Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). This devastating disease is estimated to have killed 20 million people to date, and approximately 36 million people worldwide are HIV positive.
We aim to understand cellular proteins involved in the early phase of the lifecycle of HIV-1. We have isolated a cell line mutant for retrovirus infection. It restricts infection by proteasome mediated degradation of incoming virus. This and other work lead us to hypothesize that the proteasome is directly involved in the uncoating of retroviral cores. Very little is known about the uncoating stage of HIV- 1 and this hypothesis is exploratory and novel. Specifically we hypothesize that one or more of the proteasome AAA ATPases with unfolding activity uncoat HIV-1. To test this hypothesis we propose two specific aims.
In Specific Aim 1 we will examine the in vivo interaction between the virus and the proteasome. Since proteasome mediated degradation of HIV-1 demands recognition of viral proteins by the signal dependent proteasome, we will addresses if this is a normal event in the lifecycle of HIV-1 or is promoted by mislocalization of the virus in mutant cells. To differentiate between these alternative models we will precisely determine the location and fate of incoming virus in wild-type (V79-4) and mutant (67-1) cells by fractionation of cell extracts after infection and tracking viral components. We will further track the virus visually after infection by using deconvoluting fluorescence microscopy. We will genetically test for the involvement of the AAA ATPases of the proteasome by introducing dominant negative mutants of the 6 key ATPases in the 19S regulatory particle of the proteasome in human U373 cells and examining the consequences on HIV-1 infection. A prediction of our hypothesis is one of these mutants will decrease infection.
In Specific aim 2, we will develop innovative and novel assays to examine the in vitro interaction between the virus and the proteasome. We will test the interaction of purified viral cores and the proteasome by developing a degradation assay for HIV-1 cores using cell extracts from wild type V79-4 and mutant 67-1 cells. We will also pioneer novel assays for the fate of core particles incubated with purified 19S regulatory, 20S catalytic and 26S proteasome (19S and 20S) subunits in vitro. Results from these genetic and biochemical analyses will test our hypothesis that retroviral uncoating is mediated by the proteasome, thus contributing to a greater understanding of a stage of the viral life cycle about which we currently know very little. Identification of proteins and signals that mediate interaction between viral cores and the proteasome will aid translational research for drug discovery to combat HIV-1 infection. ? ? ?
