Resumo

Nef is an accessory protein expressed early during the replication cycle of the primate lentiviruses HIV and SIV. This protein plays an essential role in viral infectivity and progression to AIDS. It has been reported that Nef interacts with different cellular partners to perform several functions in the viral replicative cycle, but the function related to increased viral infectivity in primary lymphocytes and macrophages has not been described. Nef may mediate downregulation of surface expression of CD4 membrane molecules and may prevent apoptosis in T cells infected with HIV-1. It has been reported that the cellular protein Alix/AIP1 plays a central role in directing the ESCRT machinery and this is essential for the budding of certain enveloped viruses such as HIV-1. We are investigating the role of the interaction between Nef and cellular protein Alix/AIP1. Nef interaction with Alix/AIP1 have been previously mapped to the amino acid residues YPLTF present at the positions 135-139 on the C-terminal of the Nef protein from the HIV NL4-3 isolate. The objective of this study is to elucidate the interaction between these two proteins has an influence on the increase in viral infectivity. For this, siRNA knockout assays were performed in Hek293T cell cultures. The knockout was carried out from the transfection of siRNA against Alix/AIP1. The expression of Alix was monitored by Western blotting with specific antibodies against Alix showing a 96% of knockout after 24h when it was used 50mM siRNA. So we started testing transfection of plasmids NL 4.3 and NL 4.3 ΔNef after 24h of knockout. Interestingly, the transfection of the plasmids, NL 4.3 and NL 4.3 ΔNef induce an upregulation in the expression of Alix/AIP1 even in knockout cells, and this regulation is still greater in the absence of Nef, indicating that this viral protein has essential role in the negative regulation of Alix. In addition, tests for infectivity of viruses produced in these conditions showed a dependence on Alix to increase of viral infectivity of 60% when using NL 4.3 and this increase was not observed when was used the NL 4.3 ΔNef mutant. We assume that we do not observed any difference when was used the NL 4.3 ΔNef because of the cells transfected with this plasmid recuperated the Alix levels. The results indicate that the Alix/AIP1 protein has a role in increased infectivity of HIV-1 in the presence of Nef protein.