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Show Mapping the Interactions of the Human ESCRT-I Complex Associated with HIV Budding Sanaz Ghaffarian and Professor Wesley Sundquist Department of Biochemistry ESCRT-I Complex Comprised of three proteins: Vps37p hVPS37'' Recruits protein machinery necessary for degradation ubiquitylated proteins in MVB pathway Is kidnapped by HIV - I Gag to recruit other MVB proteins forHIV budding TSG10I MVB Formation and Virus Budding m Yeast Two-Hvbrid 1111 11 I I Results 1 m HIV-l is an infectious retrovirus that attacks cells and incorporates its genetic material into the host cell's DNA. The virus kidnaps the host cell's machinery to create new viral proteins that can then be assembled into viral particles, which upon release from the cell can infect other cells. We are interested in the specific proteins that are involved in the viral release (budding) of HIV. The process of virus budding is analogous to Multivesicular Body (MVB) formation (modeled in yeast), which involves vesicles that are formed away from the cytoplasm and into the endosome. The endosome is a membrane-bound compartment located in the cytosol that sorts vesicles for degradation in the lysosome (the cell's digestive system). There are several key complexes that are involved in this process of vesicle formation at the endosomal membrane. These complexes are kidnapped by an HIV protein (HIV-l Gag) to the plasma membrane where they assist in HIV budding. The first protein complex is known as the ESCRT-I complex. In yeast, this complex is composed of three proteins: Vps23p, Vps28p, and Vps37p. The ESCRT-I complex normally functions by recruiting proteins destined for degradation to the membrane of the endosome. Other complexes required for vesicle formation are sequentially recruited. Similarly the human orthologs in the ESCRT-I complex (TSG101, hVPS28, and hVPS37) also function in MVB formation. Human ESCRT-I is also hijacked by HIV-l Gag and signals the recruitment of downstream protein complexes to facilitate virus budding. However, there has not been an acceptable human ortholog identified for yeast Vps37p. We have a putative match and are in the process of testing its interactions with the other proteins in the ESCRTI complex, namely TSG101. Various con-structs of the putative hVPS37 and TSG101 were produced and their interactions tested with the Yeast Two-Hybrid assay. The Yeast Two-Hybrid assay is a method used to determine whether two proteins interact. Furthermore, a positive interaction is signified by the presence of blue colonies of yeast. As a result of these studies, we have a preliminary interaction map established between TSG101 and hVPS37, and will repeat these experiments along with otherbiochemical assays to determine the interacting domains. The clarification of the human proteins in the ESCRT-I complex will greatly add to our knowledge on the process of HIV budding and vesicle formation, which has exciting implications in treatment of the disease. Acknowledgements Many thanks to Melissa Stuchell and the rest of the Sundquist lab for their help and support in my research. hVPS37 was originally found in a Yeast Two-Hybrid screen of TSG101 interactors conducted at Myriad Genetics, Salt Lake City. {71} |
