RESUMO
The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.
Assuntos
Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Mutação/genética , Receptores de HIV/metabolismo , Sítios de Ligação , Linhagem Celular , Enfuvirtida , Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Fusão de Membrana , Fragmentos de Peptídeos/farmacologia , Ligação ProteicaRESUMO
In this study, we report the first example of a nonpeptide chemokine receptor agonist, 2-{2-[4-(3-phenoxybenzyl)piperazin-1-yl]ethoxy}ethanol (ZK 756326), for the CC chemokine receptor CCR8. ZK 756326 inhibited the binding of the CCR8 ligand I-309 (CCL1), with an IC(50) value of 1.8 muM. Furthermore, ZK 756326 was a full agonist of CCR8, dose-responsively eliciting an increase in intracellular calcium and cross-desensitizing the response of the receptor to CCL1. In addition, ZK 756326 stimulated extracellular acidification in cells expressing human CCR8. The ability of ZK 756326 to induce a response was receptor-specific and mediated through Galpha(i), because it could be blocked by treatment with pertussis toxin. The CCR8 agonist activated cells expressing murine CCR8, eliciting their chemotaxis and inducing phosphorylation of extracellular signal-regulated kinase ERK1/2. Like CCL1, ZK 756326 inhibited human immunodeficiency virus (HIV) fusion of cells expressing CD4 and CCR8. Finally, unlike mCCL1, ZK 756326 bound to and activated a form of mCCR8 that was mutated to eliminate O-linked sulfation at tyrosines 14 and 15. Therefore, ZK 756326 is most probably not binding in the same manner as CCL1 but can activate the switch mechanism involved in transducing signaling events. In summary, we have identified a nonpeptide agonist of CCR8. This compound may be useful in evaluating the physiological role of CCR8 in HIV infection, as well as in the general study of CCR8 biology without the constraints inherent to the use of protein agonists such as its natural ligand.
Assuntos
Piperazinas/farmacologia , Receptores de Quimiocinas/agonistas , Animais , Ligação Competitiva , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos , Fosforilação , Piperazinas/química , Receptores CCR8 , Transdução de Sinais , TransfecçãoRESUMO
Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4 degrees C became sensitive to peptide within the first 5 min of elevating the temperature to 37 degrees C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.
Assuntos
Vírus da Leucose Aviária/efeitos dos fármacos , Vírus do Sarcoma Aviário/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas Virais de Fusão/química , Sequência de Aminoácidos , Animais , Vírus da Leucose Aviária/patogenicidade , Vírus do Sarcoma Aviário/patogenicidade , Fusão Celular , Linhagem Celular , Dados de Sequência Molecular , Mutação , Peptídeos/síntese química , Peptídeos/química , Sequências Repetitivas de AminoácidosRESUMO
An increasingly large number of antiviral agents that prevent entry of human immunodeficiency virus (HIV) into cells are in preclinical and clinical development. The envelope (Env) protein of HIV is the major viral determinant that affects sensitivity to these compounds. To understand how changes in Env can impact entry inhibitor sensitivity, we introduced six mutations into the conserved coreceptor binding site of the R5 HIV-1 strain YU-2 and measured the effect of these changes on CD4 and coreceptor binding, membrane fusion levels and rates, virus infection, and sensitivity to the fusion inhibitors enfuvirtide (T-20) and T-1249, the CCR5 inhibitor TAK-779, and an antibody to CD4. The mutations had little effect on CD4 binding but reduced CCR5 binding to various extents. In general, reductions in coreceptor binding efficiency resulted in slower fusion kinetics and increased sensitivity to TAK-779 and enfuvirtide. In addition, low CCR5 binding usually reduced overall fusion and infection levels. However, one mutation adjacent to the bridging sheet beta21 strand, P438A, had little effect on fusion activity, fusion rate, infectivity, or sensitivity to enfuvirtide or T-1249 despite causing a marked reduction in CCR5 binding and a significant increase in TAK-779 sensitivity. Thus, our findings indicate that changes in the coreceptor binding site of Env can modulate its fusion activity, infectivity, and entry inhibitor sensitivity by multiple mechanisms and suggest that reductions in coreceptor binding do not always result in prolonged fusion kinetics and increased sensitivity to enfuvirtide.
Assuntos
Fármacos Anti-HIV/farmacologia , Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , HIV-1/fisiologia , Fusão de Membrana , Receptores CCR5/metabolismo , Amidas/farmacologia , Sítios de Ligação , Antígenos CD4/química , Linhagem Celular , Enfuvirtida , Proteína gp120 do Envelope de HIV/fisiologia , Proteína gp41 do Envelope de HIV/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Mutação , Fragmentos de Peptídeos/farmacologia , Compostos de Amônio Quaternário/farmacologia , Receptores CCR5/química , Relação Estrutura-AtividadeRESUMO
Characterization of the mechanisms by which HIV-1 enters cells has allowed for an increased understanding of not only tropism and pathogenesis, but also the identification of new targets for rational drug design. Several classes of HIV-1 entry inhibitors have been developed. Antagonists targeting the interaction of the viral envelope protein and receptors on the cell surface, as well as peptides that target an intermediate in the fusion process, have shown promise in vitro and are currently being evaluated in clinical trials. The addition of entry inhibitors to current drug regimens has the potential to significantly improve the therapeutic options for infected individuals, which is crucial for those resistant to or failing currently available therapies.