Abstract |
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 degrees C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40 degrees 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.
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Authors | Valery Z Grdzelishvili, Sherin Smallwood, Dallas Tower, Richard L Hall, D Margaret Hunt, Sue A Moyer |
Journal | Journal of virology
(J Virol)
Vol. 79
Issue 12
Pg. 7327-37
(Jun 2005)
ISSN: 0022-538X [Print] United States |
PMID | 15919887
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, P.H.S.)
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Chemical References |
- RNA Caps
- RNA, Messenger
- RNA, Viral
- Viral Proteins
- L protein, vesicular stomatitis virus
- RNA-Dependent RNA Polymerase
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Topics |
- Amino Acid Sequence
- Amino Acid Substitution
- Animals
- Cell Line
- Cricetinae
- Humans
- Methylation
- Molecular Sequence Data
- Mutation
- RNA Caps
(metabolism)
- RNA, Messenger
(metabolism)
- RNA, Viral
(metabolism)
- RNA-Dependent RNA Polymerase
(chemistry, genetics, metabolism)
- Recombination, Genetic
- Temperature
- Vesicular stomatitis Indiana virus
(genetics, metabolism, physiology)
- Viral Proteins
(chemistry, genetics, metabolism)
- Virus Replication
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