Cellular protein interactions with a picornavirus RNA intermediate affects positive-strand RNA synthesis

Poliovirus is a member of a group of small, single-stranded positive-sense RNA viruses that replicate in the cytoplasm of the infected cell. Due to the limited coding capacity of the viral genome, poliovirus relies on sequestration and binding of host cell proteins, in addition to the non-structural proteins encoded by the single open reading frame of the viral RNA, to productively produce progeny positive-strand RNAs that will be incorporated into infectious virions. Association of host cell and viral non-structural proteins occurs on the regions of the viral RNA that do not code for protein, and allows for a local concentration of the protein components required for the necessary processes through extensive base-pairing to form stable RNA secondary structures. Examples of important viral non-coding regions (NCRs) interacting with cellular and/or viral proteins include the internal ribosome entry site or IRES located at the 5' end of the poliovirus genome, which allows for initiation of cap-independent RNA translation of the RNA genome and the cis-acting element or cre, which is required for uridylylation of the RNA polymerase protein primer VPg. In our studies we have investigated the importance of the 3' NCR and its function in poliovirus replication. Previous studies had determined that a viral mutant lacking the 3' NCR was still capable of replicating in vitro and in cell culture, albeit with a positive-strand RNA synthesis defect compared to wild type. We report here that the cellular protein hnRNP C, previously identified as binding to the 3' end of the poliovirus negative-strand RNA intermediate, also binds at the 5' end of negative-strand RNA. We demonstrate that the addition of hnRNP C proteins can stimulate viral RNA synthesis in vitro, as well as increase the production of infectious virus progeny in cell culture. Conversely, we show that removal of hnRNP C results in decreased virus yields and positive-strand RNA synthesis, suggesting that hnRNP C binding to the RNA termini of the poliovirus negative-strand may allow efficient RNA replication. Based upon these observations and the observation that the 5' end of poliovirus negative-strand RNA also binds poliovirus replication proteins, we conclude that the 3' NCR of poliovirus has an important role in positive-strand RNA synthesis via the interaction of its negative-strand RNA complement with hnRNP C and other viral and/or cellular factors.