A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation.
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.
Grdzelishvili
Valery Z
VZ
University of Florida College of Medicine, Department of Molecular Genetics and Microbiology, 1600 S.W. Archer Road, P.O. Box 100266, Gainesville, FL 32610-0266, USA.
Smallwood
Sherin
S
Tower
Dallas
D
Hall
Richard L
RL
Hunt
D Margaret
DM
Moyer
Sue A
SA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D013696:Temperature; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase