EXCEED THE SPACE PROVIDED. Developmentof new drugs that inhibit HIV-1 replication would be aided by more precise understanding of HIV-1 replication mechanisms. HIV-1 gag is an appealing target for study since it encodes proteins that play essential roles in practically every step of the viral replication cycle. Over the previous funding period, gag sequence requirements for virion assembly were extensively characterized. New Gag-interacting factors, including EFl-alpha, were discovered by screening for Matrix-interacting proteins.
Aim 1. Among new Capsid-binding factors, Hsp70 was shown to be packaged within the virion membrane of HIV-1 but not that of other viruses. Sequence requirements for the Capsid-Hsp70 interaction will be characterized in detail and this information will be exploited in experiments designed to determine the function of Hsp70 in HIV-1 replication.
In Aim 2 we will determine the functional significance of our discovery that p6 interacts with SUMO-1, Ubc-9, and Daxx. These proteins localize to nuclear PML bodies, regulate apoptosis, and are believed important for replication of DNA viruses.
Aim 3 is to develop more efficient methods for targeted gene disruption by homologous recombination in somatic cells. These methods will signficantly advance efforts to determinethe functional signficance of factors that interact with HIV-1 proteins. As a test case we will disrupt the gene encoding Daxx in Jurkat T cells.
Aim 4 concerns a new screen based on function for host factors that regulate HIV-1 replication. ModifiedHIV-1 proviruses were shown to stably propagate and express heterologouscDNAs from within the HIV-1 genome. To permit efficientcDNA cloning, the proviral plasmids were engineeredinto ~-phage. Plasmids bearing a cDNA library in the context of infectious HIV-1 provirus were excised from phage by Cre-lox recombination. Cells non-permissivefor HIV-1 replication, due to species- or cell type-specificblocks, will be infectedwith cDNA library-containingvirus, cDNAs encoding factors that promote HIV-1 replication under these conditionswill have the potential to 'clone themselves'by 'rescuing' replication of the provirus that bears them. The role these factors play in HIV-1 replication will be characterized. PERFORMANCE SITE ========================================Section End===========================================
