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Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5′-cap structure that is methylated at the guanine N-7 and the ribose 2′-OH positions of the first transcribed nucleotide, adenine (m⁷GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2′-O methylations and therefore is essential for flavivirus 5′-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m⁷GpppA-capped RNAs to m⁷GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m⁷GpppA- and m⁷GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2′-O methylation. Mutagenesis of a K₆₁-D₁₄₆-K …