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Journal of Virology, May 2003, p. 5378-5388, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5378-5388.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Manganese-Dependent Polioviruses Caused by Mutations within the Viral Polymerase

Shane Crotty,1,{dagger} David Gohara,2,{ddagger} Devin K. Gilligan,2 Sveta Karelsky,1 Craig E. Cameron,2 and Raul Andino1*

Department of Microbiology and Immunology, University of California, San Francisco, California 94143-0414,1 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802-45002

Received 7 October 2002/ Accepted 21 January 2003

Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3Dpol, we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn2+-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of California, 513 Parnassus Ave., Box 0414, San Francisco, CA 94143-0414. Phone: (415) 502-7196. Fax: (415) 476-0939. E-mail: andino{at}itsa.ucsf.edu.

{dagger} Present address: Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322.

{ddagger} Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.

Journal of Virology, May 2003, p. 5378-5388, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5378-5388.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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