Roles of Capsid-Interacting Host Factors in Multimodal Inhibition of HIV-1 by PF74

Saito, Akatsuki; Ferhadian, Damien; Sowd, Gregory A.; Serrao, Erik; Shi, Jiong; Halambage, Upul D.; Teng, Samantha; Soto, Juan; Siddiqui, Mohammad Adnan; Engelman, Alan; Aiken, Christopher; Yamashita, Masahiro

doi:10.1128/jvi.03116-15

Cited by 75 publications

(97 citation statements)

References 70 publications

(247 reference statements)

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“…Therefore, we tested whether the A77V mutation also altered the cofactor utilization for nuclear entry by using RNA interference. Transient gene knockdown by siRNA, which was successfully used in our previous study (52,56), was validated by qRT-PCR of total cellular RNA. In two independent experiments, expression of TNPO3 and NUP153 was downregulated on average by 4.8-fold and 9.5-fold, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…NUP358 was stably depleted from HeLa cells by using a puromycin-encoding lentiviral vector (pLKO.1; Addgene), which encodes the identical short hairpin RNA (shRNA) as a similar lentiviral vector used in our previous work (52). HeLa cell subclones (F1-5F and F1-8C) generated by two rounds of single-cell cloning, which were used in this study, were shown to exhibit a decreased level of NUP358 by a Western blot assay (56). In addition, silencing of each gene was validated by using quantitative reverse transcription-PCR (qRT-PCR) as described previously (51).…”

Section: Methodsmentioning

confidence: 99%

“…However, it remains unclear to what extent HIV-1 can tolerate reduced binding of CA to CPSF6 and accompanying alterations in the nuclear entry pathway and integration targeting during in vivo viral replication. This question is significant as there is growing interest in antiviral compounds that target a highly conserved pocket in HIV-1 CA bound by CPSF6 (39,40,(53)(54)(55)(56). In the present study, we utilized a unique capsid mutant virus that markedly reduced CPSF6 binding in a humanized mouse model to elucidate the in vivo requirement for optimal CA-CPSF6 interactions during viral replication.…”

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Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

Saito

Henning

Serrao

et al. 2016

J Virol

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Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4 ؉ T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains. IMPORTANCECPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for nuclear entry, and alters integration site preference. Despite these changes, virus carrying this mutation replicated in humanized mice at levels indistinguishable from those of the wild-type virus. However, in the majority of the animals, the mutant virus reverted back to the wild-type sequence, hence restoring the wild-type level of CA-CPSF6 interactions. These results suggest that optimal binding of CA to CPSF6 is not absolutely essential for HIV-1 replication in vivo but provides a fitness advantage that leads to the widespread usage of CPSF6 by HIV-1 in vivo.T he HIV-1 capsid (CA) protein forms the protein core that envelopes viral enzymes and genetic material following fusion with the plasma membrane (1-3). Uncoating of the viral core is a fine-tuned process that regulates downstream events, including reverse transcription, nuclear entry, and integration targeting (4-9). It is likely that HIV-1 exploits multiple host factors that can directly interact with the viral CA protein for proper uncoating in virus-infected cells. These host factors likely in...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

Saito

Henning

Serrao

et al. 2016

J Virol

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“…PF74 can destabilize the viral capsid, but its major antiviral effect occurs between reverse transcription and integration (19, 27, 49). The potency of PF74 antiviral activity depends on expression of host cell factors CypA, Nup153, and TNPO3 in target cells (23,28,49,50), and the PF74-binding pocket represents a binding site for host factor interactions, suggesting that the inhibitor's major effect is to alter virus-host cell interactions (19,24). While none of the currently reported CA-targeting compounds exhibits sufficient potency for clinical development, the variety of apparent antiviral mechanisms associated with such compounds suggests the intriguing possibility that the inhibitors act synergistically, and high potency may be achieved by creating bifunctional CAtargeting compounds.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein

Wang

Zhou

Halambage

et al. 2017

J Virol

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The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, BoehringerIngelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle. IMPORTANCEThe HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a mutation in the capsid protein that confers resistance to the inhibitor. This study reveals a novel mechanism by which a capsid-targeting small molecule can inhibit HIV-1 replication.

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“…Relatively high drug concentrations (~10 μM) block reverse transcription by probably accelerating capsid disassembly, whereas lower drug concentrations (~2 μM) block steps following reverse transcription, most likely nuclear entry [69, 72, 108, 140–142]. In contrast to higher PF74 concentrations that block HIV-1 independent from host molecules, antiviral activity by low doses of PF74 depends on capsid interactions with CypA, NUP153, and CPSF6 [24, 112, 141], host factors implicated in HIV-1 PIC nuclear entry. Drug resistant mutations, which mapped within the binding pocket, altered the requirement for HIV-1 nuclear entry cofactors and conferred fitness costs prominently in primary cells [143, 144].…”

Section: Figurementioning

confidence: 99%

Capsid-Dependent Host Factors in HIV-1 Infection

Yamashita

Engelman

2017

Trends in Microbiology

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112

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After invasion of a susceptible target cell, HIV-1 completes the early phase of its life cycle upon integration of reverse-transcribed viral DNA into host chromatin. The viral capsid, a conical shell encasing the viral ribonucleoprotein complex, along with its constitutive capsid protein plays essential roles at virtually every step in the early phase of the viral life cycle. Recent work has begun to reveal how the viral capsid interacts with specific cellular proteins to promote these processes. At the same time, cellular restriction factors target the viral capsid to thwart infection. Comprehensive understanding of capsid-host interactions that promote or impede HIV-1 infection may provide unique insight to exploit for novel therapeutic interventions.

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Roles of Capsid-Interacting Host Factors in Multimodal Inhibition of HIV-1 by PF74

Cited by 75 publications

References 70 publications

Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein

Capsid-Dependent Host Factors in HIV-1 Infection

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