Second-site suppressors of HIV-1 capsid mutations: restoration of intracellular activities without correction of intrinsic capsid stability defects

Yang, Ruifeng; Shi, Jiong; Byeon, In‐Ja L.; Ahn, Jin-Woo; Sheehan, Jonathan H.; Meiler, Jens; Gronenborn, Angela M.; Aiken, Christopher

doi:10.1186/1742-4690-9-30

Cited by 57 publications

(103 citation statements)

References 59 publications

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“…Infection by E45A is resistant to PF74, yet, the mutant virus retains full ability to bind to the inhibitor (32). Addition of the second-site suppressor R132T to E45A partially rescues infectivity (48) and TNPO3 dependence (40) as well as sensitivity to inhibition of PF74 (48). Like E45A, the Q63A Q67A mutant also exhibits resistance to PF74 and altered host factor dependence.…”

Section: Discussionmentioning

confidence: 97%

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: Discussionmentioning

confidence: 97%

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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“…Changes in retroviral capsids can influence uncoating, reverse transcription, nuclear entry, chromatin targeting and binding, and integration (3,4,6,7,13,14,16,18,37). Here we describe two SIVmac239 capsid mutants, A87E and A87D, that uncoat less efficiently than wild-type SIVmac239 yet are capable of reverse transcription and nuclear entry.…”

Section: Discussionmentioning

confidence: 99%

“…The relationship between uncoating and reverse transcription has been investigated most extensively. Accelerated uncoating resulting from capsid changes, restriction by TRIM5␣, or destabilizing drugs has been associated with failure to initiate and/or complete reverse transcription (3,4,7,9,11,17,19). Conversely, blocking reverse transcription was reported to inhibit functional uncoating (51).…”

Section: Discussionmentioning

confidence: 99%

“…For example, some HIV-1 CA mutants fail to negotiate passage through the nuclear pore complex and do not access the nuclear chromatin (6). Both SIV and HIV-1 utilize karyophilic host proteins to shuttle their preintegration complexes into the nucleus of the target cell (3,4,7,9,11,17,19). The A87E and A87D SIV CA mutants can form true 2-LTR circles in the presence of an integrase inhibitor, raltegravir.…”

Section: Discussionmentioning

confidence: 99%

“…These early-phase processes include uncoating of the core, reverse transcription of the RNA genome, nuclear entry of the preintegration complex (PIC), and integration. Changes in lentivirus capsid proteins have been shown to accelerate or slow the uncoating process (3)(4)(5)(6)(7). Less expectedly, alterations in the capsid protein can affect subsequent steps in the early phase of lentivirus infection.…”

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Enhanced Autointegration in Hyperstable Simian Immunodeficiency Virus Capsid Mutants Blocked after Reverse Transcription

Tipper

Sodroski

2013

J Virol

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cAfter entering a host cell, retroviruses such as simian immunodeficiency virus (SIV) uncoat, disassembling the viral capsid. Rates of uncoating that are too high and too low can be detrimental to the efficiency of infection. Rapid uncoating typically leads to blocks in reverse transcription, but the basis for replication defects associated with slow uncoating is less clear. Here we characterize the phenotypes of two SIVmac239 mutants with changes, A87E and A87D, in the helix 4/5 loop of the capsid protein. These mutant viruses exhibited normal capsid morphology but were significantly attenuated for infectivity. The infectivity of wild-type and mutant SIVmac239 was not decreased by aphidicolin-induced growth arrest of the target cells. In the cytosol of infected cells, the A87E and A87D capsids remained in particulate form longer than the wild-type SIVmac239 capsid, suggesting that the mutants uncoat more slowly than the wild-type capsid. Both mutants exhibited much higher levels of autointegrated DNA forms than wild-type SIVmac239. Thus, some changes in the helix 4/5 loop of the SIVmac239 capsid protein result in capsid hyperstability and an increase in autointegration.

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Effect of internal cleavage site mutations in human immunodeficiency virus type 1 capsid protein on its structure and function

et al. 2016

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The capsid protein of the human immunodeficiency virus type 1 has been found to be a substrate of the retroviral protease in vitro , and its processing was predicted to be strongly dependent on a pH ‐induced conformational change. Several protease cleavage sites have been identified within the capsid protein, but the importance of its cleavage by the viral protease at the early phase of infection is controversial. To confirm the relevance of this process, we aimed to design, produce, and characterize mutant capsid proteins, in which the protein susceptibility toward HIV ‐1 protease is altered without affecting other steps of the viral life cycle. Our results indicate that while the introduced mutations changed the cleavage rate at the mutated sites of the capsid protein by HIV ‐1 protease, some of them caused only negligible or moderate structural changes (A78V, L189F, and L189I). However, the effects of other mutations (W23A, A77P, and L189P) were dramatic, as assessed by secondary structure determination or cyclophilin A‐binding assay. Based on our observations, the L189F mutant capsid remains structurally and functionally unchanged and may therefore be the best candidate for use in studies aimed at better understanding the role of the protease in the early postentry events of viral infection or retrovirus‐mediated gene transduction.

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Second-site suppressors of HIV-1 capsid mutations: restoration of intracellular activities without correction of intrinsic capsid stability defects

Cited by 57 publications

References 59 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

Enhanced Autointegration in Hyperstable Simian Immunodeficiency Virus Capsid Mutants Blocked after Reverse Transcription

Effect of internal cleavage site mutations in human immunodeficiency virus type 1 capsid protein on its structure and function

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