TY - JOUR
T1 - West Nile virus 5′-cap structure is formed by sequential guanine N-7 and ribose 2′-O methylations by nonstructural protein 5
AU - Ray, Debashish
AU - Shah, Aaloki
AU - Tilgner, Mark
AU - Guo, Yi
AU - Zhao, Yiwei
AU - Dong, Hongping
AU - Deas, Tia S.
AU - Zhou, Yangsheng
AU - Li, Hongmin
AU - Shi, Pei Yong
PY - 2006/9
Y1 - 2006/9
N2 - 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 (WNY), 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 m 7GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2′-O methylation. Mutagenesis of a K61-D146-K 182-E218 tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2′-O MTase activities. The entire K61-D146-K 182-E218 motif is essential for 2′-O MTase activity, whereas N-7 MTase activity requires only D146. The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K61-D146-K 182-E218 motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.
AB - 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 (WNY), 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 m 7GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2′-O methylation. Mutagenesis of a K61-D146-K 182-E218 tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2′-O MTase activities. The entire K61-D146-K 182-E218 motif is essential for 2′-O MTase activity, whereas N-7 MTase activity requires only D146. The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K61-D146-K 182-E218 motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.
UR - http://www.scopus.com/inward/record.url?scp=33748669852&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33748669852&partnerID=8YFLogxK
U2 - 10.1128/JVI.00814-06
DO - 10.1128/JVI.00814-06
M3 - Article
C2 - 16912287
AN - SCOPUS:33748669852
SN - 0022-538X
VL - 80
SP - 8362
EP - 8370
JO - Journal of virology
JF - Journal of virology
IS - 17
ER -