A unique strategy for mRNA cap methylation used by vesicular stomatitis virus

Abstract

Nonsegmented negative-sense (nsNS) RNA viruses typically replicate within the host cell cytoplasm and do not have access to the host mRNA capping machinery. These viruses have evolved a unique mechanism for mRNA cap formation in that the guanylyltransferase transfers GDP rather than GMP onto the 5 end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype nsNS RNA virus, we now provide genetic and biochemical evidence that its mRNA cap methylase activities are also unique. Using recombinant VSV, we determined the function in mRNA cap methylation of a predicted binding site in the polymerase for the methyl donor, S-adenosyl-L-methionine. We found that amino acid substitutions to this site disrupted methylation at the guanine-N-7 (G-N-7) position or at both the G-N-7 and ribose-2-O (2-O) positions of the mRNA cap. These studies provide genetic evidence that the two methylase activities share an S-adenosyl-L-methioninebinding site and show that, in contrast to other cap methylation reactions, methylation of the G-N-7 position is not required for 2-O methylation. These findings suggest that VSV evolved an unusual strategy of mRNA cap methylation that may be shared by other nsNS RNA viruses.capping ͉ evolution ͉ methyltransferase ͉ S-adenosyl-L-methionine

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

“…In negative-sense RNA viruses, L protein is believed to possess activities required for capping, methylation and polyadenylation of mRNAs (Banerjee, 1987). Purified ribonucleoprotein (RNP) complexes from vesicular stomatitis virus (VSV) and Sendai virus containing the N, L and P proteins along with the genomic RNA have been shown to catalyse capping as well as cap methylation activities in vitro (Galloway et al, 2008;Grdzelishvili et al, 2006;Li et al, 2006;Ogino et al, 2005;Ogino & Banerjee, 2007. However, there is no direct demonstration of RTPase or GT activity associated with the RNP complex or L protein of any paramyxovirus.…”

Stratégies de formation de la structure coiffe chez les virus à ARN

“…Chez les Vésiculovirus ( Figure 2B), une polyribonucléo-tidyltransférase (PRNTase) (domaine de la protéine L), remplaçant la GTase, forme une liaison covalente avec l'extrémité 5' des ARN naissants (au lieu du GMP) via une histidine catalytique. La PRNTase liée à l'ARN transfère ensuite une molécule de GDP sur l'ARN formant ainsi la coiffe qui sera méthylée successivement en position 2'O et N7 par la 2'O-MTase et le domaine MTase de la protéine L, respectivement [11]. Chez les Alphavirus par contre, la N7-MTase virale méthyle le GTP avant de former un intermédiaire covalent avec le 7Me GMP et de le transférer à l'extrémité 5' de l'ARN ( Figure 2C) [12].…”

The TbMTr1 Spliced Leader RNA Cap 1 2 ′-O-Ribose Methyltransferase from Trypanosoma brucei Acts with Substrate Specificity

“…Region VI of the L protein in vesicular somatitis virus has similar dual MTase activities (24,25). The AcNPV MTase1 protein and HeLa cap 1 2Ј-O-ribose MTase utilize both methylated and unmethylated guanosine-capped RNA as substrates.…”

“…Recent characterization of the NS5 protein from West Nile flavivirus (WNV) demonstrated its ability to perform both guanosine N 7 and ribose 2Ј-O-ribose methylations that complete the 5Ј cap (23). Region VI of L protein in vesicular somatitis virus shows similar dual MTase activities with a single AdoMet binding pocket in the protein (24,25). Both TbMTr1 and TbMTr2 are AdoMet-dependent enzymes (17,18).…”