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dc.contributor.authorRahmeh, Amal A.
dc.contributor.authorLi, Jianrong
dc.contributor.authorKranzusch, Philip J.
dc.contributor.authorWhelan, Sean J.
dc.date.accessioned2019-10-05T16:05:58Z
dc.date.issued2009
dc.identifier.citationRahmeh, A. A., J. Li, P. J. Kranzusch, and S. P. J. Whelan. 2009. “Ribose 2’-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein.” Journal of Virology 83 (21): 11043–50. https://doi.org/10.1128/jvi.01426-09.
dc.identifier.issn0022-538X
dc.identifier.issn1070-6321
dc.identifier.issn1098-5514
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41483554*
dc.description.abstractDuring conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently at the ribose 2'-O position. For vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses, the two methylase activities share a binding site for the methyl donor S-adenosyl-L-methionine and are inhibited by individual amino acid substitutions within the C-terminal domain of the large (L) polymerase protein. This led to the suggestion that a single methylase domain functions for both 2'-O and G-N-7 methylations. Here we report a trans-methylation assay that recapitulates both ribose 2'-O and G-N-7 modifications by using purified recombinant L and in vitro-synthesized RNA. Using this assay, we demonstrate that VSV L typically modifies the 2'-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2'-O methylation of the substrate RNA. Amino acid substitutions in the C terminus of L that prevent all cap methylation in recombinant VSV (rVSV) partially retain the ability to G-N-7 methylate a pre-2'-O-methylated RNA, therefore uncoupling the effect of substitutions in the C terminus of the L protein on the two methylations. In addition, we show that the 2'-O and G-N-7 methylase activities act specifically on RNA substrates that contain the conserved elements of a VSV mRNA start at the 5' terminus. This study provides new mechanistic insights into the mRNA cap methylase activities of VSV L, demonstrates that 2'-O methylation precedes and facilitates subsequent G-N-7 methylation, and reveals an RNA sequence and length requirement for the two methylase activities. We propose a model of regulation of the activity of the C terminus of L protein in 2'-O and G-N-7 methylation of the cap structure.
dc.language.isoen_US
dc.publisherAmerican Society for Microbiology
dash.licenseLAA
dc.titleRibose 2′-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein
dc.typeJournal Article
dc.description.versionVersion of Record
dc.relation.journalJournal of Virology
dash.depositing.authorWhelan, Sean P. J.::1a236a1a655b3211832baaaf7e0e7860::600
dc.date.available2019-10-05T16:05:58Z
dash.workflow.comments1Science Serial ID 65135
dc.identifier.doi10.1128/JVI.01426-09
dash.source.volume83;21
dash.source.page11043


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