Structure and function of flavivirus NS5 methyltransferase

The plus-strand RNA genome of flavivirus contains a 5′ terminal cap 1 structure (m⁷GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2′-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA → m ⁷GpppA → m⁷GpppAm. The 2′-O methylation can be uncoupled from the N-7 methylation, since m⁷GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 A resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2′-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltraasferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2′-O cap methylations require distinct buffer conditions and different side chains within the K ₆₁-D₁₄₆-K₁₈₂-E₂₁₈ motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 27′-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.