A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin

Sowd, Gregory A.; Serrao, Erik; Wang, Hao; Wang, Weifeng; Fadel, Hind J.; Poeschla, Eric M.; Engelman, Alan

doi:10.1073/pnas.1524213113

Cited by 222 publications

(382 citation statements)

References 62 publications

Supporting

Mentioning

345

Contrasting

Unclassified

Order By: Relevance

“…As previously reported (40,41,51), CPSF6 depletion by itself did not largely affect HIV-1 infection, with an ϳ20% increase in WT virus infectivity being observed. However, we found that CPSF6 depletion counteracted the triphasic dose-response curve of the drug against the WT virus ( Fig.…”

Section: Resultssupporting

confidence: 84%

“…Dulbecco modified Eagle medium (DMEM; Cellgro) supplemented with 10% fetal bovine serum (FBS; Cellgro) and 1ϫ penicillin-streptomycin (Pen-Str; Cellgro) was used to culture adherent cell lines. CPSF6 knockout HEK293T cells that carry an empty expression vector or express the 551-amino-acid isoform of CPSF6 (CPSF6[551]) were described previously (41). RPMI medium (Cellgro) supplemented with 10% FBS (Sigma), 1ϫ Pen-Str and 2 mM 2-glutamine (Cellgro) was used to culture an immortalized T cell line (MT4).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Saito

Ferhadian

Sowd

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

The viral capsid of HIV-1 interacts with a number of host factors to orchestrate uncoating and regulate downstream events, such as reverse transcription, nuclear entry, and integration site targeting. PF-3450074 (PF74), an HIV-1 capsid-targeting low-molecular-weight antiviral compound, directly binds to the capsid (CA) protein at a site also utilized by host cell proteins CPSF6 and NUP153. Here, we found that the dose-response curve of PF74 is triphasic, consisting of a plateau and two inhibitory phases of different slope values, consistent with a bimodal mechanism of drug action. High PF74 concentrations yielded a steep curve with the highest slope value among different classes of known antiretrovirals, suggesting a dose-dependent, cooperative mechanism of action. CA interactions with both CPSF6 and cyclophilin A (CypA) were essential for the unique dose-response curve. A shift of the steep curve at lower drug concentrations upon blocking the CA-CypA interaction suggests a protective role for CypA against high concentrations of PF74. These findings, highlighting the unique characteristics of PF74, provide a model in which its multimodal mechanism of action of both noncooperative and cooperative inhibition by PF74 is regulated by interactions of cellular proteins with incoming viral capsids. IMPORTANCEPF74, a novel capsid-targeting antiviral against HIV-1, shares its binding site in the viral capsid protein (CA) with the host factors CPSF6 and NUP153. This work reveals that the dose-response curve of PF74 consists of two distinct inhibitory phases that are differentially regulated by CA-interacting host proteins. PF74's potency depended on these CA-binding factors at low doses. In contrast, the antiviral activity of high PF74 concentrations was attenuated by cyclophilin A. These observations provide novel insights into both the mechanism of action of PF74 and the roles of host factors during the early steps of HIV-1 infection.T he emergence of HIV-1 variants resistant to currently approved antiretrovirals necessitates the development of novel classes of inhibitors that possess high levels of genetic barriers to resistance (1). Among viral proteins that are not exploited as an antiviral target, the viral capsid (CA) protein is an attractive target for antiviral interventions (2, 3). CA, a genetically fragile protein (4), exhibits limited tolerance to genetic changes and, hence, would predictably temper the evolution of drug resistance (5). The mutational intolerance of CA is caused by structural and functional constraints (6-8). CA, which is the major virion core structural protein, generated by protease-mediated cleavage of the precursor Gag Pr55 protein, plays essential roles during both particle assembly and disassembly (9, 10). Perhaps it is this genetic fragility that makes CA highly vulnerable to host immune responses, such as CD8-specific adaptive immunity (11) and TRIM5␣-mediated intrinsic immunity (12), both of which target highly conserved portions of CA.Recent work discovered several novel smal...

show abstract

Section: Resultssupporting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

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

Saito

Ferhadian

Sowd

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Table S2. HIV-1 and MLV integration sites in HEK293T cells alongside the matched random control simulated data were from published work (11). HT1080, HEK293T, MRC5, or HepG2 cell line-specific gene-expression activity was reported previously (GEO accession codes GSE58968, GSE11892, GSE63577, and GSE87958) (54)(55)(56)(57).…”

Section: Methodsmentioning

confidence: 99%

“…Recent studies uncovered a hierarchical mechanism that depends on HIV-1 capsid, the major viral structural protein (a product of the viral gag gene), and integrase, which interact with cellular proteins CPSF6 and LEDGF/p75, respectively (6)(7)(8)(9)(10)(11). Ablation of the mRNA maturation factor CPSF6 results in the bulk of virus abandoning generich domains, whereas ablation of the transcriptional coactivator LEDGF leads to a reduction of integration into transcription units while largely preserving the virus's ability to locate gene-rich domains (11). Thus, the capsid-CPSF6 and integrase-LEDGF axes appear to direct HIV-1 integration on the global and local scale, respectively.…”

mentioning

confidence: 99%

Structural basis for spumavirus GAG tethering to chromatin

Lesbats

Serrao

Maskell

et al. 2017

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

Self Cite

View full text Add to dashboard Cite

The interactions between a retrovirus and host cell chromatin that underlie integration and provirus expression are poorly understood. The prototype foamy virus (PFV) structural protein GAG associates with chromosomes via a chromatin-binding sequence (CBS) located within its C-terminal region. Here, we show that the PFV CBS is essential and sufficient for a direct interaction with nucleosomes and present a crystal structure of the CBS bound to a mononucleosome. The CBS interacts with the histone octamer, engaging the H2A-H2B acidic patch in a manner similar to other acidic patch-binding proteins such as herpesvirus latency-associated nuclear antigen (LANA). Substitutions of the invariant arginine anchor residue in GAG result in global redistribution of PFV and macaque simian foamy virus (SFV mac ) integration sites toward centromeres, dampening the resulting proviral expression without affecting the overall efficiency of integration. Our findings underscore the importance of retroviral structural proteins for integration site selection and the avoidance of genomic junkyards.retrovirus | integration | nucleosome | chromatin-binding

show abstract

“…SupT1 and CEM-SS cells were propagated in similarly supplemented RPMI 1640 medium. Viruses used for infectivity measures and transmission electron microscopy were generated by plasmid DNA transfection of HEK293T cells as previously described (58).…”

Section: Methodsmentioning

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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin

Cited by 222 publications

References 62 publications

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

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

Structural basis for spumavirus GAG tethering to chromatin

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

Contact Info

Product

Resources

About