A mutation in alpha helix 3 of CA renders human immunodeficiency virus type 1 cyclosporin A resistant and dependent: rescue by a second-site substitution in a distal region of CA

The replication of many isolates of human immunodeficiency virus type 1 (HIV-1) is enhanced by binding of the host cell protein cyclophilin A (CypA) to the viral capsid protein (CA). The immunosuppressive drug cyclosporine A (CsA) and its nonimmunosuppressive analogs bind with high affinity to CypA and inhibit HIV-1 replication. Previous studies have identified two mutations, A92E and G94D, in the CypA-binding loop of CA that confer the ability of HIV-1 to replicate in the presence of CsA. Interestingly, CsA stimulates the replication of HIV-1 mutants containing either the A92E or G94D substitution in some human cell lines. Here, we show that substitution of alanine for threonine at position 54 of CA (T54A) also confers HIV-1 resistance to and dependence on CsA. Like the previously identified CsA-resistant/dependent mutants, infection by the T54A mutant was stimulated by CsA in a target cell-specific manner. RNA interference-mediated reduction of CypA expression enhanced the permissiveness of HeLa cells to infection by the T54A mutant. A suppressor mutation, encoding a substitution of threonine for alanine at position 105 of CA (A105T), was identified through adaptation of the T54A mutant virus for growth in CEM cells. A105T rescued the impaired single-cycle infectivity and replication defects of both T54A and A92E mutants. These results indicate that CA determinants outside the CypA-binding loop can modulate the dependence of HIV-1 infection on CypA.     

Replication potentials of vif variant viruses generated from monkey cell-tropic HIV-1 derivative clones NL-DT5/NL-DT5R

To obtain monkey-tropic viruses that are more closely related to HIV-1 than the original NL-DT5/NL-DT5R clones, we constructed six vif-chimeric and two site-specific vif-mutant viruses, and examined their growth ability. Different from NL-DT5/NL-DT5R, these viruses did not grow in monkey cells. We monitored the capability of the mutants to antagonize monkey APOBEC3G/F by single-cycle infectivity assays. They counteracted poorly or not at all the action of the APOBEC3G/F. Our results have indicated that the native SIVmac Vif is required to overcome the species barrier against HIV-1.     

Cyclophilin A and TRIM5alpha independently regulate human immunodeficiency virus type 1 infectivity in human cells

Cyclophilin A (CypA), a cytoplasmic, human immunodeficiency virus type 1 (HIV-1) CA-binding protein, acts after virion membrane fusion with human cells to increase HIV-1 infectivity. HIV-1 CA is similarly greeted by CypA soon after entry into rhesus macaque or African green monkey cells, where, paradoxically, the interaction decreases HIV-1 infectivity by facilitating TRIM5alpha-mediated restriction. These observations conjure a model in which CA recognition by the human TRIM5alpha orthologue is precluded by CypA. Consistent with the model, selection of a human cell line for decreased restriction of the TRIM5alpha-sensitive, N-tropic murine leukemia virus (N-MLV) rendered HIV-1 transduction of these cells independent of CypA. Additionally, HIV-1 virus-like particles (VLPs) saturate N-MLV restriction activity, particularly when the CA-CypA interaction is disrupted. Here the effects of CypA and TRIM5alpha on HIV-1 restriction were examined directly. RNA interference was used to show that endogenous human TRIM5alpha does indeed restrict HIV-1, but the magnitude of this antiviral activity was not altered by disruption of the CA-CypA interaction or by elimination of CypA protein. Conversely, the stimulatory effect of CypA on HIV-1 infectivity was completely independent of human TRIM5alpha. Together with previous reports, these data suggest that CypA protects HIV-1 from an unknown antiviral activity in human cells. Additionally, target cell permissivity increased after loading with heterologous VLPs, consistent with a common saturable target that is epistatic to both TRIM5alpha and the putative CypA-regulated restriction factor.     

Naturally occurring capsid substitutions render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in Owl monkey cells

In this study, we asked if a naturally occurring HIV-1 variant exists that circumvents CypA dependence in human cells. To address this issue, we sought viruses for CypA independence using Debio-025, a cyclosporine A (CsA) analog that disrupts CypA-capsid interaction. Surprisingly, viral variants from the Main group replicate even in the presence of the drug. Sequencing analyses revealed that these viruses encode capsid substitutions within the CypA-binding site (V86P/H87Q/I91V/M96I). When we introduced these substitutions into viruses that normally rely on CypA for replication, these mutants no longer depended on CypA, suggesting that naturally occurring capsid substitutions obviate the need for CypA. This is the first demonstration that isolates from the Main group naturally develop CypA-independent strategies to replicate in human cells. Surprisingly, we found that these capsid substitutions render HIV-1 capable of infecting Owl monkey (OMK) cells that highly restrict HIV-1. OMK cell resistance to HIV-1 is mediated via TRIM-Cyp, which arose from a retrotransposition of CypA into the TRIM5 alpha gene. Interestingly, saturation experiments suggest that the Pro86/Gln87/Val91/Ile96 capsid core is "invisible" to TRIM-Cyp. This study demonstrates that specific capsid substitutions can release HIV-1 from both CypA dependence in human cells and TRIM-Cyp restriction in monkey cells.     

Target cell cyclophilin A modulates human immunodeficiency virus type 1 infectivity

The peptidyl-prolyl isomerase cyclophilin A (CypA) increases the kinetics by which human immunodeficiency virus type 1 (HIV-1) spreads in tissue culture. This was conclusively demonstrated by gene targeting in human CD4(+) T cells, but the role of CypA in HIV-1 replication remains unknown. Though CypA binds to mature HIV-1 capsid protein (CA), it is also incorporated into nascent HIV-1 virions via interaction with the CA domain of the Gag polyprotein. These findings raised the possibility that CypA might act at multiple steps of the retroviral life cycle. Disruption of the CA-CypA interaction, either by the competitive inhibitor cyclosporine (CsA) or by mutation of CA residue G89 or P90, suggested that producer cell CypA was required for full virion infectivity. However, recent studies indicate that CypA within the target cell regulates HIV-1 infectivity by modulating Ref1- or Lv1-mediated restriction. To examine the relative contribution to HIV-1 replication of producer cell CypA and target cell CypA, we exploited multiple tools that disrupt the HIV-1 CA-CypA interaction. These tools included the drugs CsA, MeIle(4)-CsA, and Sanglifehrin; CA mutants exhibiting decreased affinity for CypA or altered CypA dependence; HeLa cells with CypA knockdown by RNA interference; and Jurkat T cells homozygous for a deletion of the gene encoding CypA. Our results clearly demonstrate that target cell CypA, and not producer cell CypA, is important for HIV-1 CA-mediated function. Inhibition of HIV-1 infectivity resulting from virion production in the presence of CsA occurs independently of the CA-CypA interaction or even of CypA.     

Cyclophilin interactions with incoming human immunodeficiency virus type 1 capsids with opposing effects on infectivity in human cells

Cyclophilin A (CypA) is a peptidyl-prolyl isomerase that binds to the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1) and by doing so facilitates HIV-1 replication. Although CypA is incorporated into HIV-1 virions by virtue of CypA-Gag interactions that occur during virion assembly, in this study we show that the CypA-CA interaction that occurs following the entry of the viral capsid into target cells is the major determinant of CypA's effects on HIV-1 replication. Specifically, by using normal and CypA-deficient Jurkat cells, we demonstrate that the presence of CypA in the target and not the virus-producing cell enhances HIV-1 infectivity. Moreover, disruption of the CypA-CA interaction with cyclosporine A (CsA) inhibits HIV-1 infectivity only if the target cell expresses CypA. The effect of CsA on HIV-1 infection of human cells varies according to which particular cell line is used as a target, and CA mutations that confer CsA resistance and dependence exert their effects only if target cells, and not if virus-producing cells, are treated with CsA. The differential effects of CsA on HIV-1 infection in different human cells appear not to be caused by polymorphisms in the recently described retrovirus restriction factor TRIM5alpha. We speculate that CypA and/or CypA-related proteins affect the fate of incoming HIV-1 capsid either directly or by modulating interactions with unidentified host cell factors.     

Cyclophilin, TRIM5, and innate immunity to HIV-1

The peptidyl-prolyl isomerase cyclophilin A (CypA) binds a proline-rich loop on the surface of HIV-1 capsid (CA). This interaction increases HIV-1 infectivity in humans but promotes an anti-HIV-1 restriction activity in non-human primates. Efforts to understand these paradoxical effects of cyclophilin, along with more targeted approaches to uncover the genetic basis for HIV-1 restriction, led to the discovery of TRIM5 (tripartite motif protein 5), a CA-specific receptor for the retroviral core. The ensuing TRIM5 publication flurry established a paradigm of innate immunity in which the protein lattice of an invading retroviral core, rather than double-stranded RNA or lipopolysaccharide, is recognized by a multimeric, cytoplasmic receptor. CypA modulates HIV-1 virion core detection by this class of innate pattern recognition molecule, apparently by inducing subtle shifts in CA conformation.     

trans-Complementation rescue of cyclophilin A-deficient viruses reveals that the requirement for cyclophilin A in human immunodeficiency virus type 1 replication is independent of its isomerase activity

Human immunodeficiency virus type 1 (HIV-1) requires the incorporation of cyclophilin A (CypA) for replication. CypA is packaged by binding to the capsid (CA) region of Gag. This interaction is disrupted by cyclosporine (CsA). Preventing CypA incorporation, either by mutations in the binding region of CA or by the presence of CsA, abrogates virus infectivity. Given that CypA possesses an isomerase activity, it has been proposed that CypA acts as an uncoating factor by destabilizing the shell of CA that surrounds the viral genome. However, because the same domain of CypA is responsible for both its isomerase activity and its capacity to be packaged, it has been challenging to determine if isomerase activity is required for HIV-1 replication. To address this issue, we fused CypA to viral protein R (Vpr), creating a Vpr-CypA chimera. Because Vpr is packaged via the p6 region of Gag, this approach bypasses the interaction with CA and allows CypA incorporation even in the presence of CsA. Using this system, we found that Vpr-CypA rescues the infectivity of viruses lacking CypA, either produced in the presence of CsA or mutated in the CypA packaging signal of CA. Furthermore, a Vpr-CypA mutant which has no isomerase activity and no capacity to bind to CA also rescues HIV-1 replication. Thus, this study demonstrates that the isomerase activity of CypA is not required for HIV-1 replication and suggests that the interaction of the catalytic site of CypA with CA serves no other function than to incorporate CypA into viruses.     

Implications for viral capsid assembly from crystal structures of HIV-1 Gag(1-278) and CA(N)(133-278)

Gag, the major structural protein of retroviruses such as HIV-1, comprises a series of domains connected by flexible linkers. These domains drive viral assembly by mediating multiple interactions between adjacent Gag molecules and by binding to viral genomic RNA and host cell membranes. Upon viral budding, Gag is processed by the viral protease to liberate distinct domains as separate proteins. The first two regions of Gag are MA, a membrane-binding module, and CA, which is a two-domain protein that makes important Gag-Gag interactions, forms the cone-shaped outer shell of the core (the capsid) in the mature HIV-1 particle, and makes an important interaction with the cellular protein cyclophilin A (CypA). Here, we report crystal structures of the mature CA N-terminal domain (CA(N)(133-278)) and a MA-CA(N) fusion (Gag(1-278)) at resolutions/R(free) values of 1.9 A/25.7% and 2.2 A/25.8%, respectively. Consistent with earlier studies, a comparison of these structures indicates that processing at the MA-CA junction causes CA to adopt an N-terminal beta-hairpin conformation that seems to be required for capsid morphology and viral infectivity. In contrast with an NMR study (Tang, C., et al. (2002) Nat. Struct. Biol. 9, 537-543), structural overlap reveals only small relative displacements for helix 6, which is located between the beta-hairpin and the CypA-binding loop. These observations argue against the proposal that CypA binding is coupled with beta-hairpin formation and support an earlier surface plasmon resonance study (Yoo, S., et al. (1997) J. Mol. Biol. 269, 780-795), which concluded that beta-hairpin formation and CypA-binding are energetically independent events.     

Human immunodeficiency virus type 1 derivative with 7% simian immunodeficiency virus genetic content is able to establish infections in pig-tailed macaques

A human immunodeficiency virus type 1 (HIV-1) derivative (HIV(NL-DT5R)) containing sequences encoding a 7-amino-acid segment of CA and the entire vif gene from simian immunodeficiency virus (SIV) was previously shown to establish spreading infections in cultured macaque peripheral blood mononuclear cells. To assess its replicative and disease-inducing properties in vivo, HIV(NL-DT5R) was inoculated into pig-tailed macaques. HIV(NL-DT5R) generated plasma viremia in all five of the monkeys and elicited humoral responses against all of the HIV-1 structural proteins but did not cause CD4(+) T-lymphocyte depletion or clinical disease. Additional adaptation will be required to optimize infectivity in vivo.     

Escape and compensation from early HLA-B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A

Certain histocompatibility leukocyte antigen (HLA) alleles are associated with improved clinical outcomes for individuals infected with human immunodeficiency virus type 1 (HIV-1), but the mechanisms for their effects remain undefined. An early CD8(+) T-cell escape mutation in the dominant HLA-B57-restricted Gag epitope TW10 (TSTLQEQIGW) has been shown to impair HIV-1 replication capacity in vitro. We demonstrate here that this T(242)N substitution in the capsid protein is associated with upstream mutations at residues H(219), I(223), and M(228) in the cyclophilin A (CypA)-binding loop in B57(+) individuals with progressive disease. In an independent cohort of epidemiologically linked transmission pairs, the presence of these substitutions in viruses encoding T(242)N was associated with significantly higher plasma viremia in donors, further suggesting that these secondary mutations compensated for the replication defect of T(242)N. Using NL4-3 constructs, we illustrate the ability of these CypA loop changes to partially restore replication of the T(242)N variant in vitro. Notably, these mutations also enhanced viral resistance to the drug cyclosporine A, indicating a reduced dependence of the compensated virus on CypA that is normally essential for optimal infectivity. Therefore, mutations in TW10 allow HIV-1 to evade a dominant early CD8(+) T-cell response, but the benefits of escape are offset by a defect in capsid function. These data suggest that TW10 escape variants undergo a postentry block that is partially overcome by changes in the CypA-binding loop and identify a mechanism for an HIV-1 fitness defect that may contribute to the slower disease progression associated with HLA-B57.     

Cyclophilin A renders human immunodeficiency virus type 1 sensitive to Old World monkey but not human TRIM5 alpha antiviral activity

TRIM5alpha is an important mediator of antiretroviral innate immunity influencing species-specific retroviral replication. Here we investigate the role of the peptidyl prolyl isomerase enzyme cyclophilin A in TRIM5alpha antiviral activity. Cyclophilin A is recruited into nascent human immunodeficiency virus type 1 (HIV-1) virions as well as incoming HIV-1 capsids, where it isomerizes an exposed proline residue. Here we show that cyclophilin A renders HIV-1 sensitive to restriction by TRIM5alpha in cells from Old World monkeys, African green monkey and rhesus macaque. Inhibition of cyclophilin A activity with cyclosporine A, or reducing cyclophilin A expression with small interfering RNA, rescues TRIM5alpha-restricted HIV-1 infectivity. The effect of cyclosporine A on HIV-1 infectivity is dependent on TRIM5alpha expression, and expression of simian TRIM5alpha in permissive feline cells renders them able to restrict HIV-1 in a cyclosporine A-sensitive way. We use an HIV-1 cyclophilin A binding mutant (CA G89V) to show that cyclophilin A has different roles in restriction by Old World monkey TRIM5alpha and owl monkey TRIM-Cyp. TRIM-Cyp, but not TRIM5alpha, recruits its tripartite motif to HIV-1 capsid via cyclophilin A and, therefore, HIV-1 G89V is insensitive to TRIM-Cyp but sensitive to TRIM5alpha. We propose that cyclophilin A isomerization of a proline residue in the TRIM5alpha sensitivity determinant of the HIV-1 capsid sensitizes it to restriction by Old World monkey TRIM5alpha. In humans, where HIV-1 has adapted to bypass TRIM5alpha activity, the effects of cyclosporine A are independent of TRIM5alpha. We speculate that cyclophilin A alters HIV-1 sensitivity to a TRIM5alpha-independent innate immune pathway in human cells.     

Gag-CA Q110D mutation elicits TRIM5-independent enhancement of HIV-1mt replication in macaque cells

HIV-1 is strictly adapted to humans, and cause disease-inducing persistent infection only in humans. We have generated a series of macaque-tropic HIV-1 (HIV-1mt) to establish non-human primate models for basic and clinical studies. HIV-1mt clones available to date grow poorly in macaque cells relative to SIVmac239. In this study, viral adaptive mutation in macaque cells, G114E in capsid (CA) helix 6 of HIV-1mt, that enhances viral replication was identified. Computer-assisted structural analysis predicted that another Q110D mutation in CA helix 6 would also increase viral growth potential. A new proviral construct MN4Rh-3 carrying CA-Q110D exhibited exquisitely enhanced growth property specifically in macaque cells. Susceptibility of MN4Rh-3 to macaque TRIM5α/TRIMCyp proteins was examined by their expression systems. HIV-1mt clones so far constructed already completely evaded TRIMCyp restriction, and further enhancement of TRIMCyp resistance by Q110D was not observed. In addition, Q110D did not contribute to evasion from TRIM5α restriction. However, the single-cycle infectivity of MN4Rh-3 in macaque cells was enhanced relative to the other HIV-1mt clones. Our results here indicate that CA-Q110D accelerates viral growth in macaque cells irrelevant to TRIM5 proteins restriction.     

Gag cytotoxic T lymphocyte escape mutations can increase sensitivity of HIV-1 to human TRIM5alpha, linking intrinsic and acquired immunity

Although laboratory-adapted HIV-1 strains are largely resistant to the human restriction factor TRIM5α (hTRIM5α), we have recently shown that some viruses carrying capsid (CA) sequences from clinical isolates can be more sensitive to this restriction factor. In this study we evaluated the contribution to this phenotype of CA mutations known to be associated with escape from cytotoxic T lymphocyte (CTL) responses. Recombinant viruses carrying HIV-1 CA sequences from NL4-3 and three different clinical isolates were prepared, along with variants in which mutations associated with CTL resistance were modified by site-directed mutagenesis, and the infectivities of these viruses in target cells expressing hTRIM5α and cells in which TRIM5α activity had been inhibited by overexpression of TRIM5γ were compared. For both hTRIM5α-sensitive viruses studied, CTL-associated mutations were found to be responsible for this phenotype. Both CTL resistance mutations occurring within HLA-restricted CA epitopes and compensatory mutations occurring outside CTL epitopes influenced hTRIM5α sensitivity, and mutations associated with CTL resistance selected in prior hosts can contribute to this effect. The impact of CTL resistance mutations on hTRIM5α sensitivity was context dependent, because mutations shown to be responsible for the TRIM5α-sensitive phenotype in viruses from one patient could have little or no impact on this parameter when introduced into another virus. No fixed relationship between changes in hTRIM5α sensitivity and infectivity was discernible in our studies. Taken together, these findings suggest that CTL mutations may influence HIV-1 replication by modifying both viral infectivity and sensitivity to TRIM5α.     

Altered HIV-1 Gag protein interactions with cyclophilin A (CypA) on the acquisition of H219Q and H219P substitutions in the CypA binding loop

HIV-1 Gag protein interaction with cyclophilin A (CypA) is critical for viral fitness. Among the amino acid substitutions identified in Gag noncleavage sites in HIV-1 variants resistant to protease inhibitors, H219Q (Gatanaga, H., Suzuki, Y., Tsang, H., Yoshimura, K., Kavlick, M. F., Nagashima, K., Gorelick, R. J., Mardy, S., Tang, C., Summers, M. F., and Mitsuya, H. (2002) J. Biol. Chem. 277, 5952-5961) and H219P substitutions in the viral CypA binding loop confer the greatest replication advantage to HIV-1. These substitutions represent polymorphic amino acid residues. We found that the replication advantage conferred by these substitutions was far greater in CypA-rich MT-2 and H9 cells than in Jurkat cells and peripheral blood mononuclear cells (PBM), both of which contained less CypA. High intracellular CypA content in H9 and MT-2 cells, resulting in excessive CypA levels in virions, limited wild-type HIV-1 (HIV-1(WT)) replication and H219Q introduction into HIV-1 (HIV-1(H219Q)), reduced CypA incorporation of HIV-1, and potentiated viral replication. H219Q introduction also restored the otherwise compromised replication of HIV-1(P222A) in PBM, although the CypA content in HIV-1(H219Q/P222A) was comparable with that in HIV-1(P222A), suggesting that H219Q affected the conformation of the CypA-binding motif, rendering HIV-1 replicative in a low CypA environment. Structural modeling analyses revealed that although hydrogen bonds are lost with H219Q and H219P substitutions, no significant distortion of the CypA binding loop of Gag occurred. The loop conformation of HIV-1(P222A) was found highly distorted, although H219Q introduction to HIV-1 restored the conformation of the loop close to that of HIV-1 (P222A). The present data suggested that the effect of CypA on HIV-1 replicative (WT) ability is bimodal (both high and low CypA content limits HIV-1 replication), that the conformation of the CypA binding region of Gag is important for viral fitness, and that the function of CypA is to maintain the conformation.     

Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors

Many mammalian species express restriction factors that confer host resistance to retroviral infection. Here we show that HIV-1 sensitivity to restriction factors is modulated by cyclophilin A (CypA), a host cell protein that binds the HIV-1 capsid protein (CA). In certain nonhuman primate cells, the CA-CypA interaction is essential for restriction: HIV-1 infectivity is increased >100-fold by cyclosporin A (CsA), a competitive inhibitor of the interaction, or by an HIV-1 CA mutation that disrupts CypA binding. Conversely, disruption of CA-CypA interaction in human cells reveals that CypA protects HIV-1 from the Ref-1 restriction factor. These findings suggest that HIV-1 has co-opted a host cell protein to counteract restriction factors expressed by human cells and that this adaptation can confer sensitivity to restriction in unnatural hosts. Manipulation of HIV-1 CA recognition by restriction factors promises to advance animal models and new therapeutic strategies for HIV-1 and AIDS.