dc.contributor.author | Rahmeh, Amal A. | |
dc.contributor.author | Li, Jianrong | |
dc.contributor.author | Kranzusch, Philip J. | |
dc.contributor.author | Whelan, Sean J. | |
dc.date.accessioned | 2019-10-05T16:05:58Z | |
dc.date.issued | 2009 | |
dc.identifier.citation | Rahmeh, 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.issn | 0022-538X | |
dc.identifier.issn | 1070-6321 | |
dc.identifier.issn | 1098-5514 | |
dc.identifier.uri | http://nrs.harvard.edu/urn-3:HUL.InstRepos:41483554 | * |
dc.description.abstract | During 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.iso | en_US | |
dc.publisher | American Society for Microbiology | |
dash.license | LAA | |
dc.title | Ribose 2′-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein | |
dc.type | Journal Article | |
dc.description.version | Version of Record | |
dc.relation.journal | Journal of Virology | |
dash.depositing.author | Whelan, Sean P. J.::1a236a1a655b3211832baaaf7e0e7860::600 | |
dc.date.available | 2019-10-05T16:05:58Z | |
dash.workflow.comments | 1Science Serial ID 65135 | |
dc.identifier.doi | 10.1128/JVI.01426-09 | |
dash.source.volume | 83;21 | |
dash.source.page | 11043 | |