The Role of Lipid Rafts in Vpu Function

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DESCRIPTION (provided by applicant): Human immunodeficiency virus type 1 (HIV-1) encodes for a small type I membrane protein known as Vpu and has two major functions in the infected cell. Vpu is known to interact with and shunt the CD4 molecule from the rough endoplasmic reticulum (RER) to the proteasome for degradation. In addition, Vpu is known to enhance virus release from infected cells. The assembly of HIV-1 viruses in CD4+ T cells lacking a vpu gene is characterized by the accumulation of and tethering of virus particles at the cell surface and the maturation of viruses into intracellular vesicles. This enhanced release function of Vpu has been associated with the transmembrane (TM) domain of the Vpu. Recently, Vpu has been shown to antagonize the antiviral activities of bone marrow stromal cell antigen 2 (BST-2) also known as CD317, HM1.24, or tetherin. BST-2 is an interferon inducible, lipid raft , type II integral membrane protein with an unusual topology. It contains a short N- terminal region followed by a transmembrane domain, a central extracellular coiled-coiled domain containing two N-liked glycosylation sites and a C-terminal glycosyl-phosphatidylinositol (GPI) anchor. We present preliminary data that the Vpu protein can be detected in lipid or membrane rafts. We further present data that Vpu can be found in lipid rafts isolated from infected cells and that cholesterol depleting drugs as lovastatin/ cyclodextrin reduce the level of Vpu in raft fractions. In the studies proposed of this application, we hypothesize that Vpu localization to lipid rafts is necessary for antagonism of BST-2 and enhancement of virus release. In Specific Aim 1, we propose to use site-directed mutagenesis to identify amino acid residues within the TM domain that are necessary for raft association and determine if Vpu proteins from other HIV-1 subtypes also associate with lipid rafts. In the Specific Aim 2, we propose to examine if raft association of Vpu is required to antagonize the antiviral activities of BST-2. We will determine time kinetics of Vpu association and if BST-2 disrupts this association, if a non-raft Vpu can still interact with BST-2, it will decrease cell surface expression of BST-2 and still cause BST-2 degradation. Finally, we will determine if a virus expressing a non-raft Vpu has impaired virus release. The results of these studies will provide novel information on Vpu association with membrane rafts and BST-2 antagonism as well as provide mechanistic insight into enhanced virus release function of Vpu. PUBLIC HEALTH RELEVANCE: The Vpu protein of HIV-1 has two important functions in the virus replication cycle. One of this functions is to down-modulate the receptor for the virus, CD4. The other function of the Vpu protein is to enhance virus release from infected cells. Recently, bone marrow stromal cell antigen 2 (BST-2) was identified as a virus restriction factor that is targeted for Vpu. The exact mechanism by which Vpu antagonizes the antiviral activities of BST-2 is still unknown. These studies will provide novel information on whether the membrane raft properties of Vpu are required for the interaction with and antagonism of BST-2. The information gained from these studies may result in novel antiviral strategies against HIV-1.