Poliovirus and related human enteroviruses cause a diverse spectrum of human diseases including acute flaccid paralysis (paralytic poliomyelitis), aseptic meningitis, myocarditis, conjunctivitis and fatal systemic infections of neonates. Poliovirus (PV), the prototypic human enterovirus, is well characterized at the molecular level and serves as a model organism for detailed studies of human enterovirus mRNA translation, RNA replication, and virus assembly. In this proposal we will exploit a remarkable cell-free replication system and tissue culture systems to investigate the mechanisms of PV mRNA stability, PV mRNA translation, and PV RNA replication. In the previous funding period we made three important discoveries: 1) we discovered mechanisms by which naturally uncapped PV mRNA assembles polysomes uncoupled from host cell mRNA turnover machinery, 2) we discovered an evolutionarily ancient telomerase-like mechanism of RNA replication that maintains the integrity of the poly(A) tail at the 3'end of viral RNA genomes, and 3) we discovered an RNA structure in PV and related group C enteroviruses that functions as a competitive inhibitor of ribonuclease L, an antiviral endoribonuclease. Experiments will be performed to: 1) determine how PV and related human enteroviruses uncouple viral mRNA translation from host cell mRNA turnover pathways, and 2) elucidate molecular features associated with the telomerase-like mechanisms of PV RNA replication. These investigations will reveal how the seemingly arcane mechanisms of viral mRNA translation and viral RNA replication represent concerted strategies shaped by the selective pressures of host mRNA turnover machinery. The telomerase-like mechanisms of PV RNA replication, if substantiated by the proposed investigation, will have significant impact on our understanding of molecular evolution, drawing attention to the common features and mechanisms of a viral RNA-dependent RNA polymerase and eukaryotic telomerase reverse-transcriptase. Insights supported by the proposed investigation will be important for designing safe and effective vaccines and novel antiviral drugs for PV and other picornaviruses.
Our investigations provide information applicable to many common human pathogens. The molecular processes under investigation in this project are shared among many picornaviruses, including hundreds of human enteroviruses and human rhinoviruses, which together afflict at one or more times every human being in the US and around the world. Our experiments are designed to determine how specific host proteins detect and destroy human enterovirus and rhinovirus mRNAs and how these viruses evade and counteract such host enzymes to maintain the integrity of their RNA genomes. This information can be used to improve poliovirus vaccines, to create new vaccines against related human picornaviruses, and to optimize antiviral drugs designed to activate specific host enzymes that destroy viral RNA. The success of the poliovirus eradication campaign may depend upon improved poliovirus vaccines and new antiviral drugs.
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