RESUMO
The evolution of HIV-1 sequences over time is the result of the selection of mutant variants that have escaped from host immune responses or the outgrowth of mutants with increased viral replication, or both. We investigated the contribution of both selection processes to the overall evolution of the Tat and Rev regulatory gene sequences from four individuals, ranging in time from just prior to seroconversion to stable asymptomatic infection. After sequencing at least 15 clones per sample per gene, we analyzed the sequence evolution of the MHC-I motifs that were predicted from the MHC-I haplotypes of these patients. For each identified Tat sequence, we tested the activity of the corresponding encoded protein in a transactivation assay in vitro. Our results suggest that the evolution of the Tat and Rev sequences from these individuals can be explained by mutational escape of the MHC-I epitopes and that no mutations that replaced the original sequences in the viral population are associated with either an increase or decrease in Tat activity. CTL-mediated selection appears to be an important determinant of HIV-1 regulatory gene sequence evolution during the early stages of infection.
Assuntos
Evolução Molecular , Produtos do Gene rev/genética , Produtos do Gene tat/genética , Infecções por HIV/genética , HIV-1/genética , Linfócitos T Citotóxicos/virologia , Sequência de Aminoácidos , Epitopos , Produtos do Gene rev/imunologia , Produtos do Gene tat/imunologia , Genes rev/genética , Genes rev/imunologia , Genes tat/imunologia , Soropositividade para HIV/genética , Soropositividade para HIV/virologia , Humanos , Dados de Sequência Molecular , Seleção Genética , Alinhamento de Sequência , Replicação Viral , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The Semliki Forest virus (SFV) vector system is a new approach for in vivo expression of heterologous proteins and can also be used to generate specific immune responses in animal models. HIV-1 envelope glycoprotein produced using the SFV expression system is correctly folded, cleaved, transported to the cell surface and exhibits functional activity. We evaluated a recombinant Semliki Forest virus naked RNA-based immunization protocol for generation of monoclonal antibodies against the HIV-1 envelope glycoprotein. In vitro-transcribed RNA encoding for the SFV replicase complex and Env protein of HIV-1 (HXB2 strain) was injected intramuscularly to mice. This approach elicited an Env-specific antibody response in four mice out of five and a monoclonal antibody, 12H2, directed against gp41 was produced. Our results show that recombinant SFV RNA immunization can potentially be used as a quick and direct method to produce monoclonal antibodies, with the particular advantage that vectored RNA, rather than purified antigen, delivers a complex oligomer produced correctly.
