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Journal of Virology, November 2009, p. 11043-11050, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01426-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Ribose 2'-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein{triangledown}

Amal A. Rahmeh, Jianrong Li,{dagger} Philip J. Kranzusch, and Sean P. J. Whelan*

Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, Massachusetts 02115

Received 10 July 2009/ Accepted 14 August 2009

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.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1923. Fax: (617) 738-7664. E-mail: swhelan{at}hms.harvard.edu

{triangledown} Published ahead of print on 26 August 2009.

{dagger} Present address: Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Road, Columbus, OH 43210.

Journal of Virology, November 2009, p. 11043-11050, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01426-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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