRIM5αrh cells.

We also examined the ability of these single-round recombinantviruses to infect primary rhesus monkey lung (PRL) fibroblasts, MK2Drhesus macaque cells and the OMK owl monkey kidney cell line. ThePRL andMK2D cells express TRIM5αrh, whereas the OMK cells expressTRIMCyp from owl monkeys, a New World monkey species (Sayahet al., 2004; Stremlau et al., 2004; Wilson et al., 2008). Wild-type HIV-1 infection of PRL cells was nearly undetectable, whereas SCA infectedthese cells very efficiently (Fig. 3D). The V86M and H87Q HIV-1mutants and, to a slightly lesser extent, the G89A and P90A mutants,infected the PRL cells more efficiently than wild-type HIV-1. In theMK2D rhesus monkey cells, infection by wild-type HIV-1 and theG89A and P90A mutants was much less efficient than infectionmediated by the V86M and H87Q mutants. In the OMK cells from theowlmonkey, only SCA and the G89A and P90AHIV-1mutants infectedefficiently. Thus, as was observed in the Cf2Th cells above, the V86Mand H87Q changes in the HIV-1 capsid confer a specific advantageduring the early phase of infection of cells expressing rhesus monkeyTRIM5α.

Competition of wild-type and mutant virus-like particles forrestriction factors

Previous studies have shown that the restriction of HIV-1 in OldWorld monkey cells can be saturated by incubation of the cells withenveloped virus-like particles (VLPs) (Cowan et al., 2002; Hatziioannouet al., 2003; Munk et al., 2002). To test whether the HIV-1 capsidmutants were able to compete for restriction factors in primary rhesusmonkey cells, we incubated PRL cells with thewild-type HIV-1 reporter

virus encoding firefly luciferase along with wild-type or mutant VLPs.The presence of wild-type HIV-1 VLPs increased the infectivity of theHIV-1 reporter virus (Fig. 4), suggesting that these VLPs are able tocompete for the binding to restriction factors. The V86M, H87Q, G89Aand P90A mutant VLPs exhibited reduced ability to compete forrestriction factors in the PRL cells, compared with the wild-type HIV-1VLPs. The YQ VLPs, which contain two changes in the HIV-1 capsid thatreduce susceptibility to Old World monkey TRIM5α without affectingrestriction factor binding (Owens et al. 2004), competed efficiently forrestricting activity. As expected, HIV-1 VLPs without envelope glyco-proteins or the SCA VLPs, which have an SIVmac capsid in an HIV-1background (Owens et al., 2003), did not compete for the PRL restrictionfactor(s). The results suggest that all of the HIV-1 VLPs with changes inthe cyclophilin-binding loop interact less efficiently with restrictionfactors following entry into PRL cells.

Capsid binding to cyclophilin A, TRIM5αrh and TRIMCyp

To determine the effects of the HIV-1 capsid changes on thebinding of cyclophilin A, we examined cyclophilin A incorporationinto HIV-1 VLPs that were produced in cyclophilin A-expressing cells.Although the major effects of cyclophilin A incorporation aremanifested during the early phase of HIV-1 infection (Hatziioannouet al., 2005), the level of cyclophilin A in the virions is a surrogate forcapsid-binding affinity. We transfected 293T cells with plasmidsexpressing the wild-type or mutant HIV-1 Gag-Pol proteins, the HA-tagged human cyclophilin A and the HIV-1 Rev protein. As one control,we used a chimeric HIV-1/SIVmac provirus (SCA) encoding the SIVmac

capsid (Owens et al., 2003). As a second control, a wild-type HIV-1VLPwas produced in the absence of HA-tagged cyclophilin A. The VLPs

Fig. 3. Infectivity of HIV-1 capsid mutants in different cell lines. Target cells were infected with single-round recombinant reporter viruses expressing firefly luciferase. Forty-eighthours after infection, cells were lysed and the luciferase activity was measured using an EG&G Berthold LB 96 V microplate luminometer. The target cells were: Cf2Th cells stablytransduced with the empty LPCX vector (A), Cf2Th cells stably expressing TRIM5αrh (B); Cf2Th cells stably expressing owl monkey TRIMCyp (C); primary rhesus monkey lung (PRL)fibroblasts (D), rhesus monkey kidney (MK2D) cells (E); and owl monkey kidney (OMK) cells (F). RLU, relative luciferase units.

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were pelleted, lysed and Western blotted with anti-HA and anti-capsid antibodies (Fig. 5A and Table 2). The V86M, H87Q and YQmutant VLPs efficiently incorporated cyclophilin A, whereas the G89Aand P90Amutant VLPs incorporated only trace amounts of cyclophilinA. The HIV-1/SIVmac chimera (SCA) with the SIVmac capsid did notdetectably incorporate cyclophilin A into VLPs. We conclude that theV86M capsid mutant retains the ability to bind cyclophilin Aefficiently.

TRIM5α specifically recognizes assembled retroviral capsids ratherthan individual capsid proteins (Dodding et al., 2005; Sebastian andLuban, 2005). To investigate the ability of TRIM5αrh to recognize themutant HIV-1 capsids, we analyzed the binding of HA-taggedTRIM5αrh to wild-type and mutant capsid–nucleocapsid (CA–NC)complexes that had been assembled in vitro, as previously described(Stremlau et al., 2006a). TRIM5αrh bound efficiently to the wild-type,YQ, G89A and P90A CA–NC complexes (Fig. 5B and Table 2). In fact,the binding of TRIM5αrh to the YQ CA–NC complexes was more

efficient than to thewild-type CA–NC complexes. By contrast, TRIM5αrh

binding to the V86M and H87Q CA–NC complexes was significantlyreduced compared to the binding observed for the wild-type CA–NCcomplexes (Table 2). Thus, diminished binding of TRIM5αrh by theV86MandH87Q capsids provides a natural explanation for the ability ofthese mutants to escape TRIM5αrh restriction.

We also tested the binding of owl monkey TRIMCyp to the wild-type and mutant CA–NC complexes (Fig. 5C and Table 2). TRIMCypbound efficiently to the wild-type, V86M and YQ CA–NC complexes,and with an intermediate level of efficiency to the H87Q variant. Inagreement with their reduced affinity for cyclophilin A, the G89A andP90A CA–NC complexes did not detectably bind TRIMCyp.

Discussion

Selection of an HIV-1 variant for replication in cells expressingTRIM5αrh allowed an assessment of the preferred viral solution to the

Fig. 4. Competition assay with HIV-1 VLPs. Target PRL cells were infected with 2,500 RTunits of the wild-type recombinant HIV-1 expressing luciferase in the presence of100,000 RT units of virus-like particles (VLPs) lacking a reporter gene. The capsidproteins of the VLPs were either wild-type HIV-1 (wt) or contained the indicatedchanges. All VLPs were pseudotyped with the VSV G glycoprotein, except control VLPswith wild-type HIV-1 capsids but with no added envelope glycoproteins (no Env).Forty-eight hours after infection, cells were lysed and the luciferase activity wasmeasured.

Table 2Interaction of ligands with HIV-1 capsid variants.

Capsidvariant

Relative cyclophilin Aincorporationa

Relative TRIM5αrh

bindingbRelative TRIMCypbindingb

wt 1.00 1.00 1.00V86M 0.84±0.39 0.05±0.05 0.88±0.32H87Q 0.75±0.61 0.18±0.18 0.46±0.14YQ 1.02±0.57 1.77±0.90 0.74 ±0.32G89A 0.08±0.04 0.84±0.30 0.03±0.008P90A 0.09±0.07 0.87±0.18 0.06±0.10SCA 0.06±0.07 ND ND

a The incorporation of HA-tagged cyclophilin A into HIV-1 virus-like particles (VLPs)was measured as described in Materials and Methods. The values for the mutant VLPswere normalized to those observed for the wild-type (wt) VLP. Themeans and standarddeviations derived from three independent experiments are shown.

b The binding of TRIM5αrh and owl monkey TRIMCyp to HIV-1 CA–NC complexeswas measured as described in Materials andMethods. The values for the mutant CA–NCcomplexes were normalized to those observed for the wild-type (wt) CA–NC complex.The means and standard deviations derived from three independent experiments areshown. ND=not determined.

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challenge of circumventing a potent restriction factor. A single aminoacid change, V86M, in the cyclophilin A-binding loop of the capsidprotein was sufficient to allow efficient HIV-1 replication in HeLa-CD4cells expressing TRIM5αrh. Of note, changes in the cyclophilin A-binding loop of capsid, including one change (V86Q) in valine 86,were previously shown to confer improved HIV-1 infectivity in OldWorld monkey target cells (Hatziioannou et al., 2004; Ikeda et al.,2004; Kootstra et al., 2003, 2007; Nagao et al., 2009; Owens et al.,2004). These changes involved valine 86, histidine 87, methionine 96and arginine 100, residues that are located near the point of departureof the cyclophilin A-binding loop from the main body of the capsidprotein (Fig. 1B). Because of this location, changes in these residueshave the potential to alter the conformation/orientation of the distalcyclophilin A-binding loop. The impact of such alterations on thestructure of the capsid surface could be amplified by the location ofthe cyclophilin A-binding loop at the end of the hexamer spoke,proximal to 2-fold and three-fold axes of pseudosymmetry (Fig. 1C).

Our results suggest that the mechanism of escape of the V86Mmutant is a reduced interaction of the capsid with TRIM5αrh. Both theV86M and the H87Q VLPs competed for restriction factors in PRL cellsless efficiently than wild-type HIV-1 VLPs. The TRIM5αrh protein incell lysates bound less to the V86M and H87Q capsid-nucleocapsidcomplexes than to wild-type HIV-1 CA–NC complexes. Previous

Fig. 5. Interaction of ligands with the HIV-1 capsid variants. (A) Cyclophilin A incorporation icoexpressed FLAG-tagged cyclophilin A (CypA) were pelleted through a 20% sucrose cushioand anti-FLAG antibodies. The incorporation of cyclophilin A into mutant VLPs, relativerepresentative experiment is shown. (B,C) The binding of TRIM5αrh or owl monkey TRIMCmeasured as previously described (Stremlau et al., 2006a). The samples were analyzed by W(Immunodiagnostics, Inc.) and an anti-HA antibody (3F10) conjugated to horseradish perorelative to the binding to wt HIV-1 CA–NC complexes, is reported in Table 2. The results of acapsid protein (Owens et al., 2004).

studies have suggested that TRIM5α recognizes assembled capsidstructures rather than individual capsid proteins (Dodding et al.,2005; Sebastian and Luban, 2005). Moreover, TRIM5α B-box 2mutants that can dimerize but not form higher-order oligomersexhibit reductions in capsid-binding ability and virus restriction(Diaz-Griffero et al., 2009; Li and Sodroski, 2008). Thus, retrovirusrestriction apparently is mediated by an array of TRIM5α dimers thatmust first assemble on the surface of the viral capsid. In light of thisrequirement, the potential of valine 86 and histidine 87 changes toaffect the conformation of the cyclophilin A-binding loop and therebyto alter the boundaries of the two- or three-fold symmetric pockets onthe capsid surface is noteworthy. To gain avidity for the capsid,TRIM5α dimers are expected to use both B30.2(SPRY) domains tocontact the capsid, perhaps via a 2-fold symmetric mode of binding.The V86M and H87Q changes could potentially disrupt this initialTRIM5αrh binding. Although the precise higher-order assembly ofTRIM5α is not understood, the formation of TRIM5α hexamers insolution has been reported (Nepveu-Traversy et al., 2009). It ispossible that alterations of the conformation of the cyclophilin A-binding loop resulting from the V86M or H87Q changes also disruptone or more aspects of the higher-order assembly of TRIM5α dimers.

The binding of cyclophilin A to the capsid contributes to thepotency of HIV-1 restriction by some TRIM5α proteins, includingTRIM5αrh (Berthoux et al., 2005; Stremlau et al., 2006b; Towers et al.,2003). Decreased binding of the V86M variant to cyclophilin A,

nto HIV-1 VLPs was measured. VLPs produced in 239T cells in the presence or absence ofn prepared in PBS. The pellets were analyzed by Western blotting with anti-p24 capsidto that of the wild-type HIV-1 VLP, is reported in Table 2. The Western blot of oneyp to HIV-1 CA–NC complexes is shown. Binding to the HIV-1 CA–NC complexes wasestern blotting with an anti-p24 antibody (4F6) conjugated to horseradish peroxidasexidase (Roche). The binding of TRIM5α and TRIMCyp to each mutant CA–NC complex,typical experiment are shown. The YQ mutant has the Q50Y/T54Q changes in the HIV-1

210 B. Pacheco et al. / Virology 408 (2010) 204–212

however, does not explain the observed decrease in susceptibility toTRIM5αrh restriction. First, the P90A mutant, which is markedlydefective in the ability to bind cyclophilin A, replicated less efficientlyin TRIM5αrh-expressing cells than the V86M or H87Q mutants; thiswas true even in HeLa cells where cyclophilin A binding is notrequired by HIV-1 for efficient infection (Li et al., 2009). Second,although valine 86 and histidine 87 are close to the cyclophilin A-binding site on the HIV-1 capsid, they do not appear to contributesignificantly to the affinity of the cyclophilin-capsid interaction.Valine 86 does not contact cyclophilin A, being just beyond a vander Waals contact with alanine 103 of cyclophilin A (Gamble et al.,1996; Gatanaga et al., 2006). The methionine substitution at capsidresidue 86 might even be expected to increase opportunities for suchcontacts with cyclophilin A. Histidine 87 makes no van der Waalscontacts with cyclophilin A; at best, the Nδ1 of histidine 87 ispredicted to form only a non-ideal hydrogen bond with the backbonecarbonyl oxygen of asparagine 71 of cyclophilin A (Gamble et al.,1996; Gatanaga et al., 2006). Consistent with these structuralobservations, the V86M change in the CA–NC complexes resulted inonly very slight decreases in the ability to interact with cyclophilin Aand TRIMCyp, relative to the wild-type CA–NC complexes. Slightlygreater decreases in cyclophilin A and TRIMCyp binding wereobserved for the H87Q CA–NC complexes. Apparently, cyclophilin Aand TRIM5αrh bind proximal but distinct structures on the HIV-1capsid, the former present on the monomer and the latter formed byquaternary interactions (Dodding et al., 2005; Sebastian and Luban,2005). In adapting to the TRIM5αrh-expressing cell, HIV-1 apparentlyseeks a path that decreases TRIM5α binding while preserving thegreater part of cyclophilin A binding. Both the binding of a restrictingTRIM5α protein and failure to bind cyclophilin A have been shown tolead to a decrease in the stability of the HIV-1 capsid in the cytosol ofinfected cells and a consequent decrease in infectivity (Li et al., 2009;Stremlau et al., 2006a).

Our results provide some insight into the mechanism wherebycyclophilin A interaction with the HIV-1 capsid in an infected cellpotentiates TRIM5αrh restriction. Both the G89A and P90A VLPs,which are deficient in cyclophilin A binding, were inefficient incompeting for restriction factors in PRL cells, compared with the wild-type HIV-1 VLPs. The deduced decrease in TRIM5αrh binding in thesecircumstances may be secondary to the decreased stability, in mostcell types, of HIV-1 capsids that do not bind cyclophilin A (Li et al.,2009). Changes in the stability of the HIV-1 capsid have been shown toaffect the ability of VLPs to saturate TRIM5α restriction (Shi andAiken, 2006). Of note, the G89A and P90A changes exert only minimaleffects on TRIM5αrh binding to CA–NC complexes in vitro; this isconsistent with previous observations that the P90A change onlyminimally decreases the in vitro stability of capsid assemblies (Liet al., 2009). Any slight direct effect of the G89A and P90A changeson TRIM5αrh binding to the HIV-1 capsid may be amplified bythe decreased ability of cyclophilin A-binding-deficient capsids topresent a higher-order quaternary structure to TRIM5αrh in thecytosol.

Future studies will investigate in greater detail the mechanisms bywhich HIV-1 crosses species barriers and explore opportunities to usethis information in creating animal models for HIV-1 infection.

Materials and methods

Cell lines

OMK, MK2D, 293T and Cf2Th cells were obtained from theAmerican Type Culture Collection and maintained in DMEM supple-mented with 10% fetal bovine serum (DMEM-10). HeLa-CD4 cellsexpressing TRIM5αrh have been previously described (Stremlau et al.,2004) and were maintained in DMEM-10. Primary rhesus lung (PRL)fibroblasts have been previously described (Hofmann et al., 1999) and

were maintained in DMEM-10. Rhesus monkey peripheral bloodmononuclear cells (PBMC) were isolated from rhesus monkey bloodprovided by the New England Primate Research Center (NEPRC).After isolation with Ficoll-Paque Plus, PBMCs were activated for3 days with 1 μg of PHA-P and cultured in complete lymphocytemedium (RPMI-1640 supplemented with 20% FBS, 10% IL-2, 25 mMHEPES, 2 mM glutamine and antibiotics). The rhesus monkey T-cellline 221-98 (Alexander et al., 1997), kindly provided by Dr. RonaldDesrosiers (NEPRC), was maintained in complete lymphocytemedium.

Cf2Th cell lines expressing TRIM5αrh from rhesus macaques orTRIMCyp from owlmonkeyswere prepared by transducing Cf2Th cellswith murine leukemia virus (MLV) vectors harboring TRIM5α orTRIMCyp cDNA. Recombinant TRIM-expressing MLV viruses pseudo-typed with VSV-G were generated by cotransfecting 293T cells with4 μg of the pVSV-G plasmid, 4 μg of the pLPCX-TRIM vector plasmidand 12 μg of the pcPack-B-MLV plasmid using the calcium phosphatemethod (Invitrogen). The resulting viral particles were used totransduce canine Cf2Th cells. Transduced Cf2Th cells were selectedwith 5 μg/ml of puromycin.

Virus replication

Replication-competentHIV-1 viruseswere generated by transfecting20 μg of the pNL4-3 plasmid, which contains an infectious HIV-1NL4-3provirus (Adachi et al., 1986), into 2×106 293T cells using the calciumphosphate transfection method (Invitrogen). Forty-eight hours aftertransfection, supernatants containing viruses were harvested andclearedby low-speed centrifugation. The level of virus in the supernatantwas determined by measuring reverse transcriptase (RT), as describedpreviously (Rho et al., 1981).

Cells were infected with 30,000 RT units of HIV-1NL4-KB9 variantsfor 14 h and then washed once with PBS. Every 3 or 4 days, cellsupernatants were removed and used for RT assays. Cells weretrypsinized, diluted 1/10 in fresh medium and replated.

Selected gag mutations were introduced into the HIV-1NL4-KB9provirus encoding the SEMQ Vif variant (Schrofelbauer et al., 2006).Infectious viruses were generated and tested for replicative ability asdescribed above.

Analysis of Gag and Pol sequences

The gag and pol genes of the adapted viruses were amplified bypolymerase chain reaction (PCR) of genomic DNA isolated frominfected cells with the QIAamp® DNA Blood Mini Kit (QIAGEN). A4.5-kb fragment containing the full gag-pol sequence was generatedby PCR with PfuUltra™ High-Fidelity DNA polymerase (Stratagene)and the primers gag-pol-forward: 5′-CTCTCGACGCAGGACTCG-3′ andgag-pol-reverse: 5′-AAACCAGTCCTTAGCTTTCCTTG-3′. The 4.5 kb frag-ment was cloned into the pCR®4blunt-TOPO plasmid (Invitrogen).The inserts from five individual clones were sequenced to obtain theconsensus sequence in the gag and pol region of the adapted virus.

Site-directed mutagenesis

DNA sequence changes specifying the V86M alteration in capsid,which was associated with viral adaptation to TRIM5αrh, wereintroduced by site-directed mutagenesis using the QuikChange II XLSite-Directed Mutagenesis protocol (Stratagene) into thepCMVΔP1ΔenvpA plasmid containing the gag, pol and vif regions ofNL4-3 HIV-1. DNA sequences specifying the previously describedcapsid variants H87Q, Q50Y/T54Y, G89A and P90A (Bukovsky et al.,1997; Franke et al., 1994; Owens et al., 2004) were also introduced bysite-directed mutagenesis into this plasmid for comparison purposes.The presence of the desiredmutationswas verified by automated DNAsequencing.

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Single-round infection assay

The efficiency of a single round of HIV-1 infection was measuredby using recombinant reporter viruses expressing firefly luciferase inplace of Nef and pseudotyped with the vesicular stomatitis virus Gglycoprotein (VSV-G). Recombinant luciferase-expressing HIV-1viruses (Helseth et al., 1990) were generated by transfecting 293Tcells using the calcium phosphate transfection method (Invitrogen)with 2 μg of the pHCMV-G plasmid expressing the VSV envelopeglycoprotein G, 2 μg of a Rev-expressing plasmid, 4 μg of thepCMVΔP1ΔenvpA packaging plasmid expressing the correspondingCA mutant, and 12 μg of an HIV-1 vector plasmid. The HIV-1 vectorplasmid expresses an RNA that can be packaged into virions, reversetranscribed, and integrated into a target cell, where it encodes fireflyluciferase. Forty-eight hours after transfection, supernatants contain-ing reporter viruses were harvested and cleared by low-speedcentrifugation. The amounts of virus in the supernatants werequantified by measuring RT activity.

Target cells were seeded at a density of 6,000 cells/well in 96-wellluminometer-compatible tissue culture plates. Twenty-four hourslater, medium was changed and different amount of viruses wereadded to the cells. After 14 h of incubation, the supernatant wasremoved and fresh medium was added. Forty-eight hours later, themedium was removed and cells were lysed with 30 μl of passive lysisbuffer (Promega). Luciferase activity was measured using an EG&GBerthold LB 96 V microplate luminometer in accordance with theluciferase assay system technical bulletin (Promega).

Competition for restriction factors with virus-like particles (VLPs)

For the competition assay with VLPs, the PRL cells were infectedwith 2,500 RT units of wild-type luciferase reporter virus in thepresence of 100,000 RT units of virus-like particles lacking a reporter.These VLPs were generated by co-transfecting 293T cells with 4 μg ofthe pHCMV-G plasmid expressing the VSV envelope glycoprotein G,4 μg of a Rev-expressing plasmid (pRev), and 12 μg of thepCMVΔP1ΔenvpA packaging plasmid expressing the correspondingCA mutant. VLPs were concentrated by pelleting through a 20%sucrose cushion.

Cyclophilin A incorporation into virions

Eight million 293T cells were transfected with 3 μg of pcDNA-CypA-FLAG, 1 μg of pRev, and 8 μg of pCMVΔP1ΔenvpA harboringdifferent capsid changes. Forty-eight hours after transfection, super-natants containing the viral particles were collected and spun at lowspeed to remove cell debris. Cleared supernatants were pelletedthrough a 20% sucrose cushion prepared in PBS for 2 h at 30,000 rpmin an SW41 rotor at 4 °C. Pelleted VLPs were resuspended in RTsuspension buffer. Similar amount of VLPs, normalized for RT activity,were analyzed by SDS-PAGE and Western blotting with anti-p24 andanti-FLAG antibodies. Western blots were exposed to film for timeperiods that allowed detection of signals but avoided saturation of thefilm.

TRIM5αrh and TRIMCyp binding to HIV-1 CA–NC complexes

Purification of recombinantly expressed HIV-1 CA–NC proteinfrom Escherichia coli was carried out as described previously (Ganseret al., 1999). High-molecular-weight HIV-1–capsid complexes wereassembled using 0.3 mM CA–NC protein and 60 μM (TG)50 DNAoligonucleotide in a volume of 50 μl of 50 mM Tris–HCl, pH 8.0 and500 mM NaCl. The reaction was allowed to proceed overnight at 4 °C,and the assembled CA–NC complexes were stored at 4 °C untilneeded.

Binding of TRIM5α and TRIMCyp to HIV-1 CA–NC complexes wasmeasured as previously described (Stremlau et al., 2006a). For asource of TRIM5 protein, about 4 x 107 Cf2Th cells stably expressingTRIM5αrh or TRIMCyp were lysed in 1 ml of hypotonic lysis buffer(10 mM Tris·HCl pH 8.0, 10 mM KCl, 1 mM EDTA) and placed on icefor 15 min. The cells were frozen/thawed once and the cell debriswere removed by centrifugation at 4 °C for 10 min at maximum speed(14,000g) in an Eppendorf microfuge. The concentration of NaCl wasadjusted to 150 mM. One hundredmicroliters of the cleared cell lysatewas combinedwith 5 μl of HIV-1 CA–NC complexes from the assemblyreaction. Themixture was incubated for 1 h at room temperature withgentle mixing. After incubation, the mixture was layered onto a 4.5 ml70% sucrose cushion (prepared in PBS) and centrifuged at 110,000×gfor 2 h at 4 °C in a Beckman SW55Ti rotor. The pellet was resuspendedin 1x SDS sample buffer and subjected to SDS/PAGE and Westernblotting with an anti-p24 antibody (4F6) conjugated to horseradishperoxidase (Immunodiagnostics, Inc.) and an anti-HA antibody(3F10) conjugated to HRP (Roche). Western blots were exposed tofilm for time periods that allowed detection of signals but avoidedsaturation of the film.

Acknowledgments

We thank YvetteMcLaughlin and Elizabeth Carpelan for manuscriptpreparation. We acknowledge Angela Carville and the New EnglandPrimate Research Center for providing the rhesus monkey blood. Weacknowledge Dr. Ronald Desrosiers from the New England PrimateResearch Center for kindly providing the 221-89 rhesus cell line. Weacknowledge ATCC for the 293T, Cf2Th, MK2D and OMK cell lines. Thiswork was supported by grants from the National Institutes of Health(AI063987 and a Center for AIDS Research Award AI060354) and a giftfrom the late William F. McCarty-Cooper.

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