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Journal of Virology, December 2004, p. 13007-13018, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.13007-13018.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

Christopher T. Cornell, Jo Ellen Brunner, and Bert L. Semler^*

Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California

Received 3 October 2003/ Accepted 22 August 2004

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.

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* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Med. Sci. B240, University of California, Irvine, CA 92697-4025. Phone: (949) 824-7573. Fax: (949) 824-2694. E-mail: blsemler{at}uci.edu.

Present address: Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037.

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Journal of Virology, December 2004, p. 13007-13018, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.13007-13018.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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