263 Characterization of Endocytic Proteins Involved in HIV-1 Budding A. Oztan*, O.A. Weisz, D. Stolz, S.C. Watkins, L. Traub, R.C. Montelaro Univ of Pittsburgh, PA
Background: Retroviral budding mechanistically resembles both endocytic vesicle formation at the plasma membrane and intracellular vesicle formation in late endosomes. Recent studies from our lab and others have indicated a critical link between retroviral budding and various components of these two processes. Based on these initial experiments, we hypothesize that retroviral budding is mediated by a highly concerted sequential interaction of Gag proteins with early endocytic proteins.
Methods: To test this hypothesis, we used a combination of microscopic imaging and biochemical techniques to identify candidate cellular endocytic proteins recruited by HIV-1 Gag proteins to achieve viral budding and release at the plasma membrane.
Results: Double-label immunofluorescence staining of HIV-1 producer T-lymphocytes with antibodies against selected endocytic proteins and HIV-1 Gag indicates co-localization. In addition to fluorescence labeling, we used immuno-gold labeling and TEM to examine protein localization at the sites of budding particles. We also analyzed co-immunoprecipitation of cellular proteins and HIV-1 Gag. In these studies, the use of producer cell lines provides a unique opportunity to study the cellular factors involved in late stages of HIV-1 life cycle in natural target cells in the presence of full length viral genome.
Conclusions: Studies to date indicate a specific role of selected early endocytic proteins in HIV-1 budding.
