Distinct RNA elements confer specificity to flavivirus RNA cap methylation events.
The 5' end of the flavivirus plus-sense RNA genome contains a type 1 cap (m(7)GpppAmG), 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 m(7)GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m(7)Gppp-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.
Dong
Hongping
H
Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12201, USA.
Ray
Debashish
D
Ren
Suping
S
Zhang
Bo
B
Puig-Basagoiti
Francesc
F
Takagi
Yuko
Y
Ho
C Kiong
CK
Li
Hongmin
H
Shi
Pei-Yong
PY
D020029:Base Pairing; D001483:Base Sequence; D018983:DNA Footprinting; D019175:DNA Methylation; D017931:DNA Primers; D008780:Methyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D020320:Oligoribonucleotides, Antisense; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism*; D014902:West Nile virus / Q000235:genetics*
D017931:DNA Primers; D020320:Oligoribonucleotides, Antisense; D012315:RNA Caps; D012367:RNA, Viral; D008780:Methyltransferases