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
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.
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