Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex.
The 5'-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5'-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2'-O positions, catalyzed by nsp14 N7-MTase and nsp16 2'-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2'-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.
State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, P. R. China.
Yu
Y
Chen
Ceyang
C
Su
Min
M
Ke
Xu
X
Jin
Lirong
L
Xu
Zhou
Z
Zhang
Andong
A
Wu
Ying
Y
Sun
Zhouning
Z
Yang
Po
P
Tien
Tero
T
Ahola
Yi
Y
Liang
Xinqi
X
Liu
Deyin
D
Guo
D018360:Crystallography, X-Ray; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012314:RNA Cap Analogs / Q000378:metabolism; D012315:RNA Caps / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C558878:Nsp10 protein, SARS virus; D012314:RNA Cap Analogs; D012315:RNA Caps; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C509442:m7GpppA; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C558879:Nsp16 protein, SARS virus; C434266:S-adenosyl-L-methionine-dependent N-methyltransferase