Molecular Analysis of Enterovirus 71 Replication by Yeast Two-Hybrid

The replication cycle of picornaviruses occurs entirely in the cytoplasm. Their mechanisms of action are not fully elucidated, including the a block in protein secretion from the ER and Golgi complex, the formation of cytoplasmic vesicles which harbor the viral replication complex, and alterations in intracellular calcium content in the cell. While the viral proteins responsible for some of these changes have been identified, their mechanisms of action are not known. Many consequences of viral infection are likely to be affected by interactions between viral and host cell proteins. Despite this assumption, very few such interactions have been identified. Enterovirus 71 (EV71) is an enterovirus of the family Picornaviridae. Previous studies have shown that the 2C protein of poliovirus (PV), a relative of EV71, was essential for virus replication. The PV 2C protein is associated host membrane vesicles that form viral replication complexes where viral RNA synthesis takes place. We have now identified a host-encoded 2C binding protein called Reticulon 3 (RTN3). RTN3 was demonstrated to be associated with the replication complex through direct interaction with the EV71-encoded 2C protein. We observed that the N-terminus of the 2C protein that previously has been demonstrated to encode RNA and membrane binding activity interacted with RTN3. This region of interaction in the RTN3 protein was mapped to amino acids 69-168 while that of 2C was encoded by the 25th amino acid isoleucine. It was further observed that RTN3 could also interact with the 2C proteins encoded by poliovirus (PV), and coxsackievirus A16, implying that RTN3 is a common factor for the replication of other enteroviruses. Reduction in the expression of RTN3 by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and double stranded RNA. Furthermore, reintroduction of non-degradable RTN3 into these knockdown cells, rescued EV71 infectivity and viral protein and RNA synthesis. These data suggest that RTN3 is an important component of the EV71 replication process, through its potential role in the modulation of the sequential interactions between EV71 viral RNA and the replication complex. We therefore propose to study the mechanism of viral replication. First, it has been shown that the conserved 25th amino acid of PV 2C is critical for (-)RNA synthesis. We will determine whether the reduced dsRNA is due to (-)RNA synthesis defect. Second, RTN3 is involved in the vesicular transport between ER and Golgi complex. These vesicles have the characteristics of replication complex associated vesicles. We will ask whether the reduced dsRNA synthesis is resulted from the block of replication complex associated vesicle formation in RTN3 knockdown cells. 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:PA9706-0225
External Project ID:NSC95-2311-B182-007-MY3