title: Differential rescue of poliovirus RNA replication functions by genetically modified RNA polymerase precursors.
abstract: We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.
authors.affiliation: Department of Microbiology and Molecular Genetics, Med. Sci. B240, University of California, Irvine, CA 92697-4025, USA.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Cornell
authors.forename: Jo Ellen
authors.initials: JE
authors.lastname: Brunner
authors.forename: Bert L
authors.initials: BL
authors.lastname: Semler
mesh_terms: D020121:5' Untranslated Regions; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000502:physiology*; D029822:Enterovirus B, Human / Q000235:genetics*; D004792:Enzyme Precursors / Q000235:genetics / Q000502:physiology*; D017955:Poliovirus / Q000235:genetics*; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis*
chemical_list: D020121:5' Untranslated Regions; D004792:Enzyme Precursors; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases