RESUMEN
UNLABELLED: The emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibits in vitro replication of HIV-1, including strains resistant to licensed protease, reverse transcriptase, and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24(Gag) production was unaffected, but virion release (measured as extracellular p24(Gag)) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111 but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to that of licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity. IMPORTANCE: New inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild-type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of SM111 and similar compounds may allow more detailed pharmacological studies of HIV-1 egress and provide opportunities to develop new treatments for HIV-1.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Antígenos CD/genética , Antígenos CD4/genética , Línea Celular , Proteínas Ligadas a GPI/genética , Humanos , Mutación/efectos de los fármacos , Virión/efectos de los fármacos , Liberación del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
HLA-restricted immune escape mutations that persist following HIV transmission could gradually spread through the viral population, thereby compromising host antiviral immunity as the epidemic progresses. To assess the extent and phenotypic impact of this phenomenon in an immunogenetically diverse population, we genotypically and functionally compared linked HLA and HIV (Gag/Nef) sequences from 358 historic (1979-1989) and 382 modern (2000-2011) specimens from four key cities in the North American epidemic (New York, Boston, San Francisco, Vancouver). Inferred HIV phylogenies were star-like, with approximately two-fold greater mean pairwise distances in modern versus historic sequences. The reconstructed epidemic ancestral (founder) HIV sequence was essentially identical to the North American subtype B consensus. Consistent with gradual diversification of a "consensus-like" founder virus, the median "background" frequencies of individual HLA-associated polymorphisms in HIV (in individuals lacking the restricting HLA[s]) were â¼ 2-fold higher in modern versus historic HIV sequences, though these remained notably low overall (e.g. in Gag, medians were 3.7% in the 2000s versus 2.0% in the 1980s). HIV polymorphisms exhibiting the greatest relative spread were those restricted by protective HLAs. Despite these increases, when HIV sequences were analyzed as a whole, their total average burden of polymorphisms that were "pre-adapted" to the average host HLA profile was only â¼ 2% greater in modern versus historic eras. Furthermore, HLA-associated polymorphisms identified in historic HIV sequences were consistent with those detectable today, with none identified that could explain the few HIV codons where the inferred epidemic ancestor differed from the modern consensus. Results are therefore consistent with slow HIV adaptation to HLA, but at a rate unlikely to yield imminent negative implications for cellular immunity, at least in North America. Intriguingly, temporal changes in protein activity of patient-derived Nef (though not Gag) sequences were observed, suggesting functional implications of population-level HIV evolution on certain viral proteins.
Asunto(s)
Adaptación Fisiológica/genética , Infecciones por VIH/genética , VIH-1/genética , Secuencia de Aminoácidos , Genotipo , Antígenos HLA/genética , Humanos , Masculino , Datos de Secuencia Molecular , América del Norte , Filogenia , Polimorfismo Genético/genéticaRESUMEN
UNLABELLED: Host and viral factors influence the HIV-1 infection course. Reduced Nef function has been observed in HIV-1 controllers during the chronic phase, but the kinetics and mechanisms of Nef attenuation in such individuals remain unclear. We examined plasma RNA-derived Nef clones from 10 recently infected individuals who subsequently suppressed viremia to less than 2,000 RNA copies/ml within 1 year postinfection (acute controllers) and 50 recently infected individuals who did not control viremia (acute progressors). Nef clones from acute controllers displayed a lesser ability to downregulate CD4 and HLA class I from the cell surface and a reduced ability to enhance virion infectivity compared to those from acute progressors (all P<0.01). HLA class I downregulation activity correlated inversely with days postinfection (Spearman's R=-0.85, P=0.004) and positively with baseline plasma viral load (Spearman's R=0.81, P=0.007) in acute controllers but not in acute progressors. Nef polymorphisms associated with functional changes over time were identified in follow-up samples from six controllers. For one such individual, mutational analyses indicated that four polymorphisms selected by HLA-A*31 and B*37 acted in combination to reduce Nef steady-state protein levels and HLA class I downregulation activity. Our results demonstrate that relative control of initial HIV-1 viremia is associated with Nef clones that display reduced function, which in turn may influence the course of HIV-1 infection. Transmission of impaired Nef sequences likely contributed in part to this observation; however, accumulation of HLA-associated polymorphisms in Nef that impair function also suggests that CD8+ T-cell pressures play a role in this phenomenon. IMPORTANCE: Rare individuals can spontaneously control HIV-1 viremia in the absence of antiretroviral treatment. Understanding the host and viral factors that contribute to the controller phenotype may identify new strategies to design effective vaccines or therapeutics. The HIV-1 Nef protein enhances viral pathogenesis through multiple mechanisms. We examined the function of plasma HIV-1 RNA-derived Nef clones isolated from 10 recently infected individuals who subsequently controlled HIV viremia compared to the function of those from 50 individuals who failed to control viremia. Our results demonstrate that early Nef clones from HIV controllers displayed lower HLA class I and CD4 downregulation activity, as well as a reduced ability to enhance virion infectivity. The accumulation of HLA-associated polymorphisms in Nef during the first year postinfection was associated with impaired protein function in some controllers. This report highlights the potential for host immune responses to modulate HIV pathogenicity and disease outcome by targeting cytotoxic T lymphocyte (CTL) epitopes in Nef.
Asunto(s)
Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Viremia/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/deficiencia , Antígenos CD4/análisis , Regulación hacia Abajo , Genotipo , VIH-1/genética , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Datos de Secuencia Molecular , Plasma/virología , Polimorfismo Genético , ARN Viral/genética , ARN Viral/aislamiento & purificación , Análisis de Secuencia de ADN , Carga Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: Impaired HIV-1 Gag, Pol, and Env function has been described in elite controllers (EC) who spontaneously suppress plasma viremia to < 50 RNA copies/mL; however, activity of the accessory protein Nef remains incompletely characterized. We examined the ability of 91 Nef clones, isolated from plasma of 45 EC and 46 chronic progressors (CP), to down-regulate HLA class I and CD4, up-regulate HLA class II invariant chain (CD74), enhance viral infectivity, and stimulate viral replication in PBMC. RESULTS: In general, EC Nef clones were functional; however, all five activities were significantly lower in EC compared to CP. Nef clones from HLA-B*57-expressing EC exhibited poorer CD4 down-regulation function compared to those from non-B*57 EC, and the number of EC-specific B*57-associated Nef polymorphisms correlated inversely with 4 of 5 Nef functions in these individuals. CONCLUSION: Results indicate that decreased HIV-1 Nef function, due in part to host immune selection pressures, may be a hallmark of the EC phenotype.
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VIH-1/patogenicidad , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Regulación hacia Abajo , VIH-1/inmunología , VIH-1/fisiología , Antígenos HLA/genética , Antígenos HLA/inmunología , Antígenos HLA/metabolismo , Humanos , Polimorfismo Genético , Análisis de Secuencia de Proteína , Viremia , Virión/genética , Virión/patogenicidad , Replicación Viral/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: The highly genetically diverse HIV-1 group M subtypes may differ in their biological properties. Nef is an important mediator of viral pathogenicity; however, to date, a comprehensive inter-subtype comparison of Nef in vitro function has not been undertaken. Here, we investigate two of Nef's most well-characterized activities, CD4 and HLA class I downregulation, for clones obtained from 360 chronic patients infected with HIV-1 subtypes A, B, C or D. RESULTS: Single HIV-1 plasma RNA Nef clones were obtained from N=360 antiretroviral-naïve, chronically infected patients from Africa and North America: 96 (subtype A), 93 (B), 85 (C), and 86 (D). Nef clones were expressed by transfection in an immortalized CD4+ T-cell line. CD4 and HLA class I surface levels were assessed by flow cytometry. Nef expression was verified by Western blot. Subset analyses and multivariable linear regression were used to adjust for differences in age, sex and clinical parameters between cohorts. Consensus HIV-1 subtype B and C Nef sequences were synthesized and functionally assessed. Exploratory sequence analyses were performed to identify potential genotypic correlates of Nef function. Subtype B Nef clones displayed marginally greater CD4 downregulation activity (p = 0.03) and markedly greater HLA class I downregulation activity (p < 0.0001) than clones from other subtypes. Subtype C Nefs displayed the lowest in vitro functionality. Inter-subtype differences in HLA class I downregulation remained statistically significant after controlling for differences in age, sex, and clinical parameters (p < 0.0001). The synthesized consensus subtype B Nef showed higher activities compared to consensus C Nef, which was most pronounced in cells expressing lower protein levels. Nef clones exhibited substantial inter-subtype diversity: cohort consensus residues differed at 25% of codons, while a similar proportion of codons exhibited substantial inter-subtype differences in major variant frequency. These amino acids, along with others identified in intra-subtype analyses, represent candidates for mediating inter-subtype differences in Nef function. CONCLUSIONS: Results support a functional hierarchy of subtype B > A/D > C for Nef-mediated CD4 and HLA class I downregulation. The mechanisms underlying these differences and their relevance to HIV-1 pathogenicity merit further investigation.
Asunto(s)
Antígenos CD4/biosíntesis , VIH-1/fisiología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Interacciones Huésped-Patógeno , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Adulto , África , Linfocitos T CD4-Positivos/virología , Línea Celular , Regulación hacia Abajo , Femenino , Genotipo , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Masculino , América del NorteRESUMEN
BACKGROUND: Although HIV can infect several cellular subsets, such as CD4⺠T lymphocytes and macrophages, it remains unclear whether an HIV infection in macrophages supports cytotoxic T lymphocyte (CTL) escape. Here, we tested two naturally-arising mutations located in the well-conserved polyproline region of Nef for their effects on CTL recognition, Nef's functionality, and viral replication capacity in macrophages. These mutations were selected because they are known to cause CTL escape in the context of T lymphocytes. FINDINGS: Monocyte-derived macrophages (MDMs) infected with the wild-type virus, but not with variant viruses, were efficiently killed by CTL clones targeting Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). The CTL-escape mutation, Arg75Thr, or Arg75Thr/Tyr85Phe double mutation, reduced the HLA class I down-regulation activity and, interestingly, increased the susceptibility of virus-infected MDMs to recognition by CTLs targeting a different epitope. The same mutations reduced the CCR5, but not CD4, down-regulation activity. Moreover, the Nef variants were impaired for Hck activation and enhancement of viral replication in MDMs. CONCLUSIONS: These results suggest that HIV-infected MDMs are killed by CTLs targeting Nef epitopes, contributing to selection and adaptation of CTL-escape viral variants.
Asunto(s)
VIH/inmunología , Macrófagos/inmunología , Macrófagos/virología , Mutación Missense , Linfocitos T Citotóxicos/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Antígenos CD4/biosíntesis , Epítopos de Linfocito T/inmunología , VIH/genética , VIH/fisiología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Evasión Inmune , Receptores CCR5/biosíntesis , Replicación ViralRESUMEN
HIV-1 Nef is a key factor for pathogenesis and is known to down-regulate functionally important molecules, including viral entry co-receptor CCR5 and CXCR4, from the surface of HIV-infected cells. Some of these Nef activities are mediated by the well-conserved proline-rich region of Nef, and this region is highly targeted by cytotoxic T lymphocytes (CTLs). In the present study, we asked whether Nef variants selected under CTL-mediated selective pressure in vivo may constrain these important Nef activities. The analysis of autologous nef sequences isolated from a cohort of total 235 subjects in Japan revealed that the subjects showing amino acid variations, such as Arg75Thr and Tyr85Phe, located within the proline-rich region were significantly over-represented by those having HLA-B*3501. CTL assays corroborated that these mutations conferred escape from HLA-B(∗)3501-restricted CTLs. The Arg75Thr variant Nef selectively impaired CCR5, but not CXCR4, down-regulation activity from the cell surface; whereas the Tyr85Phe variant Nef affected neither CCR5 nor CXCR4 down-regulation activity. Moreover, the cells expressing the Arg75Thr variant Nef significantly impaired protection from superinfection by CCR5-tropic, but not CXCR4-tropic, viruses. These results highlighted the importance of certain Nef-specific CTLs in modulation of viral co-receptor down-regulation activity and protection from HIV-1 superinfection, providing us with additional insight into vaccine design.
Asunto(s)
VIH-1/inmunología , Sobreinfección/inmunología , Linfocitos T Citotóxicos/inmunología , Internalización del Virus , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/inmunología , Sustitución de Aminoácidos , Análisis Mutacional de ADN , Regulación hacia Abajo , VIH-1/genética , Antígenos HLA-B/inmunología , Antígeno HLA-B35 , Humanos , Mutación , Receptores CCR5/inmunología , Receptores CXCR4/inmunología , Sobreinfección/virología , Linfocitos T Citotóxicos/virología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
HIV-1 inhibitors that act by mechanisms distinct from existing antiretrovirals can provide novel insights into viral replication and potentially inform development of new therapeutics. Using a multi-cycle HIV-1 replication assay, we screened 252 pure compounds derived from marine invertebrates and microorganisms and identified 6 (actinomycin Z2, bastadin 6, bengamide A, haliclonacyclamine A + B, keramamine C, neopetrosiamide B) that inhibited HIV-1 with 50% effective concentrations (EC50s) of 3.8⯵M or less. The most potent inhibitor, bengamide A, blocked HIV-1 in a T cell line with an EC50 of 0.015⯵M and in peripheral blood mononuclear cells with an EC50 of 0.032⯵M. Bengamide A was previously described to inhibit NF-κB signaling. Consistent with this mechanism, bengamide A suppressed reporter expression from an NF-κB-driven minimal promoter and an HIV-1 long terminal repeat (LTR) with conserved NF-κB response elements, but lacked activity against an LTR construct with mutation of these elements. In single-cycle HIV-1 infection assays, bengamide A also suppressed viral protein expression when viruses encoded an intact LTR but exhibited minimal activity against those with mutated NF-κB elements. Finally, bengamide A did not inhibit viral DNA accumulation, indicating that it likely acts downstream of this step in HIV-1 replication. Our study identifies multiple new antiviral compounds including an unusually potent inhibitor of HIV-1 gene expression.
Asunto(s)
Fármacos Anti-VIH/farmacología , Productos Biológicos/farmacología , Infecciones por VIH/metabolismo , VIH-1/fisiología , FN-kappa B/metabolismo , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/química , Organismos Acuáticos/química , Productos Biológicos/química , Evaluación Preclínica de Medicamentos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/genética , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH/efectos de los fármacos , VIH-1/genética , Humanos , Leucocitos Mononucleares/virología , FN-kappa B/genéticaRESUMEN
UNLABELLED: HIV-1 Nef binds to the cytoplasmic region of HLA-A and HLA-B and downregulates these molecules from the surface of virus-infected cells, thus evading immune detection by CD8(+) T cells. Polymorphic residues within the HLA cytoplasmic region may affect Nef's downregulation activity. However, the effects of HLA polymorphisms on recognition by primary Nef isolates remain elusive, as do the specific Nef regions responsible for downregulation of HLA-A versus HLA-B. Here, we examined 46 Nef clones isolated from chronically HIV-1 subtype B-infected subjects for their ability to downregulate various HLA-A, HLA-B, and HLA-C molecules on the surface of virus-infected cells. Overall, HLA-B exhibited greater resistance to Nef-mediated downregulation than HLA-A, regardless of the cell type examined. As expected, no Nef clone downregulated HLA-C. Importantly, the differential abilities of patient-derived Nef clones to downregulate HLA-A and HLA-B correlated inversely with the sensitivities of HIV-infected target cells to recognition by effector cells expressing an HIV-1 Gag-specific T cell receptor. Nef codon function analysis implicated amino acid variation at position 202 (Nef-202) in differentially affecting the ability to downregulate HLA-A and HLA-B, an observation that was subsequently confirmed by experiments using Nef mutants constructed by site-directed mutagenesis. The in silico and mutagenesis analyses further suggested that Nef-202 may interact with the C-terminal Cys-Lys-Val residues of HLA-A, which are absent in HLA-B. Taken together, the results show that natural polymorphisms within Nef modulate its interaction with natural polymorphisms in the HLA cytoplasmic tails, thereby affecting the efficiency of HLA downregulation and consequent recognition by HIV-specific T cells. These results thus extend our understanding of this complex pathway of retroviral immune evasion. IMPORTANCE: Recognition of genetically diverse pathogens by the adaptive immune system represents a primary strategy for host defense; however, pathogens such as HIV-1 can evade these responses to achieve persistent infection. The HIV-1 nef gene and the HLA class I locus rank among the most diverse genes of virus and host, respectively. The HIV-1 Nef protein interacts with the cytoplasmic region of HLA-A and HLA-B and downregulates these molecules to evade cellular immunity. By combining molecular, genetic, and in silico analyses, we demonstrate that patient-derived Nef clones downregulate HLA-A more effectively than HLA-B molecules. This in turn modulates the ability of HIV-specific T cells to recognize HIV-infected cells. We also identify a naturally polymorphic site at Nef codon 202 and HLA cytoplasmic motifs (GG314,315 and CKV339-341) that contribute to differential HLA downregulation by Nef. Our results highlight new interactions between HIV-1 and the human immune system that may contribute to pathogenesis.
Asunto(s)
Regulación hacia Abajo , VIH-1/fisiología , Antígenos HLA-B/metabolismo , Interacciones Huésped-Patógeno , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Variación Genética , Antígenos HLA-A/metabolismo , Antígenos HLA-C/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Human Immunodeficiency Virus (HIV) strains resistant to licensed anti-retroviral drugs (ARVs) continue to emerge. On the African continent, uneven access to ARVs combined with occurrence of side-effects after prolonged ARV therapy have led to searches for traditional medicines as alternative or complementary remedies to conventional HIV/AIDS management. AIM OF THE STUDY: Here we characterize a specific three-step traditional HIV/AIDS treatment regimen consisting of Cassia sieberiana root, Vitex doniana root, and Croton megalobotrys bark by combining qualitative interviews of traditional medical knowledge users in Botswana with in vitro HIV replication studies. MATERIALS AND METHODS: Crude extracts from a total of seven medicinal plants were tested for in vitro cytotoxicity and inhibition of wild-type (NL4.3) and ARV-resistant HIV-1 replication in an immortalized GFP-reporter CD4+ T-cell line. RESULTS: C. sieberiana root, V. doniana root, and C. megalobotrys bark extracts inhibited HIV-1NL4.3 replication with dose-dependence and without concomitant cytotoxicity. C. sieberiana and V. doniana extracts inhibited HIV-1 replication by 50% at 84.8µg/mL and at 25µg/mL, respectively, while C. megalobotrys extracts inhibited HIV-1 replication by a maximum of 45% at concentrations as low as 0.05µg/mL. Extracts did not interfere with antiviral activities of licensed ARVs when applied in combination and exhibited comparable efficacies against viruses harboring major resistance mutations to licensed protease, reverse-transcriptase, or integrase inhibitors. CONCLUSIONS: We report for the first time a three-step traditional HIV/AIDS regimen, used alone or in combination with standard ARV regimens, where each step exhibited more potent ability to inhibit HIV replication in vitro. Our observations support the "reverse pharmacology" model where documented clinical experiences are used to identify natural products of therapeutic value.
Asunto(s)
Fármacos Anti-VIH/farmacología , Cassia/química , Croton/química , VIH-1/efectos de los fármacos , Medicinas Tradicionales Africanas , Extractos Vegetales/farmacología , Replicación Viral/efectos de los fármacos , Vitex/química , Fármacos Anti-VIH/aislamiento & purificación , Fármacos Anti-VIH/toxicidad , Población Negra , Botswana , Cassia/toxicidad , Línea Celular , Croton/toxicidad , Características Culturales , Relación Dosis-Respuesta a Droga , Farmacorresistencia Viral , Quimioterapia Combinada , Etnobotánica , Etnofarmacología , VIH-1/genética , VIH-1/crecimiento & desarrollo , Conocimientos, Actitudes y Práctica en Salud/etnología , Humanos , Entrevistas como Asunto , Fitoterapia , Corteza de la Planta/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/toxicidad , Raíces de Plantas/química , Plantas Medicinales , Transfección , Vitex/toxicidadRESUMEN
The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world.
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Aporfinas/farmacología , Productos Biológicos/química , VIH-1/efectos de los fármacos , Proantocianidinas/farmacología , Aporfinas/química , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/virología , Línea Celular , Farmacorresistencia Viral , Guanidinas/farmacología , VIH-1/fisiología , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/virología , Simulación del Acoplamiento Molecular , Proantocianidinas/química , Pirazoles/farmacología , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Nef plays a major role in HIV-1 pathogenicity. We studied HIV-1 subtype C infected individuals in acute/early (n = 120) or chronic (n = 207) infection to investigate the relationship between Nef-mediated CD4/HLA-I down-regulation activities and disease progression, and the influence of immune-driven sequence variation on these Nef functions. A single Nef sequence per individual was cloned into an expression plasmid, followed by transfection of a T cell line and measurement of CD4 and HLA-I expression. In early infection, a trend of higher CD4 down-regulation ability correlating with higher viral load set point was observed (r = 0.19, p = 0.05), and higher HLA-I down-regulation activity was significantly associated with faster rate of CD4 decline (p = 0.02). HLA-I down-regulation function correlated inversely with the number HLA-associated polymorphisms previously associated with reversion in the absence of the selecting HLA allele (r = -0.21, p = 0.0002). These data support consideration of certain Nef regions in HIV-1 vaccine strategies designed to attenuate the infection course.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Genes MHC Clase I/fisiología , Infecciones por VIH/virología , VIH-1/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Linfocitos T CD4-Positivos , Genes MHC Clase I/genética , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Humanos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
HIV-1 Nef is required for efficient viral replication and pathogenesis. However, the extent to which Nef's functions are maintained in natural sequences during chronic infection, and their clinical relevance, remains incompletely characterized. Relative to a control Nef from HIV-1 strain SF2, HLA class I and CD4 down-regulation activities of 46 plasma RNA Nef sequences derived from unique chronic infected individuals were generally high and displayed narrow dynamic ranges, whereas Nef-mediated virion infectivity, PBMC replication and CD74 up-regulation exhibited broader dynamic ranges. 80% of patient-derived Nefs were active for at least three functions examined. Functional co-dependencies were identified, including positive correlations between CD4 down-regulation and virion infectivity, replication, and CD74 up-regulation, and between CD74 up-regulation and PBMC replication. Nef-mediated virion infectivity inversely correlated with patient CD4(±) T-cell count. Strong functional co-dependencies and the polyfunctional nature of patient-derived Nef sequences suggest a phenotypic requirement to maintain multiple Nef functions during chronic infection.
Asunto(s)
Infecciones por VIH/virología , VIH-1/patogenicidad , Factores de Virulencia/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Antígenos de Diferenciación de Linfocitos B/biosíntesis , Recuento de Linfocito CD4 , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Leucocitos Mononucleares/virología , Regulación hacia Arriba , Replicación ViralRESUMEN
BACKGROUND: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. METHODS AND RESULTS: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. CONCLUSION: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness.
Asunto(s)
Adenina/análogos & derivados , Fármacos Anti-VIH/farmacología , Infecciones por VIH/virología , VIH-1/genética , Organofosfonatos/farmacología , Cremas, Espumas y Geles Vaginales/farmacología , Adenina/administración & dosificación , Adenina/farmacología , Fármacos Anti-VIH/administración & dosificación , Ensayos Clínicos como Asunto , Femenino , Infecciones por VIH/prevención & control , Infecciones por VIH/transmisión , VIH-1/efectos de los fármacos , VIH-1/enzimología , Humanos , Organofosfonatos/administración & dosificación , Filogenia , Análisis de Secuencia de ADN , Tenofovir , Cremas, Espumas y Geles Vaginales/administración & dosificación , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
In the original manuscript, the text in figure 1 is illegible. Furthermore, there is an unnecessary carriage return (page 1716, ~line 18) "crystallographic ... methods".
Asunto(s)
VIH-1/patogenicidad , Antígenos de Histocompatibilidad Clase I/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Infecciones por VIH/virología , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
HIV-1 causes a chronic infection in humans that is characterized by high plasma viremia, progressive loss of CD4+ T lymphocytes, and severe immunodeficiency resulting in opportunistic disease and AIDS. Viral persistence is mediated in part by the ability of the Nef protein to down-regulate HLA molecules on the infected cell surface, thereby allowing HIV-1 to evade recognition by antiviral CD8+ T lymphocytes. Extensive research has been conducted on Nef to determine protein domains that are required for its immune evasion activities and to identify critical cellular co-factors, and our mechanistic understanding of this process is becoming more complete. This review highlights our current knowledge of Nef-mediated HLA class I down-regulation and places this work in the context of naturally occurring sequence variation in this protein. We argue that efforts to fully understand the critical role of Nef for HIV-1 pathogenesis will require greater analysis of patient-derived sequences to elucidate subtle differences in immune evasion activity that may alter clinical outcome.
Asunto(s)
Regulación hacia Abajo , Variación Genética , VIH-1/patogenicidad , Antígenos de Histocompatibilidad Clase I/biosíntesis , Factores de Virulencia/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Linfocitos T CD8-positivos/inmunología , Humanos , Evasión Inmune , Factores de Virulencia/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef(+) wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencias de Aminoácidos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Evaluación Preclínica de Medicamentos , Células HEK293 , VIH-1/metabolismo , Humanos , Modelos Moleculares , Prolina , Proteínas Proto-Oncogénicas c-hck/química , Proteínas Proto-Oncogénicas c-hck/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Dominios Homologos srcRESUMEN
Untreated human immunodeficiency virus (HIV) infections usually lead to death from AIDS, although the rate of the disease progression varies widely among individuals. The cytotoxic T lymphocyte (CTL) response, which is restricted by highly polymorphic MHC class I alleles, plays a central role in controlling HIV replication. It is now recognized that the antiviral efficacy of CTLs at the single cell level is dependent on their antigen specificity and is important in determining the quality of host response to viruses so that the individual will remain asymptomatic. However, because of the extreme mutational plasticity of HIV, HIV-specific CTL responses are continuously and dynamically changing. In order to rationally design an effective vaccine, the questions as to what constitutes an effective antiviral CTL response and what characterizes a potent antigenic peptide to induce such responses are becoming highlighted as needing to be answered.
Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Animales , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Epítopos de Linfocito T , Evolución Molecular , Variación Genética , Infecciones por VIH/inmunología , VIH-1/genética , VIH-1/patogenicidad , Interacciones Huésped-Patógeno , Humanos , Epítopos Inmunodominantes , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virología , Replicación ViralRESUMEN
HIV-1 Nef plays multiple roles in modulating immune responses, even though it is a dominant CTL target itself. How Nef accomplishes the balance between such conflicting selective pressures remains elusive. By genetic and functional studies, we found that Arg75Thr and Tyr85Phe mutations, located in a well-conserved proline-rich region in Nef, were differently associated with escape from CTL responses specific for two overlapping HLA-B35-restricted epitopes. CTLs specific for an epitope, that selected Tyr85Phe, were elicited earlier and had more potent functional avidities than did those that selected Arg75Thr. Although the double mutant could escape from both CTLs, the mutations are rarely observed in combination naturally. Introduction of both mutations reduced Nef's HLA class I down-regulation activity and increased the susceptibility of virus-infected cells to recognition by CTLs targeting other epitopes. Moreover, the mutant Nef was impaired in the association with activated cellular kinases and in the enhancement of viral replication. These results highlight CTL immunosurveillance as important modulators of Nef's biological activity in the infected host.