Mechanistic Studies of Enterovirus 71 Replication as a Model for (+) RNA Viruses

In this proposal, we will study the biological roles of two cellular proteins, reticulon 3 (RTN3) and G protein-coupled receptor 125 (GPR125), in the replication of positive strand [(+)] RNA viruses, such as hepatitis C virus (HCV) and enterovirus 71 (EV71). The 2C protein of poliovirus, a relative of enterovirus 71, is essential for viral replication. The poliovirus 2C protein is associated with host membrane vesicles, which form viral replication complexes where viral RNA synthesis takes place. We have now identified a host-encoded 2C binding protein called RTN3 by yeast two-hybrid assay, which we found to be associated with the replication complex and is implicated in EV71 replication (Journal of Biological Chemistry, 282:5888–5898, 2007). RTN3 also interacted with amphipathic helix containing NS4B of HCV by co-immunoprecipitation (Co-IP) and immunofluorescent microscopy (IF). Viral RNA synthesis was reduced by 5-fold in RTN3 knockdown Huh7 cells infected by HCV by quantitative RT-PCR. We therefore propose that RTN3 is involved in the replication of (+) RNA viruses. In addition, we also indentified another EV71 2C-interacting protein, GPR125. The interaction between GPR125 and 2C was confirmed by co-IP and IF. Viral protein synthesis was reduced in GPR125 knockdown RD cells infected by EV71 by western blot. We suggest that GPR125 is involved in EV71 replication. We therefore propose to study the role of these two proteins in replication of (+) RNA viruses. First, we will study the interacting domain of GPR125. Second, we will investigate the biological role of RTN3 in HCV replication; third, GPR125 in the replication of EV71. Last but not least, we have also identified several other 2C-interacting proteins. The characterization of these proteins is ongoing. We hope to identify all the molecules involved in the viral replication and build up the entire replication pathway in the future.

Project ID:PA9808-0187
External Project ID:NSC98-2311-B182-003-MY3