Identification of aromatic amino acid residues in conserved region VI of the large polymerase of vesicular stomatitis virus is essential for both guanine-N-7 and ribose 2'-O methyltransferases.
Non-segmented negative-sense RNA viruses possess a unique mechanism for mRNA cap methylation. For vesicular stomatitis virus, conserved region VI in the large (L) polymerase protein catalyzes both guanine-N-7 (G-N-7) and ribose 2'-O (2'-O) methyltransferases, and the two methylases share a binding site for the methyl donor S-adenosyl-l-methionine. Unlike conventional mRNA cap methylation, the 2'-O methylation of VSV precedes subsequent G-N-7 methylation. In this study, we found that individual alanine substitutions in two conserved aromatic residues (Y1650 and F1691) in region VI of L protein abolished both G-N-7 and 2'-O methylation. However, replacement of one aromatic residue with another aromatic residue did not significantly affect the methyltransferase activities. Our studies provide genetic and biochemical evidence that conserved aromatic residues in region VI of L protein essential for both G-N-7 and 2'-O methylations. In combination with the structural prediction, our results suggest that these aromatic residues may participate in RNA recognition.
College of Animal Science, Zhejiang University, Hangzhou, Zhejiang, China.
Xiaodong
X
Zhang
Yongwei
Y
Wei
Yuanmei
Y
Ma
Songhua
S
Hu
Jianrong
J
Li
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000737:chemistry; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012315:RNA Caps / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D018116:Vesiculovirus / Q000201:enzymology* / Q000235:genetics*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D024322:Amino Acids, Aromatic; D012315:RNA Caps; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D008780:Methyltransferases; D012321:DNA-Directed RNA Polymerases; C512399:L polymerase protein, Vesicular stomatitis-Indiana virus