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Journal of Virology, September 2005, p. 11962-11973, Vol. 79, No. 18
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.18.11962-11973.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

An Authentic 3' Noncoding Region Is Necessary for Efficient Poliovirus Replication

David M. Brown,{dagger} Christopher T. Cornell,{ddagger} Genevieve P. Tran, Joseph H. C. Nguyen, and Bert L. Semler*

Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California 92697

Received 13 April 2005/ Accepted 29 June 2005

Picornavirus RNA replication involves the specific synthesis of negative-strand intermediates followed by an accumulation of positive-strand viral RNA in the presence of a multitude of cellular mRNAs. Previously, in an effort to identify cis-acting elements required for initiation of negative-strand RNA synthesis, we deleted the entire 3' noncoding regions from human rhinovirus and poliovirus genomic RNAs. These deletion mutation transcripts displayed a severe delay in RNA accumulation following transfection of HeLa cells. Interestingly, in subsequent infection of HeLa cells, the deletion-mutant poliovirus displayed only a moderate deficiency in RNA synthesis. These data suggested that the delay in the production of cytopathic effects after transfection may have been due to an RNA replication defect overcome by the accumulation of a compensatory mutation(s) generated during initial rounds of RNA synthesis. In this study, we have sequenced the entire genome of the deletion-mutant virus and found only two nucleotide changes from the parental clone. Transfection analysis of these sequence variants revealed that the sequence changes did not provide compensatory functions for the 3' noncoding region deletion mutation replication defect. Further examination of the deletion mutant phenotype revealed that the severe replication defect following RNA transfection is due, in part, to nonviral terminal sequences present in the in vitro-derived deletion mutation transcripts. Our data suggest that poliovirus RNA harboring a complete 3' noncoding region deletion mutation is infectious (not merely quasi-infectious).

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

{dagger} Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544.

{ddagger} Present address: Department of Molecular and Integrative Neuroscience, The Scripps Research Institute, La Jolla, CA 92037.

Journal of Virology, September 2005, p. 11962-11973, Vol. 79, No. 18
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.18.11962-11973.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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