HIV is a complex retrovirus containing a number of genes not commonly found in other retroviruses. We are focusing on the analysis of two of these genes, vpu and vif. Vpu is encoded only by HIV-1 and encodes a small integral membrane protein. Vpu regulates virus release from the cell surface and degradation of CD4 in the endoplasmic reticulum. These two biological activities of Vpu are based on two independent and distinct molecular mechanisms that can be attributed to separable structural domains of Vpu. CD4 degradation involves a direct interaction with Vpu via the cytoplasmic domains of both proteins. The precise mechanism of degradation is still unclear; however, we have evidence that it is an energy-dependent process that requires phosphorylation of Vpu. On the other hand, Vpu-regulated virus release is sensitive to changes in the transmembrane (TM) domain of Vpu. HIV-2 which is very closely related to HIV-1 lacks a vpu gene. However, we recently found that in the absence of the HIV-2 Env glycoprotein, Vpu can enhance HIV-2 virus release. In the presence of the HIV-2 Env product, efficient virus release is observed which was not further enhanced by Vpu. We subsequently found that the HIV-2 Env product encodes a Vpu-like function that regulates, similar to Vpu, virus particle release. Vif is a 23 kDa basic protein which has an important function in regulating infectivity of progeny virions. The biochemical mechanism of Vif function is obscure. We analyzed the role of Vif by studying its subcellular distribution by cell fractionation as well as confocal microscopy. We found that a substantial portion of intracellular Vif protein is associated with the cytoskeleton, specifically intermediate filaments. The association of Vif with intermediate filaments is specific and can result in the reorganization of the cytoskeletal network. In addition, we found that Vif is incorporated into virus particles where it associates with the core of the virions. Mutational analysis of Vif suggests that a domain located near the N-terminus of Vif is involved in the interaction with vimentin. The interaction of Vif with vimentin results in the partial or complete reorganization of vimentin and, in extreme cases, culminates in the perinuclear aggregation of vimentin and Vif. Vif-dependent collapse of vimentin is cell-cycle dependent and occurs during late S to G2/M phase. The purpose of this project is to investigate the biological and biochemical functions of the HIV accessory proteins Vif and Vpu. One of our goals is to characterize the involvement of cellular factors in the activities of Vif or Vpu which will require a variety of virological and cell-biological techniques. From our studies on Vpu we expect to gain insights into the general principles of protein degradation from the ER and into mechanisms involving late stages of virus production, in particular the involvement of ion channels in export mechanisms. From our studies on Vif we not only expect to gain insights into the function of this viral factor but we hope to learn more about the role of the cytoskeleton in virus replication. Finally, we hope our studies will contribute not only to the general understanding of retrovirus function, but will also provide a basis for the assessment of viral proteins as potential antiviral targets.
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