RESUMO
The HIV-1 capsid executes essential functions that are regulated by capsid stability and host factors. In contrast to increasing knowledge on functional roles of capsid-interacting host proteins during postentry steps, less is known about capsid stability and its impact on intracellular events. Here, using the antiviral compound PF-3450074 (PF74) as a probe for capsid function, we uncovered a novel phenotype of capsid stability that has a profound effect on innate sensing of viral DNA by the DNA sensor cGAS. A single mutation, R143A, in the capsid protein conferred resistance to high concentrations of PF74, without affecting capsid binding to PF74. A cell-free assay showed that the R143A mutant partially counteracted the capsid-destabilizing activity of PF74, pointing to capsid stabilization as a resistance mechanism for the R143A mutant. In monocytic THP-1 cells, the R143A virus, but not the wild-type virus, suppressed cGAS-dependent innate immune activation. These results suggest that capsid stabilization improves the shielding of viral DNA from innate sensing. We found that a naturally occurring transmitted founder (T/F) variant shares the same properties as the R143A mutant with respect to PF74 resistance and DNA sensing. Imaging assays revealed delayed uncoating kinetics of this T/F variant and the R143A mutant. All these phenotypes of this T/F variant were controlled by a genetic polymorphism located at the trimeric interface between capsid hexamers, thus linking these capsid-dependent properties. Overall, this work functionally connects capsid stability to innate sensing of viral DNA and reveals naturally occurring phenotypic variation in HIV-1 capsid stability.IMPORTANCE The HIV-1 capsid, which is made from individual viral capsid proteins (CA), is a target for a number of antiviral compounds, including the small-molecule inhibitor PF74. In the present study, we utilized PF74 to identify a transmitted/founder (T/F) strain that shows increased capsid stability. Interestingly, PF74-resistant variants prevented cGAS-dependent innate immune activation under a condition where the other T/F strains induced type I interferon. These observations thus reveal a new CA-specific phenotype that couples capsid stability to viral DNA recognition by cytosolic DNA sensors.
Assuntos
Capsídeo/metabolismo , DNA Viral , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Nucleotidiltransferases/metabolismo , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Resistência à Doença , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Indóis/farmacologia , Mutação , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Estabilidade ProteicaRESUMO
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, Boehringer-Ingelheim 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.IMPORTANCE The 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.
Assuntos
Fármacos Anti-HIV/farmacologia , Benzimidazóis/farmacologia , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , HIV-1/crescimento & desenvolvimento , Montagem de Vírus/efeitos dos fármacos , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Proteínas do Capsídeo/genética , Linhagem Celular Tumoral , Proliferação de Células , Farmacorresistência Viral/genética , Células HEK293 , HIV-1/genética , Humanos , Estrutura Terciária de Proteína , Transcrição Reversa/efeitos dos fármacosRESUMO
UNLABELLED: 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. IMPORTANCE: PF74, 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.
Assuntos
Fármacos Anti-HIV/farmacologia , Capsídeo/metabolismo , HIV-1/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Indóis/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenilalanina/análogos & derivados , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sítios de Ligação , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/metabolismo , Ciclofilina A/metabolismo , Ciclofilina A/farmacologia , Células HEK293 , HIV-1/fisiologia , Células HeLa , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fenilalanina/farmacologia , Transcrição Reversa/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
UNLABELLED: 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.
Assuntos
Proteínas do Capsídeo/genética , Capsídeo/efeitos dos fármacos , Farmacorresistência Viral/fisiologia , HIV-1/efeitos dos fármacos , Indóis/farmacologia , Fenilalanina/análogos & derivados , Substituição de Aminoácidos , Fármacos Anti-HIV/farmacologia , Sítios de Ligação/genética , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/virologia , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenilalanina/farmacologia , Ligação Proteica/genética , Conformação Proteica , Interferência de RNA , RNA Interferente Pequeno , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , beta Carioferinas/genética , beta Carioferinas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
UNLABELLED: The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes in vitro and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes. IMPORTANCE: The HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.
Assuntos
Substituição de Aminoácidos , Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Proteína do Núcleo p24 do HIV/genética , HIV-1/fisiologia , Indóis/farmacologia , Fenilalanina/análogos & derivados , Replicação Viral , Células Cultivadas , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Macrófagos/virologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Fenilalanina/farmacologia , Seleção Genética , Supressão GenéticaRESUMO
The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/química , Infecções por HIV/virologia , HIV-1/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Infecções por HIV/tratamento farmacológico , HIV-1/genética , HIV-1/metabolismo , Humanos , Indóis/farmacologia , Ligantes , Modelos Moleculares , Modelos Estruturais , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Compostos Policíclicos/farmacologia , Polimerização , Ligação Proteica , Estrutura Terciária de Proteína , Transcrição Reversa/efeitos dos fármacos , Vírion , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity.