Study the Mechanism of Nef Induced Endosome Re-Distribution and the Role in HIV Immune Evasion

Nef, a determinant of human immunodeficiency virus (HIV) pathogenicity, is required for the maximal virus replication and disease progression in vivo. The HIV persistency is attributed to the ability of Nef in immune evasion, including MHC-I downregulation, anti-apoptosis activity, FasL upregulation, and prevention of the formation of immune synapse. Nef also directly or indirectly modulates the expression of the level of many cell surface proteins to influence viral pathogenesis. So far, more than 20 cell surface proteins are up- or down-modulated by Nef but the understanding on the mechanisms, which modulate these proteins, are limited. Among these proteins, MHC-I has been studied extensively. Our study revealed that Nef used PACS-1/Src/PI3K signaling pathway to usurp an ARF6-dependent endocytic pathway, accompany with the blocking of MHC-I recycling, to downregulate cell-surface MHC-I molecules to the trans-Golgi network (TGN). My current work observes that the endosome in the Nef-expressing cell is concentrated at the perinuclear region rather than dispersed punctate distribution in control cells. My study indicates that the Nef EEEE65 motif and PACS-1 are involved in the process of Nef-induced endosome movement. Since changing the distribution of endosome may be accompanied with the functional changes of endosome, resulting in global changes of vesicular trafficking pathway and modulating surface expression of proteins, the objectives of this project are to investigate whether Nef-induced endosome movement changes the normal functions of endosome and leads to the changes in the surface expression of many proteins. Alix and KIF16B siRNA, which causes endosome aggregation, will be used to mimic the situation of Nef-induced endosome aggregation. The kinetics of vesicular trafficking will be study in details, including the efficiency of protein transport to plasma membrane, the rate of endocytosis and recycling, and the rate of protein degradation. Furthermore, this study will determine the involvement of Nef motifs and corresponded interacting proteins in Nef-induced endosome movement. Specifically, this study will focus on the study of the interaction among Nef, PACS-1 and molecular motors of microtubule on the control of the movement of endosome. The interaction and regulation between Nef and Alix in the endosome movement will also be investigated. The downstream targets of Nef-induced PACS-1/Src/PI3K signaling affected by endosome aggregation will be determined. These results will provide a better understanding of the Nef functions and pathogenesis of AIDS. From these studies, the global change induced by Nef can provide a good target for future therapy.

Project ID:PC9709-0440
External Project ID:NSC97-2320-B182-028