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-->m7GpppA-->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 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 K61-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 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.
Zhou
Yangsheng
Y
Wadsworth Center, New York State Department of Health, 120 New Scotland Ave., Albany, NY 12208, USA.
Ray
Debashish
D
Zhao
Yiwei
Y
Dong
Hongping
H
Ren
Suping
S
Li
Zhong
Z
Guo
Yi
Y
Bernard
Kristen A
KA
Shi
Pei-Yong
PY
Li
Hongmin
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003716:Dengue Virus / Q000737:chemistry; D005416:Flavivirus; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012315:RNA Caps / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*; D037521:Virulence Factors / Q000737:chemistry / Q000378:metabolism; D014901:West Nile Fever / Q000821:virology; D014902:West Nile virus / Q000737:chemistry* / Q000472:pathogenicity / Q000502:physiology*; D015005:Yellow fever virus / Q000737:chemistry
C096041:NS5 protein, flavivirus; D012315:RNA Caps; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D037521:Virulence Factors; D012436:S-Adenosylmethionine; D008780:Methyltransferases