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Distinct RNA Elements Confer Specificity to Flavivirus RNA Cap Methylation Events

Hongping Dong,^1, Debashish Ray,^1, Suping Ren,² Bo Zhang,¹ Francesc Puig-Basagoiti,¹ Yuko Takagi,³ C. Kiong Ho,³ Hongmin Li,^1,2 and Pei-Yong Shi^1,2*

Wadsworth Center, New York State Department of Health,¹ Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12201,² Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260³

Received 7 November 2006/ Accepted 5 February 2007

The 5' end of the flavivirus plus-sense RNA genome contains a type 1 cap (m⁷GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5' terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2'-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5' stem-loop structure; in contrast, 2'-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5' viral RNA of 20 nucleotides. The cap analogues GpppA and m⁷GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m⁷Gppp-terminated RNAs suggest that the 5' termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.

Published ahead of print on 14 February 2007.

H.D. and D.R. contributed equally to this study.

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