Structure and Function of Flavivirus NS5 Methyltransferase

• DOI: 10.1128/jvi.02704-06
• Publication Date: 2007-02-03T02:42:26Z
• Authors: Yangsheng Zhou; Debashish Ray; Yiwei Zhao; Hongping Dong; Suping Ren; Zhong Li; Yi Guo; Kristen A. Bernard; Pei-Yong Shi; Hongmin Li
• Keywords: Models, Molecular; RNA Caps; 0301 basic medicine; Mice, Inbred C3H; S-Adenosylmethionine; Binding Sites; Virulence Factors; Flavivirus; Molecular Sequence Data; Methyltransferases; Dengue Virus; Viral Nonstructural Proteins; Crystallography, X-Ray; Protein Structure, Tertiary; 3. Good health; Mice; 03 medical and health sciences; Mutagenesis, Site-Directed; Animals; RNA, Viral; Amino Acid Sequence; West Nile virus; West Nile Fever

ABSTRACT

The plus-strand RNA genome of flavivirus contains a 5′ terminal cap 1 structure (m 7 GpppAmG). 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 7 GpppA → m 7 GpppAm. The 2′-O methylation can be uncoupled from the N-7 methylation, since m 7 GpppA-RNA can be readily methylated to m 7 GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å 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 methyltransferase 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 -D 146 -K 182 -E 218 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 2′-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.