RESUMO
Whereas gp120 CD4-induced structures have been largely documented and at least in part elucidated by crystallization, information about gp120 coreceptor-induced structures remains incomplete despite numerous studies. In this work, mutations were carried out in a selected internal region of HIV-1/YU2 gp120, proximal to the CD4-binding site, because of its highly conserved nature among retroviruses and its high structural stability. The targeted residues, belonging to the beta16/beta17 beta-hairpin, modulate gp120 binding to CD4 and gp120-CD4 complex binding to CCR5. Thus, it appears that this gp120 structure acts as a hinge between the CD4-binding site and the putative coreceptor binding structure. Substitution of amino acid residues like E381A did not affect gp120 binding to CD4 and did not induce significant structural changes in gp120, as demonstrated by epitope analysis, BIACORE analysis, and circular dichroism. Nevertheless, E381 has a critical influence on the maintenance of CCR5 coreceptor binding by forming a salt bridge with K207. Another important element of the beta-hairpin in this interaction is the probable hydrophobic link between F383 and I420. Altogether, these results suggest that the beta-hairpin structure likely governs interactions between the surface of gp120 with native CCR5 or the CCR5 amino-terminal domain (CCR5-Nt). The mutations within the beta-hairpin had a direct effect on the proximal surface of the bridging sheet, the putative CCR5 surface, and the gp120 YU2 HIV-1-CD4 binding site. These results on the gp120-CCR5-Nt binding mechanism contribute to our understanding of CCR5 and HIV-1 gp120 association and HIV-1 entry; they may also contribute to designing novel inhibitors.
Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/química , HIV-1/metabolismo , Receptores CCR5/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígenos CD4/metabolismo , Dicroísmo Circular , Citometria de Fluxo , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Secundária de Proteína , Homologia de SequênciaRESUMO
OBJECTIVES: The soluble HIV-1 gp120 envelope glycoprotein, after being shed from infected cells, can cross-link its receptors on both HIV-1 infected and non-infected target cells, leading to their activation. We have assessed the impact of soluble gp120 on viral replication in CD4+/CXCR4+ T cells, via its effects on Tat-mediated transactivation of the HIV-1/LTR. MATERIALS AND METHODS: Primary cord blood-derived CD4+/CXCR4+ T cells were stimulated with soluble recombinant gp120 (rgp120) from the HIV-1/HXB2 clone. The level of gene or protein expression was assessed by serial analysis gene expression (SAGE), reverse transcriptase-polymerase chain reaction, western blotting or flow-cytometry analysis. Cellular division of rgp120-stimulated T cells was assessed by CFDA-SE labeling. Long terminal repeat (LTR) activity and HIV infection level were respectively measured by a chemiluminescent beta-gal Reporter Gene Assay and by p24 determination. RESULTS: We have demonstrated that rgp120 activates both PKCepsilon and its upstream effector PI3K/Akt, involved in the HIV-1 replication process. Moreover, rgp120 enhances the gene, as well as protein expression of the cellular Tat cofactors Tat-Sf1 and SPT5 in primary CD4+/CXCR4+ T cells. Finally, stimulation of HIV-1 infected T cells with rgp120 was found to result in both a higher LTR-activity and an increased production of viral particles. CONCLUSION: Taken together, these results show that soluble gp120 contributes to HIV-1 replication and dissemination, via the activation of multiple cell signaling pathways and the induction of Tat-cofactor expression, underscoring its potential as a therapeutic target in HIV-1-mediated pathogenesis.
Assuntos
Linfócitos T CD4-Positivos/virologia , Produtos do Gene tat/metabolismo , Proteína gp120 do Envelope de HIV/farmacologia , HIV-1/fisiologia , Replicação Viral/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Repetição Terminal Longa de HIV/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Receptores CXCR4/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais/imunologia , Solubilidade , Ativação Transcricional/efeitos dos fármacos , Replicação Viral/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
BACKGROUND: CCL28 (MEC) binds to CCR3 and CCR10 and recruits IgA-secreting plasma cells (IgA-ASC) in the mucosal lamina propria (MLP). Mucosal HIV-specific IgA are detected in HIV-infection and exposure. The CCL28 circuit was analyzed in HIV-infected and-exposed individuals and in HIV-unexposed controls; the effect of CCL28 administration on gastrointestinal MLP IgA-ASC was verified in a mouse model. METHODOLOGY/FINDINGS: CCL28 was augmented in breast milk (BM) plasma and saliva of HIV-infected and -exposed individuals; CCR3+ and CCR10+ B lymphocytes were increased in these same individuals. Additionally: 1) CCL28 concentration in BM was associated with longer survival in HIV vertically-infected children; and 2) gastro-intestinal mucosal IgA-ASC were significantly increased in VSV-immunized mice receiving CCL28. CONCLUSIONS: CCL28 mediates mucosal immunity in HIV exposure and infection. CCL28-including constructs should be considered in mucosal vaccines to prevent HIV infection of the gastro-intestinal MLP via modulation of IgA-ASC.