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Journal of Virology, June 2005, p. 7327-7337, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7327-7337.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Single Amino Acid Change in the L-Polymerase Protein of Vesicular Stomatitis Virus Completely Abolishes Viral mRNA Cap Methylation

Valery Z. Grdzelishvili,¹ Sherin Smallwood,¹ Dallas Tower,¹ Richard L. Hall,^1, D. Margaret Hunt,² and Sue A. Moyer^1*

Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32610,¹ Department of Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina 29208²

Received 9 November 2004/ Accepted 8 February 2005

The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5'-cap structures methylated at the guanine-N7 and 2'-O-adenosine positions (7mGpppA^m). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the L-polymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5'-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-L-methionine-binding domain of the L protein.

Present address: Vaccinex, Inc., Rochester, NY 14620.

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