Understanding how HIV-1 Vpu and HIV-2 Env stimulate virus release

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Primate lentiviruses code for a conserved function that is necessary for the efficient production of viral particles and in vivo pathogenicity. For HIV-1 this is carried out by the accessory protein Vpu, while for HIV-2, the function has been mapped to the Env glycoprotein. In addition, our studies with the simple retrovirus, murine leukemia virus (MLV), have identified a similar activity in its Env protein. We have found that all three of these enhancers of virus release (EVRs) can boost the production of heterologous retroviruses, suggesting that they act on the general cellular pathways involved in retrovirus budding. Analysis of EVR-responsive and non-responsive cell lines has led to the suggestion that human cells contain a natural restriction factor that suppresses retrovirus budding and which the EVRs have evolved to counteract. Therefore, it is our central hypothesis that EVR function is a conserved and important activity for retroviruses in general. Although the EVR activity of Vpu was first described in 1989, the mechanism whereby it enhances virus release is still unknown and represents a significant gap in our knowledge of HIV biology. Even less is understood about the HIV-2 Env activity, which shows both similarities and striking differences with Vpu. The research plan outlined in this proposal will take a comprehensive approach to studying retroviral EVRs, both in order to identify the functional domains of the viral proteins involved, and to understand the host cell processes that are the targets of their activity. Using the three different EVRs that we have identified, we will precisely define the functional domains within these proteins and assess whether they have the same cellular target and activity, or whether a common EVR phenotype is achieved through different pathways. We will also take advantage of a non-Vpu-responsive HeLa cell line that we have identified, which retains the ability to respond to the HIV-2 Env EVR. Preliminary studies of this cell lines are already suggesting a mechanism for Vpu `s activity. We will directly examine how EVRs affect virus assembly and budding and their influence on cellular trafficking pathways, using a combination of microarray, biochemical and immunofluorescence approaches. Finally, we will attempt to clone the human cell restriction factor using approaches based on the microarray analysis of complex cDNA and siRNA libraries. Relevance of research to public health: HIV infection and AIDS continue to be a major public health problem, with anti-HIV drugs only available against limited viral targets. The HIV-1 Vpu protein is essential for HIV pathogenicity but its mechanism of action is unclear. It is our goal to investigate the mechanism behind this important HIV function and thereby identify new targets for drug development.