Sendai Virus RNA-dependent RNA Polymerase L Protein Catalyzes Cap Methylation of Virus-specific mRNA

Kiyohisa Mizumoto,Masaki Kobayashi,Minako Iwama,Tomoaki Ogino

doi:10.1074/jbc.m411167200

Kiyohisa Mizumoto, Masaki Kobayashi

Open Access

https://doi.org/10.1074/jbc.m411167200

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Abstract

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.

Highlights

Many viruses that replicate in the cytoplasm synthesize capped mRNAs by their own transcription systems
The Sendai virus (SeV) ribonucleoprotein-M protein complex (RNP-M), which is mainly composed of genomic RNA, N, P, L, and M proteins [23], was purified from virus particles and used as an enzyme source. 32P-Cap-labeled G(5Ј)ppp(5Ј)ApGpGpGpU was incubated with the RNP-M complex in the presence of AdoMet (Fig. 2)
Recombinant cellular G-7-MTase that produces the m7GpppA cap structure was assayed (Fig. 2, A and B, lanes 4). These results indicate that the SeV RNP-M complex contains AdoMet-dependent G-7-MTase activity that can be assayed independently of viral mRNA synthesis

Summary

Introduction

Many viruses that replicate in the cytoplasm synthesize capped mRNAs by their own transcription systems (reviewed in Refs. 1 and 2). 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.

Results

Conclusion