RESUMO
BACKGROUND: The CCR5 antagonist maraviroc (MVC) inhibits human immunodeficiency virus type 1 (HIV-1) entry by altering the CCR5 extracellular loops (ECL), such that the gp120 envelope glycoproteins (Env) no longer recognize CCR5. The mechanisms of HIV-1 resistance to MVC, the only CCR5 antagonist licensed for clinical use are poorly understood, with insights into MVC resistance almost exclusively limited to knowledge obtained from in vitro studies or from studies of resistance to other CCR5 antagonists. To more precisely understand mechanisms of resistance to MVC in vivo, we characterized Envs isolated from 2 subjects who experienced virologic failure on MVC. RESULTS: Envs were cloned from subjects 17 and 24 before commencement of MVC (17-Sens and 24-Sens) and after virologic failure (17-Res and 24-Res). The Envs cloned during virologic failure showed broad divergence in resistance levels, with 17-Res Env exhibiting a relatively high maximal percent inhibition (MPI) of ~90% in NP2-CD4/CCR5 cells and peripheral blood mononuclear cells (PBMC), and 24-Res Env exhibiting a very low MPI of ~0 to 12% in both cell types, indicating relatively "weak" and "strong" resistance, respectively. Resistance mutations were strain-specific and mapped to the gp120 V3 loop. Affinity profiling by the 293-Affinofile assay and mathematical modeling using VERSA (Viral Entry Receptor Sensitivity Analysis) metrics revealed that 17-Res and 24-Res Envs engaged MVC-bound CCR5 inefficiently or very efficiently, respectively. Despite highly divergent phenotypes, and a lack of common gp120 resistance mutations, both resistant Envs exhibited an almost superimposable pattern of dramatically increased reliance on sulfated tyrosine residues in the CCR5 N-terminus, and on histidine residues in the CCR5 ECLs. This altered mechanism of CCR5 engagement rendered both the resistant Envs susceptible to neutralization by a sulfated peptide fragment of the CCR5 N-terminus. CONCLUSIONS: Clinical resistance to MVC may involve divergent Env phenotypes and different genetic alterations in gp120, but the molecular mechanism of resistance of the Envs studied here appears to be related. The increased reliance on sulfated CCR5 N-terminus residues suggests a new avenue to block HIV-1 entry by CCR5 N-terminus sulfopeptidomimetic drugs.
Assuntos
Fármacos Anti-HIV/farmacologia , Cicloexanos/farmacologia , Proteína gp120 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação de Sentido Incorreto , Triazóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Cicloexanos/uso terapêutico , Variação Genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Maraviroc , Dados de Sequência Molecular , Análise de Sequência de DNA , Falha de Tratamento , Triazóis/uso terapêuticoRESUMO
Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro-generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop-CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.
Assuntos
Cicloexanos/farmacologia , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Macrófagos/virologia , Receptores CCR5/metabolismo , Triazóis/farmacologia , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos , Maraviroc , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Tropismo ViralRESUMO
BACKGROUND: Maraviroc (MVC) and other CCR5 antagonists are HIV-1 entry inhibitors that bind to- and alter the conformation of CCR5, such that CCR5 is no longer recognized by the viral gp120 envelope (Env) glycoproteins. Resistance to CCR5 antagonists results from HIV-1 Env acquiring the ability to utilize the drug-bound conformation of CCR5. Selecting for HIV-1 resistance to CCR5-antagonists in vitro is relatively difficult. However, the CCR5-using CC1/85 strain appears to be uniquely predisposed to acquiring resistance to several CCR5 antagonists in vitro including MVC, vicriviroc and AD101. FINDINGS: Here, we show that Env derived from the parental CC1/85 strain is inherently capable of a low affinity interaction with MVC-bound CCR5. However, this phenotype was only revealed in 293-Affinofile cells and NP2-CD4/CCR5 cells that express very high levels of CCR5, and was masked in TZM-bl, JC53 and U87-CD4/CCR5 cells as well as PBMC, which express comparatively lower levels of CCR5 and which are more commonly used to detect resistance to CCR5 antagonists. CONCLUSIONS: Env derived from the CC1/85 strain of HIV-1 is inherently capable of a low-affinity interaction with MVC-bound CCR5, which helps explain the relative ease in which CC1/85 can acquire resistance to CCR5 antagonists in vitro. The detection of similar phenotypes in patients may identify those who could be at higher risk of virological failure on MVC.
Assuntos
Antagonistas dos Receptores CCR5 , Cicloexanos/farmacologia , Farmacorresistência Viral , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , Triazóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Linhagem Celular , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Maraviroc , Ligação Proteica/efeitos dos fármacos , Receptores CCR5/metabolismoRESUMO
Maraviroc (MVC) is an allosteric inhibitor of human immunodeficiency virus type 1 (HIV-1) entry, and is the only CCR5 antagonist licensed for use as an anti-HIV-1 therapeutic. It acts by altering the conformation of the CCR5 extracellular loops, rendering CCR5 unrecognizable by the HIV-1 envelope (Env) glycoproteins. This study aimed to understand the mechanisms underlying the development of MVC resistance in HIV-1-infected patients. To do this, we obtained longitudinal plasma samples from eight subjects who experienced treatment failure with phenotypically verified, CCR5-tropic MVC resistance. We then cloned and characterized HIV-1 Envs (n = 77) from plasma of pretreatment (n = 36) and treatment failure (n = 41) samples. Our results showed variation in the magnitude of MVC resistance as measured by reductions in maximal percent inhibition of Env-pseudotyped viruses, which was more pronounced in 293-Affinofile cells compared to other cells with similar levels of CCR5 expression. Amino acid determinants of MVC resistance localized to the V3 Env region and were strain specific. We also observed minimal cross-resistance to other CCR5 antagonists by MVC-resistant strains. We conclude that 293-Affinofile cells are highly sensitive for detecting and measuring MVC resistance through a mechanism that is CCR5-dependent yet independent of CCR5 expression levels. The strain-specific nature of resistance mutations suggests that sequence-based diagnostics and prognostics will need to be more sophisticated than simple position scoring to be useful for managing resistance in subjects taking MVC. Finally, the minimal levels of cross-resistance suggests that recognition of the MVC-modified form of CCR5 does not necessarily lead to recognition of other antagonist-modified forms of CCR5.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Antagonistas dos Receptores CCR5/uso terapêutico , Cicloexanos/uso terapêutico , Farmacorresistência Viral/genética , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/tratamento farmacológico , Receptores CCR5/efeitos dos fármacos , Triazóis/uso terapêutico , Adulto , Contagem de Linfócito CD4 , Linhagem Celular , Feminino , Células HEK293 , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Masculino , Maraviroc , Pessoa de Meia-Idade , Falha de Tratamento , Internalização do Vírus/efeitos dos fármacosAssuntos
Antagonistas dos Receptores CCR5/uso terapêutico , Infecções por HIV/tratamento farmacológico , Maraviroc/uso terapêutico , Receptores CCR5/fisiologia , Receptores CXCR4/fisiologia , Contagem de Linfócito CD4 , Método Duplo-Cego , Farmacorresistência Viral , HIV-1/genética , HIV-1/imunologia , Humanos , Placebos , RNA Viral/análiseRESUMO
Human immunodeficiency virus type 1 (HIV-1) resistance to CCR5 antagonists, including maraviroc (MVC), results from alterations in the HIV-1 envelope glycoproteins (Env) enabling recognition of antagonist-bound CCR5. Here, we characterized tropism alterations for CD4+ T-cell subsets and macrophages by Envs from two subjects who developed MVC resistance in vivo, which displayed either relatively efficient or inefficient recognition of MVC-bound CCR5. We show that MVC-resistant Env with efficient recognition of drug-bound CCR5 displays a tropism shift for CD4+ T-cell subsets associated with increased infection of central memory T-cells and reduced infection of effector memory and transitional memory T-cells, and no change in macrophage infectivity. In contrast, MVC-resistant Env with inefficient recognition of drug-bound CCR5 displays no change in tropism for CD4+ T-cell subsets, but exhibits a significant reduction in macrophage infectivity. The pattern of HIV-1 tropism alterations for susceptible cells may therefore be variable in subjects with MVC resistance.