Sendai virus RNA-dependent RNA polymerase L protein catalyzes cap methylation of virus-specific mRNA.
The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5'-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756-2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1-1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis.
Ogino
Tomoaki
T
Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Kobayashi
Masaki
M
Iwama
Minako
M
Mizumoto
Kiyohisa
K
D016367:Baculoviridae / Q000378:metabolism; D015153:Blotting, Western; D018076:DNA, Complementary / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism; D008957:Models, Genetic; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012315:RNA Caps; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism / Q000502:physiology*; D029082:Sendai virus / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism / Q000502:physiology*
D018076:DNA, Complementary; D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D012965:Sodium Chloride; D012313:RNA; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; C064365:L protein, Sendai virus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases