RESUMO
Impacts of mutations at position E138 (A/G/K/Q/R/V) alone or in combination with M184I in HIV-1 reverse transcriptase (RT) were investigated. We also determined why E138K is the most prevalent nonnucleoside reverse transcriptase inhibitor mutation in patients failing rilpivirine (RPV) therapy. Recombinant RT enzymes and viruses containing each of the above-mentioned mutations were generated, and drug susceptibility was assayed. Each of the E138A/G/K/Q/R mutations, alone or in combination with M184I, resulted in decreased susceptibility to RPV and etravirine (ETR). The maximum decrease in susceptibility to RPV was observed for E138/R/Q/G by both recombinant RT assay and cell-based assays. E138Q/R-containing enzymes and viruses also showed the most marked decrease in susceptibility to ETR by both assays. The addition of M184I to the E138 mutations did not significantly change the levels of diminution in drug susceptibility. These findings indicate that E138R caused the highest level of loss of susceptibility to both RPV and ETR, and, accordingly, E138R should be recognized as an ETR resistance-associated mutation. The E138K/Q/R mutations can compensate for M184I in regard to both enzymatic fitness and viral replication capacity. The favored emergence of E138K over other mutations at position E138, together with M184I, is not due to an advantage in either the level of drug resistance or viral replication capacity but may reflect the fact that E138R and E138Q require two distinct mutations to occur, one of which is a disfavorable G-to-C mutation, whereas E138K requires only a single favorable G-to-A hypermutation. Of course, other factors may also affect the concept of barrier to resistance.
Assuntos
Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Nitrilas/farmacologia , Piridazinas/farmacologia , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Células Cultivadas , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Mutação , Rilpivirina , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: The host protein APOBEC3G (A3G) can limit HIV-1 replication. Its protective effect is overcome by the HIV-1 'viral infectivity factor' (Vif), which targets A3G for proteosomal degradation. Although Vif is considered to be essential for HIV-1 replication, the effect of Vif variability among commonly used HIV-1 molecular clones of different genetic backgrounds on viral infectiousness and pathogenesis has not been fully determined. METHODS: We cloned the intact Vif coding regions of available molecular clones of different subtypes into expression vectors. Δvif full-length HIV-1 clonal variants were generated from corresponding subtype-specific full-length molecular clones. Replication-competent viruses were produced in 293T cells in the presence or absence of A3G, with Vif being supplied by the full-length HIV-1 clone or in trans. The extent of A3G-mediated restriction was then determined in a viral replication assay using a reporter cell line. RESULTS AND CONCLUSIONS: In the absence of A3G, Vif subtype origin did not impact viral replication. In the presence of A3G the subtype origin of Vif had a differential effect on viral replication. Vif derived from a subtype C molecular clone was less effective at overcoming A3G-mediated inhibition than Vif derived from either subtype B or CRF02_AG molecular clones.
Assuntos
Citidina Desaminase/metabolismo , Variação Genética , HIV-1/fisiologia , Replicação Viral , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminase APOBEC-3G , Linhagem Celular , Citidina Desaminase/imunologia , HIV-1/genética , HIV-1/imunologia , Humanos , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
Antiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cells in vitro in the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , HIV-1/efeitos dos fármacos , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Mutação , Organofosfonatos/farmacologia , TenofovirRESUMO
BMS-599793 is a small molecule entry inhibitor that binds to human immunodeficiency virus type 1 (HIV-1) gp120, resulting in the inhibition of CD4-dependent entry into cells. Since BMS-599793 is currently considered a candidate microbicide drug, we evaluated its efficacy against a number of primary patient HIV isolates from different subtypes and circulating recombinant forms (CRFs) and showed that activity varied between â¼3 ρM and 7 µM at 50% effective concentrations (EC(50)s). Interestingly, CRF01_AE HIV-1 isolates consistently demonstrated natural resistance against this compound. Genotypic analysis of >1,600 sequences (Los Alamos HIV sequence database) indicated that a single amino acid polymorphism in Env, H375, may account for the observed BMS-599793 resistance in CRF01_AE HIV-1. Results of site-directed mutagenesis experiments confirmed this hypothesis, and in silico drug docking simulations identified a drug resistance mechanism at the molecular level. In addition, CRF01_AE viruses were shown to be resistant to multiple broadly neutralizing monoclonal antibodies. Thus, our results not only provide insight into how Env polymorphisms may contribute to entry inhibitor resistance but also may help to elucidate how HIV can evade some broadly neutralizing antibodies. Furthermore, the high frequency of H375 in CRF01_AE HIV-1, and its apparent nonoccurrence in other subtypes, could serve as a means for rapid identification of CRF01_AE infections.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , Proteína gp120 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , Piperidinas/farmacologia , Pirazinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Anticorpos Neutralizantes/imunologia , Linhagem Celular Tumoral , Genes env , Genótipo , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/genética , HIV-1/imunologia , HIV-1/isolamento & purificação , HIV-2/efeitos dos fármacos , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Piperidinas/química , Piperidinas/metabolismo , Polimorfismo Genético , Estrutura Quaternária de Proteína , Pirazinas/química , Pirazinas/metabolismo , Alinhamento de SequênciaRESUMO
HIV entry inhibitors, such as maraviroc (MVC), prevent cell-free viruses from entering the cells. In clinical trials, patients who were treated with MVC often displayed viral loads that were above the limit of conventional viral load detection compared to efavirenz-based regimens. We hypothesize that viruses blocked by entry inhibitors may be redistributed to plasma, where they artificially increase viral load measurements compared to those with the use of antiretroviral drugs (ARVs) that act intracellularly. We infected PM-1 cells with CCR5-tropic HIV-1 BaL or CXCR4-tropic HIV-1 NL4-3 in the presence of inhibitory concentrations of efavirenz, raltegravir, enfuvirtide, maraviroc, and AMD3100, the latter three being entry inhibitors. Supernatant viral load, reverse transcriptase enzyme activity, and intracellular nucleic acid levels were measured at times up to 24 h postinfection. Infectivity of redistributed dual-tropic HIV-1 was assessed using TZM-bl cells. Extracellular viral load analysis revealed that entry inhibitor-treated cells had higher levels of virus in the supernatant than the cells treated with other ARVs at 8 h postinfection. By 24 h, the supernatant viral load was still higher for entry inhibitors than other ARVs. We observed a correlation between viral load and the step of entry inhibition. Dual-tropic virus infectivity was undiminished utilizing the CCR5 coreceptor following redistribution by CXCR4 entry inhibition. This in vitro model indicates that entry inhibitors exhibit a redistribution effect unseen with intracellular ARV drugs. Based on these results, the effectiveness of some entry inhibitors may be underestimated if plasma viral load is used as a sole indicator of clinical success.
Assuntos
Cicloexanos/farmacologia , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Triazóis/farmacologia , Carga Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Alcinos , Fármacos Anti-HIV/farmacologia , Benzoxazinas/farmacologia , Benzilaminas , Linhagem Celular , Ciclamos , Ciclopropanos , DNA Viral/análise , Farmacorresistência Viral , Enfuvirtida , Proteína gp41 do Envelope de HIV/farmacologia , Transcriptase Reversa do HIV/análise , Compostos Heterocíclicos/farmacologia , Humanos , Maraviroc , Fragmentos de Peptídeos/farmacologia , Pirrolidinonas/farmacologia , RNA Viral/análise , Raltegravir Potássico , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMO
Current antiretroviral therapy (ART) increases the survival of HIV-infected individuals, yet it is not curative. The major barrier to finding a definitive cure for HIV is our inability to identify and eliminate long-lived cells containing the dormant provirus, termed viral reservoir. When ART is interrupted, the viral reservoir ensures heterogenous and stochastic HIV viral gene expression, which can reseed infection back to pre-ART levels. While strategies to permanently eradicate the virus have not yet provided significant success, recent work has focused on the management of this residual viral reservoir to effectively limit comorbidities associated with the ongoing viral transcription still observed during suppressive ART, as well as limit the need for daily ART. Our group has been at the forefront of exploring the viability of the block-and-lock remission approach, focused on the long-lasting epigenetic block of viral transcription such that without daily ART, there is no risk of viral rebound, transmission, or progression to AIDS. Numerous studies have reported inhibitors of both viral and host factors required for HIV transcriptional activation. Here, we highlight and review some of the latest HIV transcriptional inhibitor discoveries that may be leveraged for the clinical exploration of block-and-lock and revolutionize the way we treat HIV infections.
Assuntos
Infecções por HIV , HIV-1 , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Provírus/genética , Latência ViralRESUMO
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
HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we have developed a cell-based 'gain of function' assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. We developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu; however, this assay may be slightly modified to include more significant Vpu strains in the future.
Assuntos
Fármacos Anti-HIV , HIV-1 , Proteínas do Vírus da Imunodeficiência Humana , Proteínas Virais Reguladoras e Acessórias , Proteínas Viroporinas , Fármacos Anti-HIV/química , Antígeno 2 do Estroma da Médula Óssea/metabolismo , Proteínas Ligadas por GPI/metabolismo , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Vírus da Leucemia do Macaco Gibão/metabolismo , Bibliotecas de Moléculas Pequenas , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Viroporinas/antagonistas & inibidoresRESUMO
Human immunodeficiency virus type 1 (HIV-1) continues to spread, principally by heterosexual sex, but no vaccine is available. Hence, alternative prevention methods are needed to supplement educational and behavioural-modification programmes. One such approach is a vaginal microbicide: the application of inhibitory compounds before intercourse. Here, we have evaluated the microbicide concept using the rhesus macaque 'high dose' vaginal transmission model with a CCR5-receptor-using simian-human immunodeficiency virus (SHIV-162P3) and three compounds that inhibit different stages of the virus-cell attachment and entry process. These compounds are BMS-378806, a small molecule that binds the viral gp120 glycoprotein and prevents its attachment to the CD4 and CCR5 receptors, CMPD167, a small molecule that binds to CCR5 to inhibit gp120 association, and C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion. In vitro, all three compounds inhibit infection of T cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, significant protection was achieved using each compound alone and in combinations. CMPD167 and BMS-378806 were protective even when applied 6 h before challenge.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/prevenção & controle , HIV/efeitos dos fármacos , Macaca mulatta/virologia , Fusão de Membrana/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vagina/virologia , Administração Intravaginal , Animais , Fármacos Anti-HIV/administração & dosagem , Antagonistas dos Receptores CCR5 , Antígenos CD4/metabolismo , Fusão Celular , Quimioterapia Combinada , Feminino , HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/transmissão , Infecções por HIV/virologia , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Receptores CCR5/metabolismo , Receptores Virais/antagonistas & inibidores , Receptores Virais/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/metabolismo , Fatores de Tempo , Vagina/efeitos dos fármacos , Valina/administração & dosagem , Valina/análogos & derivados , Valina/farmacologiaRESUMO
Recent clinical trials have shown that the use of the HIV-1 integrase (IN) inhibitor raltegravir (RAL) results in drops in the viral load that are more rapid than those achieved by use of the reverse transcriptase (RT) inhibitor efavirenz. Previously, mathematical modeling of viral load decay that takes into account the stage of viral replication targeted by a drug has yielded data that closely approximate the clinical trial results. This model predicts greater inhibition of viral replication by drugs that act later in the viral replication cycle. In the present study, we have added drugs that target entry, reverse transcription, integration, or proteolytic processing to acutely infected cells and have shown modest viral inhibition by entry inhibitors, intermediate levels of inhibition by RT and IN inhibitors, and high levels of inhibition by protease inhibitors relative to the levels of growth for the no-drug controls. When dual or triple combinations of these drugs were added to acutely infected cells, we found that the levels of inhibition achieved by any given combination were comparable to those achieved by the latest-acting drug in the combination. In single-round infections in which the kinetics of reverse transcription and integration had been determined by quantitative PCR, addition of IN inhibitors at various times postinfection resulted in levels of inhibition equal to or greater than those achieved by addition of RT inhibitors. Collectively, our data provide in vitro evidence of the stage-dependent inhibition of HIV-1 by clinically relevant drugs. We discuss how stage-dependent inhibition helps to explain the unique viral load decay dynamics observed clinically with RAL.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Inibidores da Transcriptase Reversa/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , DNA Viral/análise , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/genética , Humanos , Modelos Biológicos , Reação em Cadeia da Polimerase , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The amino acid at position 36 of the HIV-1 protease differs among various viral subtypes, in that methionine is usually found in subtype B viruses but isoleucine is common in other subtypes. This polymorphism is associated with higher rates of treatment failure involving protease inhibitors (PIs) in non-subtype B-infected patients. To investigate this, we generated genetically homogeneous wild-type viruses from subtype B, subtype C, and CRF02_AG full-length molecular clones and showed that subtype C and CRF02_AG I36 viruses exhibited higher levels of resistance to various PIs than their respective M36 counterparts, while the opposite was observed for subtype B viruses. Selections for resistance with each variant were performed with nelfinavir (NFV), lopinavir (LPV), and atazanavir (ATV). Sequence analysis of the protease gene at week 35 revealed that the major NFV resistance mutation D30N emerged in NFV-selected subtype B viruses and in I36 subtype C viruses, despite polymorphic variation. A unique mutational pattern developed in subtype C M36 viruses selected with NFV or ATV. The presence of I47A in LPV-selected I36 CRF02_AG virus conferred higher-level resistance than L76V in LPV-selected M36 CRF02_AG virus. Phenotypic analysis revealed a >1,000-fold increase in NFV resistance in I36 subtype C NFV-selected virus with no apparent impact on viral replication capacity. Thus, the position 36 polymorphism in the HIV-1 protease appears to have a differential effect on both drug susceptibility and the viral replication capacity, depending on both the viral subtype and the drug being evaluated.
Assuntos
Fármacos Anti-HIV/farmacologia , Códon/genética , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Polimorfismo Genético/genética , Fármacos Anti-HIV/uso terapêutico , Sulfato de Atazanavir , Linhagem Celular , Células Cultivadas , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Lopinavir , Nelfinavir/uso terapêutico , Oligopeptídeos/uso terapêutico , Piridinas/uso terapêutico , Pirimidinonas/uso terapêuticoRESUMO
The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.
Assuntos
Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/genética , Fármacos Anti-HIV/farmacologia , Sequência de Bases , Linhagem Celular , DNA Viral/biossíntese , DNA Viral/genética , Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/fisiologia , Humanos , Mutação , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Etravirine (ETR) is a second-generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) active against common human immunodeficiency virus type 1 (HIV-1) drug-resistant strains. This study was designed to determine the extent to which each of the Y181C or G190A mutations in RT might confer resistance to ETR and other members of the NNRTI family of drugs. Recombinant HIV-1 RT enzymes containing either the Y181C or the G190A mutation, or both mutations in tandem, were purified. Both RNA- and DNA-dependent DNA polymerase assays were performed in order to determine the extent to which each of these mutations might confer resistance in cell-free biochemical assays against each of ETR, efavirenz, and nevirapine. Both the biochemical and the cell-based phenotypic assays confirmed the susceptibility of G190A-containing enzymes and viruses to ETR. The results of this study indicate that the G190A mutation is not associated with resistance to ETR.
Assuntos
Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , Mutação , Piridazinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Farmacorresistência Viral/genética , Testes de Sensibilidade Microbiana , Nitrilas , Pirimidinas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Vpx encoded by HIV-2 and SIVsm enhances retroviral reverse transcription in macrophages in vitro by mediating the degradation of the host SAMHD1 protein that hydrolyzes dNTPs and by elevating cellular dNTP levels. Here we employed RT-SHIV constructs (SIV encoding HIV-1 RT) to investigate the contribution of Vpx to the potency of NRTIs, which compete against dNTPs, in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells. Relative to HIV-1, both SIV and RT-SHIV exhibited reduced sensitivities to AZT, 3TC and TDF in MDMs but not in activated CD4(+) T cells. However, when SIV and RT-SHIV constructs not coding for Vpx were utilized, we observed greater sensitivities to all NRTIs tested using activated CD4(+) T cells relative to the Vpx-coding counterparts. This latter phenomenon was observed for AZT only when using MDMs. Our data suggest that Vpx in RT-SHIVs may underestimate the antiviral efficacy of NRTIs in a cell type dependent manner.
Assuntos
Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Macrófagos/virologia , Inibidores da Transcriptase Reversa/farmacologia , Vírus da Imunodeficiência Símia/fisiologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Farmacorresistência Viral , Expressão Gênica , Genes Reporter , Vetores Genéticos/genética , HIV-1/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Ativação Linfocitária/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Testes de Sensibilidade Microbiana , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/imunologiaRESUMO
OBJECTIVE: To evaluate the candidate antiretroviral microbicide compounds, dapivirine (DAP) and tenofovir (TFV), alone and in combination against the transmission of wild-type and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 from different subtypes. DESIGN AND METHODS: We determined single-drug efficacy of the RTIs, DAP and TFV, against subtype B and non-B wild-type and NNRTI-resistant HIV-1 in vitro. To assess breadth of activity, compounds were tested alone and in combination against wild-type and NNRTI-resistant subtype C primary HIV-1 isolates and complimentary clonal HIV-1 from subtypes B, C and CRF02_AG to control for viral variation. Early infection was quantified by counting light units emitted from TZM-bl cells less than 48-h postinfection. Combination ratios were based on drug inhibitory concentrations (IC(50)s) and combined effects were determined by calculating combination indices. RESULTS: Both candidate microbicide antiretrovirals demonstrated potent anti-NNRTI-resistant HIV-1 activity in vitro, albeit the combination protected better than the single-drug treatments. Of particular interest, the DAP with TFV combination exhibited synergy (50% combination index, CI(50) = 0.567) against subtype C NNRTI-resistant HIV-1, whereas additivity (CI(50) = 0.987) was observed against the wild-type counterpart from the same patient. The effect was not compounded by the presence of subdominant viral fractions, as experiments using complimentary clonal subtype C wild-type (CI(50) = 0.968) and NNRTI-resistant (CI(50) = 0.672) HIV-1, in lieu of the patient quasispecies, gave similar results. CONCLUSION: This study supports the notion that antiretroviral drug combinations may retain antiviral activity against some drug-resistant HIV-1 despite subtype classification and quasispecies diversity.
Assuntos
Adenina/análogos & derivados , Farmacorresistência Viral/efeitos dos fármacos , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Organofosfonatos/farmacologia , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Adenina/farmacologia , Quimioterapia Combinada , Infecções por HIV/genética , HIV-1/genética , Humanos , TenofovirRESUMO
OBJECTIVE: a) To assess the suitability of the curriculum content and didactical quality of information delivered to educate journalists in the J2J program in HIV/AIDS (process evaluation) and b) to explore the effects of such programs on journalists' reporting of HIV/AIDS related information (outcome evaluation). DESIGN: Descriptive study. METHODS: For the process evaluation, each J2J program curriculum was evaluated for accuracy and pertinence by individuals with high familiarity with HIV/AIDS research. For the outcome evaluation, a survey of J2J attendees and evaluations of the program lectures by attendees were performed in chronological order to determine their perception on usefulness of the program. RESULTS: Overall, the J2J curriculum is successful in providing journalists with a clear understanding of the current HIV/AIDS medical research objectives and issues with most journalists reporting an increased ability to better investigate and disseminate accurate information on this subject. Furthermore, the journalists surveyed reported positive community responses directly as a result of the J2J training. CONCLUSION: The J2J program helps to increase global awareness of pertinent HIV/AIDS concepts. Through this professional development strategy, journalists from around the world may help to amplify efforts to prevent new HIV infections and quench the dissemination of inaccurate information and folklore.
RESUMO
Inhibitors of viral entry are under consideration as topical microbicides to prevent HIV-1 sexual transmission. Small molecules targeting HIV-1 gp120 (BMS-378806) or CCR5 (CMPD167), and a peptide fusion inhibitor (C52L), each blocks vaginal infection of macaques by a SHIV. A microbicide, however, must be active against multiple HIV-1 variants. We therefore tested BMS-C (a BMS-378806 derivative), CMPD167, C52L and the CXCR4 ligand AMD3465, alone and in combination, against 25 primary R5, 12 X4 and 7 R5X4 isolates from subtypes A-G. At high concentrations (0.1-1 microM), the replication of most R5 isolates in human donor lymphocytes was inhibited by >90%. At lower concentrations, double and triple combinations were more effective than individual inhibitors. Similar results were obtained with X4 viruses when AMD3465 was substituted for CMPD167. The R5X4 viruses were inhibited by combining AMD3465 with CMPD167, or by the coreceptor-independent compounds. Thus, combining entry inhibitors may improve microbicide effectiveness.