Molecular Mechanism Of Regulation Of PolyC Binding Protein1on Enterovirus71Gene Expression And Replication

Enterovirus71(EV71) is one causative agent of hand, foot, and mouth disease (HFMD), which may lead to severe neurological disorders and mortality in children. EV71, belonging to the genus Enterovirus of Picornaviridae family, is a non-enveloped virus with positive and single-stranded RNA of about7400nt that encodes a large polyprotein with a single open reading frame (ORF) flanked by5'-untranslated region (5'UTR) and3'UTR. The5'UTR of EV71RNA is about745nt and consists of two secondary structures:a cloverleaf structure involving in viral RNA replication and an internal ribosome entry site (IRES) directing initiation of translation, which requires a membrane-associated replication complex of viral and cellular proteins along with a viral RNA template.Human poly(C)-binding protein1(PCBP1), also called heterogeneous nuclear ribonucleoprotein E1(hnRNP E1), is a protein of38kDa and one of four isoforms of the hnRNP E protein family. Similar to other members of hnRNP E family, PCBP1has three RNA-binding domains called K-homologous1,2, and3(KH1, KH2, and KH3), of which KH1and KH3bind specifically to poly(C) homopolymers. This protein can bind to the3'-noncoding region (NCR) of cellular mRNAs and play a role in the stabilization or translational control of mRNAs. PCBP1also interacts with the5'UTR of poliovirus (PV) RNA to facilitate the viral RNA replication. Although the effects of PCBP1on translation and RNA replication of several viruses have been investigated, including PV, coxsackievirus (CAV), Kaposi's sarcoma-associated herpesvirus (KSHV), and porcine reproductive and respiratory syndrome virus (PRRSV), the mechanism involved in such regulation is largely unclear. In addition, the role of PCBP1in the regulation of EV71replication is still unknown.In this study, we at the first time investigated the effect of PCBP1in control of EV71replication and revealed the mechanism involved in such regulation. We showed that PCBP1can specifically interact with the5'UTR of EV71RNA. Detailed studies further revealed that PCBP1binds to the stem-loops ? and ? of EV715'UTR and the KH1domain of PCBPl is responsible for such binding. Moreover, we demonstrated that the binding of PCBP1to5'UTR resulted in enhancing EV71IRES activity, viral protein expression, viral RNA replication, and virus production.Interestingly, we showed that PCBP1is distributed in the nucleus and cytoplasm of uninfected cells, but mainly localized in the cytoplasm of EV71-infected cells due to interaction and co-localization with the viral RNA. Furthermore, sub-cellular distribution analysis indicated that PCBPl is located in ER-derived membrane, in where virus replication occurred in the cytoplasm of EV71-infected cells, suggesting PCBP1is recruited in a membrane-associated replication complex to facilitate the viral replication.Thus, our results demonstrate that PCBP1plays a crucial role as a positive regulator in EV71protein translation and RNA replication and also provide insights into the mechanism involved in regulation of the viral replication. In addition, we revealed a novel mechanism in which PCBP1regulates EV71replication in the host specialized membrane-associated replication complex.