Ribose 2'-O methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine-N-7 methylation by the large polymerase protein.
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.
Rahmeh
Amal A
AA
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115, USA.
Li
Jianrong
J
Kranzusch
Philip J
PJ
Whelan
Sean P J
SP
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism*; D008745:Methylation; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012266:Ribose / Q000378:metabolism*; D018116:Vesiculovirus / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D012315:RNA Caps; D012333:RNA, Messenger; D014764:Viral Proteins; D006147:Guanine; D012266:Ribose; D008780:Methyltransferases; D012321:DNA-Directed RNA Polymerases; C512399:L polymerase protein, Vesicular stomatitis-Indiana virus