Enterovirus 71 (EV71), a member of the family Picornavirus, genus enterovirus, is the highly infectious major causative agent of hand, foot, and mouth disease (HFMD). EV71-infected children develop severe neurological complications that lead to rapid clinical deterioration and death. With the emergence of EV71 all over the world as the major causative agent of HFMD fatalities in recent years and in the absence of any effective anti-enteroviral therapy, there is clearly a need to find a specific antiviral therapy. The internal ribosomal entry site (IRES) of EV71 is a good target for antiviral therapy as viral replication can be restricted by the inhibition of IRES-mediated translation. The 5' UTR of EV71 RNA contains two secondary structures, a cloverleaf structure and an internal ribosomal entry site (IRES) which directs initiation of translation. The IRES-dependent translation requires both canonical translation initiation factors and IRES-specific trans-acting factors (ITAFs). The identification of ITAFs and their roles in viral RNA translation will not only help to understand the mechanism of how EV71 virus uses cellular proteins to promote its own protein synthesis but also lead to novel therapy for EV71 and related viruses. In preliminary studies we identified 15 cellular proteins that interact with EV71-IRES. Four of these had been shown earlier to interact with picornavirus IRES. Among the 11 novel proteins, we chose to further study the far upstream element (FUSE) binding protein (FBP) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) because there is no report indicating the interaction of these two proteins with any enterovirus IRES. FBP is a transcription regulator of c-myc; hnRNP A1 relocalizes from nucleus to cytoplasm during the course of poliovirus infection. To further investigate the mechanisms of how these novel proteins regulate EV71 translation, we propose to identify the binding sites involved in the interactions between EV71-IRES and FBP and hnRNP A1 (Aim 1). Specifically, we will map the FBP and hnRNP A1 binding sites on the EV71-IRES and the EV71-IRES binding sites on FBP and hnRNP A1. Furthermore, we will determine the roles of FBP and hnRNP A1 in EV71 IRES- dependent translation (Aim 2). Specifically we shall use siRNA to knock down levels of endogenous FBP and hnRNP A1 in EV71-infected cells and investigate the effect of depletion of these proteins on the translation of EV71. ? ? ?
