Identification and characterization of vaccinia A26 protein in membrane fusion regulation

• Main Authors: Shu-Jung Chang, 張書蓉
• Other Authors: Wen Chang
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/39079822464086411235

博士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 101 === Vaccinia virus has a wide host range and infects different cell types in vitro and animals in vivo. It belongs to the Orthopoxvirus genus of Poxviridae and replicates in cytoplasm of the host cells. Vaccinia virus produces two forms of infectious particles, mature virus (MV) and extracellular enveloped virus (EV) in the infected cells. MV is released upon cell lysis and resistant to heat and environmental stresses. Previous studies showed that MV enters cells through either endocytosis or plasma membrane fusion; however, it was not known how vaccinia virus chooses between these two entry pathways. Here using genetic approaches, we found that vaccinia viruses containing A25 and A26 enter HeLa, L, and CHO-K1 cells through low-pH dependent endocytosis whereas viruses without A25 and A26 enter cells through plasma membrane fusion at neutral pH. The data suggested a possible model in which A25 and A26 act as fusion suppressor on MV at neutral pH and, only after being internalized into acidic vesicles, the fusion suppressor activity of A25-A26 on MV becomes neutralized leading to de-repression of viral entry fusion complex (EFC) to execute membrane fusion during endocytosis. To investigate the molecular mechanism of A26 protein in fusion regulation process we tested whether A26 protein binds to EFC proteins and, by immunoprecipitation and GST pull down analyses, we found that A26 interacts with A16 and G9 subunits of vaccinia EFC supporting our hypothesis that A26 suppresses EFC activation on the MV at the neutral pH. When MV was treated with acid, A26 and A27 was partially released from MV particles implying a possibility that environmental pH change could induce conformational alteration of A26 and A27 protein complexes leading to subsequent membrane fusion event. Finally, based on the crystal structure of A27 protein, we demonstrated that A27 trimer/hexamer formation regulates viral fusion indirectly by acting as an adaptor of A26 protein incorporation into MV. This study reinforced our idea that A26-A27 protein complex assembly acts as the molecular basis of membrane fusion regulation.