RESUMO
Balanced processing of HIV-1 RNA is critical to virus replication and is regulated by host factors. In this report, we demonstrate that overexpression of either Tra2α or Tra2ß results in a marked reduction in HIV-1 Gag/Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA. A natural isoform of Tra2ß (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing. The functional differences between the Tra2ß isoforms were also observed in the context of another RNA substrate indicating that these factors have distinct functions within the cell. Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA. Together, the findings indicate that Tra2α/ß can play important roles in regulating HIV-1 RNA metabolism and expression.
Assuntos
HIV-1/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Regulação da Expressão Gênica , Células HEK293 , HIV-1/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sítios de Splice de RNA , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina , Transdução de Sinais , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
In order to analyze the role of alpha(v)beta(3) and alpha(v)beta(5) integrins in gene transfer by adenovirus-based vectors, an RGD4C motif was inserted into the HI-loop of wild type or shortened fiber protein of human adenovirus of serotype 5, thereby creating Ad5RGD4C and Ad5Delta639RGD4C vectors, respectively. Infection by the latter is independent of the Coxsackie B adenovirus receptor. Internalization and transduction of these vectors and were investigated in several stably transfected cell clones derived from a human laryngeal carcinoma cell line (HEp2) expressing different ratios of alpha(v)beta(5) and alpha(v)beta(3) integrins. We show that alpha(v)beta(3) is more successful than alpha(v)beta(5) in: (i) mediating adenovirus internalization and transduction when the RGD motif is present only in the penton base and (ii) mediating internalization and transduction by RGD4C-fiber modified adenoviruses. The highest amount of internalized virus was found in the cell clone in which alpha(v)beta(3) integrin predominated over alpha(v)beta(5) integrin (as judged by the % of cells expressing alpha(v)beta(3) and alpha(v)beta(5) integrins). However the level of transgene expression in this cell line was even lower than that in parental HEp2 cells which do not express alpha(v)beta(3) integrin. This discrepancy between internalization and transgene expression (transduction) is likely due to the crucial role of alpha(v)beta(5) in membrane permeabilization, indicating that alpha(v)beta(5) integrin is a limiting factor for Ad5-mediated gene transfer. We conclude that alpha(v)beta(3) integrin is an efficient adenovirus internalization receptor, but cannot functionally replace alpha(v)beta(5) in endosomal release.