Early work on HIV focused on understanding proteins unique to this virus and led to the discovery of anti- retroviral agents that target these viral proteins. Recent years have seen a shift in research towards understanding host factors that act at a number of stages in the HIV life cycle. Further understanding of these cellular proteins could lead to the development of a new class of anti-retroviral agents that target such viral-host interactions. One important stage of the viral life cycle in which viral-host interactions have not yet been identified is viral capsid assembly. We have recently identified a nucleotide-binding host protein (HP68) that appears to be essential for post-translational events in immature capsid formation. HP68 is associated with assembling HIV Gag chains in a variety of systems, including HIV-I-infected cells. When a truncation mutant in HP68 is expressed in mammalian cells, HIV particle formation is dramatically reduced through a dominant negative mechanism. Furthermore, immunodepletion of HP68 from a cell-free assembly system results in post-translational blockade of capsid assembly, and recoristitution with purified HP68 restores formation of completed capsids. Together, these data indicate that HP68 plays an important role in assembly. However, to understand the role of this host factor for pathogenesis, many additional questions need to be answered. Previously, HP68 was identified as an RNase L inhibitor that is upregulated by HIV-1 and blocks viral RNA degradation. Our data indicate that HP68 has a second function in which it also acts post translationally to promote capsid assembly. 'To test this hypothesis, we will first define the effect of HP68 constructs on viral RNA levels, Gag synthesis, and 1124 release using quantitative assays. Secondly, basic biochemical studies will be performed to identify whether HP68 binds ATP or GTP, associates with membranes, and binds to RNA. Mutagenesis will be used to map domains in HP68 as well as domains in Gag that are involved in the HP68-Gag interaction and capsid formation. From these studies we will be able to determine the role of ATP in HP68 function. Finally, we will study whether HP68 acts by promoting a conformational change in capsid assembly intermediates. Information (concerning the binding of HP68 to HIV-1 Gag will be utilized to pilot methods for blocking capsid formation. The possibility that IiP68 is a co-factor for virus production is an exciting one, given the importance of finding new anti-retroviral targets and extending the gains obtained with highly active anti-retroviral therapy (HAART). The studies proposed here will define the role of this novel transacting factor in HIV disease, and lay the foundation for assays that could be used for anti-retroviral drug screening in the future.
