Poliovirus is the prototypic member of the Picornavirus family of positive-strand RNA viruses, a large group of related viruses that cause a diverse spectrum of human illnesses. Contrary to previous dogma, poliovirus RNA templates for negative-strand RNA synthesis require cis-active RNA elements from both the 5'- and 3'-terminal regions of the viral genome. The applicant hypothesizes that the 5'- and 3'-terminal cis-active RNA elements of poliovirus coordinately regulate viral RNA stability, RNA translation, and negative-strand RNA synthesis via temporally dynamic ribonucleoproteins.
The specific aims are to: 1) Determine if cellular poly(C) binding protein bound to the 5'-terminus of poliovirus RNA mediates poliovirus RNA stability; 2) Determine how the 5'-terminal ribonucleoprotein of poliovirus participates in negative-strand RNA synthesis, a biosynthetic event initiated at the 3'-terminus of poliovirus RNA; 3) Determine why the 5'-terminal internal ribosome entry site and/or the initiation of translation is required to allow viral RNA molecules to serve as templates for negative-strand RNA synthesis; 4) Identify the RNA sequences, structures, and ribonucleoprotein complexes within the 3Dpol coding region that are required for negative-strand RNA synthesis. Technical advantages of cell-free virus replication reactions will be exploited to assay poliovirus RNA stability, RNA translation, and negative-strand RNA synthesis. Recombinant DNA technology will be used to create viral RNAs with specific mutations for structural and functional analyses. Temporally dynamic ribonucleoproteins composed of cellular proteins, viral protein, and cis-active poliovirus RNA elements will be functionally characterized to determine which step(s) of poliovirus replication they mediate and/or regulate. These studies will characterize the dynamic transformation of poliovirus mRNA into a template for negative-strand RNA synthesis, a fundamental event required for the replication of all positive-strand RNA viruses. This wok may help establish a new paradigm for RNA replication in which both 5'- and 3'-terminal cis-active RNA elements of genomic RNA coordinately regulate numerous steps of viral replication.
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