The V86M mutation in HIV-1 capsid confers resistance to TRIM5α by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import

Veillette, Maxime; Bichel, Katsiaryna; Pawlica, Paulina; Freund, Stefan; Plourde, Mélodie B.; Pham, Quang Toan; Reyes‐Moreno, Carlos; James, Leo C.; Berthoux, Lionel

doi:10.1186/1742-4690-10-25

Cited by 33 publications

(29 citation statements)

References 69 publications

(96 reference statements)

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“…In contrast, 4Mut substitutions did not alter the affinity for recombinant CypA, while 5Mut hexamer exhibited a 3-fold decreased affinity (41 M versus 12 M for WT and 4Mut). The latter effect is likely due to the H87P substitution in 5Mut CA, which results in the loss of a hydrogen bond between H87 in capsid and N71 in CypA and may alter the conformation of the CypA binding loop, as has been reported for a substitution at the adjacent position 86 (44).…”

Section: Resultsmentioning

confidence: 94%

HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry

Zhou

Price

Halambage

et al. 2015

J Virol

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During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4 ؉ T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution. IMPORTANCE The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages-cellular targets of infection in vivo.The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.T he HIV-1 capsid plays multiple roles during the early stages of infection, all of which are critical for optimal infectivity. The conical viral capsid, composed of the CA protein, consists of a lattice of hexamers interspersed with 12 pentamers, which provide curvature and closure of the ends of the cone (reviewed in reference 1). Studies of the viral capsid have received much attention of late, with the identification of inhibitory host proteins that directly target the viral capsid (2-8) and elucidation of high-resolution structures of the CA hexamer and pentamer and tubular assemblies of CA resulting in working structural models of the entire viral capsid (9-11). Several positive-acting host factors have been reported to bind the viral capsid, including cyclophilin A, CPSF6, TNPO3, Nup153, and RanBP2 (12-21); in general, these appear to facilitate infection by coordinating entry of the viral preintegration complex into the nucleus, where they may also play a role in chromatin targeting during integration. Moreover, the host factors CPSF6 and cyclophilin A contribut...

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Section: Resultsmentioning

confidence: 94%

HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry

Zhou

Price

Halambage

et al. 2015

J Virol

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“…The mechanism by which CypA modulates the viral infectivity is complex and poorly understood, being dependent on the CA protein primary sequence and the host cell type (4)(5)(6). For example, it is known that mutations in the CypA-binding loop of the CA protein dramatically reduce virus infectivity (7,8). The A92E and G94D escape mutants bind CypA with similar affinity to wild-type CA, but exhibit only 10% of the activity of wild-type CA in the presence of CypA, and full infectivity can be restored if CypA is inhibited with cyclosporin A in the host cells (8), as shown schematically in SI Appendix, Fig.…”

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confidence: 99%

Dynamic allostery governs cyclophilin A–HIV capsid interplay

Hou

Zhang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

149

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Host factor protein Cyclophilin A (CypA) regulates HIV-1 viral infectivity through direct interactions with the viral capsid, by an unknown mechanism. CypA can either promote or inhibit viral infection, depending on host cell type and HIV-1 capsid (CA) protein sequence. We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone 1H-15N and 1H-13C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories. Remarkably, the CypA loop dynamics of wild-type CA HXB2 assembly is significantly attenuated upon CypA binding, and the dynamics profiles of the A92E and G94D CypA escape mutants closely resemble that of wild-type CA assembly in complex with CypA. These results suggest that CypA loop dynamics is a determining factor in HIV-1's escape from CypA dependence.

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“…3). It was previously reported that V86M CA allows HIV-1 to escape from Rh (Pacheco et al, 2010) and mutant human TRIM5a (Veillette et al, 2013). On the other hand, virus harbouring only the V86E mutation exhibits decreased Rh TRIM5a sensitivity (Soll et al, 2013).…”

Section: Discussionmentioning

confidence: 96%

Novel mutant human immunodeficiency virus type 1 strains with high degree of resistance to cynomolgus macaque TRIMCyp generated by random mutagenesis

Sultana

Nakayama

Tobita

et al. 2016

Journal of General Virology

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Old World monkey TRIM5a strongly suppresses human immunodeficiency virus type 1 (HIV-1) replication. A fusion protein comprising cynomolgus macaque (CM) TRIM5 and cyclophilin A (CM TRIMCyp) also potently suppresses HIV-1 replication. However, CM TRIMCyp fails to suppress a mutant HIV-1 that encodes a mutant capsid protein containing a SIVmac239-derived loop between a-helices 4 and 5 (L4/5). There are seven amino acid differences between L4/5 of HIV-1 and SIVmac239. Here, we investigated the minimum numbers of amino acid substitutions that would allow HIV-1 to evade CM TRIMCypmediated suppression. We performed random PCR mutagenesis to construct a library of HIV-1 variants containing mutations in L4/5, and then we recovered replication-competent viruses from CD4 + MT4 cells that expressed high levels of CM TRIMCyp. CM TRIMCyp-resistant viruses were obtained after three rounds of selection in MT4 cells expressing CM TRIMCyp and these were found to contain four amino acid substitutions (H87R, A88G, P90D and P93A) in L4/5. We then confirmed that these substitutions were sufficient to confer CM TRIMCyp resistance to HIV-1. In a separate experiment using a similar method, we obtained novel CM TRIM5a-resistant HIV-1 strains after six rounds of selection and rescue. Analysis of these mutants revealed that V86A and G116E mutations in the capsid region conferred partial resistance to CM TRIM5a without substantial fitness cost when propagated in MT4 cells expressing CM TRIM5a. These results confirmed and further extended the previous notion that CM TRIMCyp and CM TRIM5a recognize the HIV-1 capsid in different manners.

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The V86M mutation in HIV-1 capsid confers resistance to TRIM5α by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import

Cited by 33 publications

References 69 publications

HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry

HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry

Dynamic allostery governs cyclophilin A–HIV capsid interplay

Novel mutant human immunodeficiency virus type 1 strains with high degree of resistance to cynomolgus macaque TRIMCyp generated by random mutagenesis

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