RESUMO
Antiretroviral therapy has revolutionized the treatment of AIDS, turning a deadly disease into a manageable chronic condition. Life-long treatment is required because existing drugs do not eradicate HIV-infected cells. The emergence of drug-resistant viral strains and uncertain vaccine prospects highlight the pressing need for new therapeutic approaches with the potential to clear the virus. The HIV-1 accessory protein Nef is essential for viral pathogenesis, making it a promising target for antiretroviral drug discovery. Nef enhances viral replication and promotes immune escape of HIV-infected cells but lacks intrinsic enzymatic activity. Instead, Nef works through diverse interactions with host cell proteins primarily related to kinase signaling pathways and endosomal trafficking. This review emphasizes the structure, function, and biological relevance of Nef interactions with host cell protein-tyrosine kinases in the broader context of Nef functions related to enhancement of the viral life cycle and immune escape. Drug discovery targeting Nef-mediated kinase activation has allowed identification of promising inhibitors of multiple Nef functions. Pharmacological inhibitors of Nef-induced MHC-I down-regulation restore the adaptive immune response to HIV-infected cells in vitro and have the potential to enhance immune recognition of latent viral reservoirs as part of a strategy for HIV clearance.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Antígenos CD4/metabolismo , Cristalografia por Raios X , Regulação para Baixo , HIV-1/patogenicidade , Evasão da Resposta Imune , Complexo Principal de Histocompatibilidade , Proteínas de Membrana/metabolismo , Proteínas Quinases/efeitos dos fármacos , Transporte Proteico , Relação Estrutura-Atividade , Produtos do Gene nef do Vírus da Imunodeficiência Humana/químicaRESUMO
The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef triggers rapid down-regulation of CD4 via the endocytic adaptor protein 2 (AP-2) complex, a process linked to enhanced viral infectivity and immune escape. Here, we describe a bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of Nef with AP-2 and CD4 in living cells. Interacting protein pairs were fused to complementary non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with both CD4 and AP-2 resulted in complementation of YFP and a bright fluorescent signal by confocal microcopy that localized to the cell periphery. Co-expression of the AP-2 α subunit enhanced the Nef·AP-2 σ2 subunit BiFC signal and vice versa, suggesting that the AP-2 α-σ2 hemicomplex interacts cooperatively with Nef. Mutagenesis of Nef amino acids Arg-134, Glu-174, and Asp-175, which stabilize Nef for AP-2 α-σ2 binding in a recent co-crystal structure, substantially reduced AP-2 interaction without affecting CD4 binding. A dimerization-defective mutant of Nef failed to interact with either CD4 or AP-2 in the BiFC assay, indicating that Nef quaternary structure is required for CD4 and AP-2 recruitment as well as CD4 down-regulation. A small molecule previously shown to bind the Nef dimerization interface also reduced Nef interactions with AP-2 and CD4 and restored CD4 expression to the surface of HIV-infected cells. Our findings provide a mechanistic explanation for previous observations that dimerization-defective Nef mutants fail to down-regulate CD4 and validate the Nef dimerization interface as a target site for antiretroviral drug development.
Assuntos
Antígenos CD4/metabolismo , Regulação para Baixo , HIV-1/metabolismo , Fator de Transcrição AP-2/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Dimerização , Fluorescência , Humanos , Modelos Moleculares , Ligação Proteica , Frações Subcelulares/metabolismoRESUMO
The HIV-1 Nef accessory factor enhances viral replication and promotes immune system evasion of HIV-infected cells, making it an attractive target for drug discovery. Recently we described a novel class of diphenylpyrazolodiazene compounds that bind directly to Nef in vitro and inhibit Nef-dependent HIV-1 infectivity and replication in cell culture. However, these first-generation Nef antagonists have several structural liabilities, including an azo linkage that led to poor oral bioavailability. The azo group was therefore replaced with either a one- or two-carbon linker. The resulting set of non-azo analogs retained nanomolar binding affinity for Nef by surface plasmon resonance, while inhibiting HIV-1 replication with micromolar potency in cell-based assays without cytotoxicity. Computational docking studies show that these non-azo analogs occupy the same predicted binding site within the HIV-1 Nef dimer interface as the original azo compound. Computational methods also identified a hot spot for inhibitor binding within this site that is defined by conserved HIV-1 Nef residues Asp108, Leu112, and Pro122. Pharmacokinetic evaluation of the non-azo B9 analogs in mice showed that replacement of the azo linkage dramatically enhanced oral bioavailability without substantially affecting plasma half-life or clearance. The improved oral bioavailability of non-azo diphenylpyrazolo Nef antagonists provides a starting point for further drug lead optimization in support of future efficacy testing in animal models of HIV/AIDS.
Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Administração Oral , Animais , Fármacos Anti-HIV/administração & dosagem , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , HIV-1/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacosRESUMO
The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent Hck activity in biochemical assays and potently blocks HIV replication in vitro. In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated Hck that result from Nef binding, as well as the impact of DFP-4AB on these changes. Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by Hck, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain. No changes in hydrogen exchange were observed in the Hck SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding. When HX MS was performed in the presence of DFP-4AB, the effect of Nef on Hck N-lobe dynamics was completely reversed. These results show that constitutive activation of Hck by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure. DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells.
Assuntos
Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-hck/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Ativação Enzimática/efeitos dos fármacos , HIV-1/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-hck/química , Replicação Viral/efeitos dos fármacos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Domínios de Homologia de srcRESUMO
In the companion paper to this work, we described development of a new type of hydrogen exchange (HX) mass spectrometry (MS) measurement that integrates Langmuir monolayers. With Langmuir monolayers, the lipid packing density can be reproducibly controlled and changed as desired. Analysis of HX in proteins that may undergo conformational changes as a function of lipid packing (for example, conformational rearrangements after insertion into a lipid layer) are then possible. We previously used neutron reflection to characterize just such a conformational change in the myristoylated HIV-1 Nef protein (myrNef): at high lipid packing density, myrNef could not insert into the lipids and maintained a compact conformation adjacent to the monolayer, whereas at lower lipid packing density, myrNef was able to insert N-terminal arm residues, causing displacement of the core domain away from the monolayer. In order to locate where conformation may have been altered by lipid association, we applied the HX MS Langmuir monolayer method to myrNef associated with monolayers of packing densities identical to those used for the prior neutron reflection measurements. The results show that the N-terminal region and the C-terminal unstructured loop undergo conformational changes when associated with a low density lipid monolayer. The results are not consistent with the hypothesis of myrNef dimerization upon membrane association in the absence of other myrNef binding partners. The HX MS Langmuir monolayer method provides new and meaningful information for myrNef that helps explain necessary conformational changes required for function at the membrane.
Assuntos
HIV-1/metabolismo , Hidrogênio/química , Espectrometria de Massas , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Medição da Troca de Deutério , Ácidos Graxos Monoinsaturados/química , Humanos , Membranas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
OBJECTIVE: Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to nontrophoblast cells. Can trophoblasts protect nontrophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection? STUDY DESIGN: Isolated primary term human trophoblasts were cultured for 48-72 hours. Diverse nonplacental human cell lines (U2OS, human foreskin fibroblast, TZM-bl, MeWo, and Caco-2) were preexposed to either trophoblast conditioned medium, nonconditioned medium, or miR-517-3p for 24 hours. Cells were infected with several viral and nonviral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student t test or analysis of variance with Bonferroni correction. RESULTS: Infection by rubella and other togaviruses, human immunodeficiency virus-1, and varicella zoster was attenuated in cells preexposed to trophoblast-conditioned medium (P < .05), and a partial effect by the chromosome 19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes. CONCLUSION: Our findings indicate that medium conditioned by primary human trophoblasts attenuates viral infection in nontrophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.
Assuntos
Resistência à Doença , Doenças Fetais/virologia , Trofoblastos/fisiologia , Viroses/imunologia , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Recém-NascidoRESUMO
HIV-1 Nef is a critical AIDS progression factor yet underexplored target for antiretroviral drug discovery. A recent high-throughput screen for pharmacological inhibitors of Nef-dependent Src-family kinase activation identified a diphenylpyrazolodiazene hit compound with submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound, known as 'B9', binds directly to Nef and inhibits its dimerization in cells as a possible mechanism of action. Here were synthesized a diverse set of B9 analogs and identified structural features essential to antiretroviral activity. Chemical modifications to each of the three rings present in the parent compound were identified that did not compromise antiviral action. These analogs will guide the development of next-generation compounds with appropriate pharmacological profiles for assessment of antiretroviral activity in vivo.
Assuntos
Fármacos Anti-HIV/farmacologia , Compostos Azo/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Pirazóis/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/efeitos dos fármacos , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Compostos Azo/síntese química , Compostos Azo/química , Linhagem Celular , Relação Dose-Resposta a Droga , Infecções por HIV/virologia , HIV-1/genética , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-Atividade , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
The HIV-1 Nef accessory factor enhances the viral life cycle in vivo, promotes immune escape of HIV-infected cells, and represents an attractive antiretroviral drug target. However, Nef lacks enzymatic activity and an active site, complicating traditional occupancy-based drug development. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on an existing hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the cereblon E3 ubiquitin ligase thalidomide-binding domain in vitro and triggered Nef degradation in a T cell expression system. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo.
Assuntos
HIV-1 , Linfócitos T , Regulação para Baixo , Membrana Celular , Replicação Viral , Proteólise , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. RESULTS: Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. CONCLUSIONS: Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.
Assuntos
Fármacos Anti-HIV/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Quinoxalinas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Sulfonamidas/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Fármacos Anti-HIV/isolamento & purificação , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-hck/genética , Quinoxalinas/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sulfonamidas/isolamento & purificação , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , BenzenossulfonamidasRESUMO
The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo where it promotes immune escape of HIV-infected cells and viral persistence. While these features identify Nef as an attractive antiretroviral drug target, Nef lacks enzymatic activity and an active site, complicating development of occupancy-based drugs. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on a previously reported hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the Cereblon E3 ubiquitin ligase, resulting in ubiquitylation of Nef and proteolytic degradation. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation of Nef is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo .
RESUMO
While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Antirretrovirais/uso terapêutico , Descoberta de Drogas , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Fatores de Virulência , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
HIV-1 Nef is an attractive target for antiretroviral drug discovery because of its role in promoting HIV-1 infectivity, replication, and host immune system avoidance. Here, we applied a screening strategy in which recombinant HIV-1 Nef protein was coupled to activation of the Src-family tyrosine kinase Hck, which enhances the HIV-1 life cycle in macrophages. Nef stimulates recombinant Hck activity in vitro, providing a robust assay for chemical library screening. High-throughput screening of more than 730â¯000 compounds using the Nef·Hck assay identified six unique hit compounds that bound directly to recombinant Nef by surface plasmon resonance (SPR) in vitro and inhibited HIV-1 replication in primary macrophages in the 0.04 to 5 µM range without cytotoxicity. Eighty-four analogs were synthesized around an isothiazolone scaffold from this series, many of which bound to recombinant Nef and inhibited HIV-1 infectivity in the low to submicromolar range. Compounds in this series restored MHC-I to the surface of HIV-infected primary cells and disrupted a recombinant protein complex of Nef with the C-terminal tail of MHC-I and the µ1 subunit of the AP-1 endocytic trafficking protein. Nef inhibitors in this class have the potential to block HIV-1 replication in myeloid cells and trigger recognition of HIV-infected cells by the adaptive immune system in vivo.
Assuntos
HIV-1 , Regulação para Baixo , HIV-1/metabolismo , Macrófagos/metabolismo , Replicação Viral , Quinases da Família src/metabolismoRESUMO
Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific member of the Src family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen-exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured, so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel-filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of activating Lck kinase activity strongly and was multiply phosphorylated by Lck. Hydrogen-exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogen atoms became deuterated after only 10 s of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium-labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that, although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.
Assuntos
Herpesvirus Saimiriíneo 2/enzimologia , Fosfoproteínas/química , Proteínas Virais/química , Espectrometria de Massas , Peptídeos/química , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Prótons , Proteínas Recombinantes/genética , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismoRESUMO
Eradication of the HIV-1 latent reservoir represents the current paradigm to developing a cure for AIDS. HIV-1 has evolved multiple mechanisms to evade CD8 T cell responses, including HIV-1 Nef-mediated downregulation of MHC-I from the surface of infected cells. Nef transcripts and protein are detectable in samples from aviremic donors, suggesting that Nef expression in latently HIV-1-infected CD4 T cells protects them from immune-mediated clearance. Here, we tested 4 small molecule inhibitors of HIV-1 Nef in an in vitro primary CD4 T cell latency model and measured the ability of autologous ex vivo or HIV-1 peptide-expanded CD8 T cells to recognize and kill latently infected cells as a function of inhibitor treatment. Nef inhibition enhanced cytokine secretion by autologous CD8 T cells against latently HIV-1-infected targets in an IFN-γ release assay. Additionally, CD8 T cell-mediated elimination of latently HIV-1-infected cells was significantly enhanced following Nef blockade, measured as a reduction in the frequency of infected cells and Gag protein in cultures following viral outgrowth assays. We demonstrate for the first time to our knowledge that Nef blockade, in combination with HIV-specific CD8 T cell expansion, might be a feasible strategy to target the HIV-1 latent reservoir that should be tested further in vivo.
Assuntos
Fármacos Anti-HIV/farmacologia , Produtos do Gene nef/antagonistas & inibidores , HIV-1/metabolismo , Latência Viral , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Regulação para Baixo , Produtos do Gene nef/genética , Produtos do Gene nef/metabolismo , HIV-1/efeitos dos fármacos , Humanos , Complexo Principal de Histocompatibilidade/imunologiaRESUMO
HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of nonenzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.
Assuntos
Antirretrovirais/química , Antirretrovirais/farmacologia , HIV-1/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Linhagem Celular , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Simulação de Acoplamento Molecular , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-hck/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Replicação Viral/efeitos dos fármacos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Human immunodeficiency virus-1 (HIV-1) has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host cell enzymes. HIV-1 Vif [viral (also called virion) infectivity factor], one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and downregulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of the protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method that is well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation, suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion, indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution, including the APOBEC3G/F binding site and the HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.