Biochemical and Structural Insights into the Mechanisms of SARS Coronavirus RNA Ribose 2′-O-Methylation by nsp16/nsp10 Protein Complex
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Open Access
- 13 October 2011
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLoS Pathogens
- Vol. 7 (10), e1002294
- https://doi.org/10.1371/journal.ppat.1002294
Abstract
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. The distinctive feature of eukaryotic mRNAs is the presence of methylated cap structure that is required for mRNA stability and protein translation. As all viruses employ cellular ribosomes for protein translation, most cytoplasmically replicating eukaryotic viruses including coronaviruses have evolved strategies to cap their RNAs. It was shown very recently that ribose 2′-O-methylation in the cap structure of viral RNAs plays an important role in viral escape from innate immune recognition. The 2′-O-methyltransferase (2′-O-MTase) encoded by SARS coronavirus is composed of two subunits, the catalytic subunit nsp16 and the stimulatory subunit nsp10, which is different from all other known 2′-O-MTases that are partner-independent. Here we show that the role of nsp10 is to promote nsp16 to bind capped RNA substrate and the methyl donor S-adenosyl-L-methionine (SAM). We solved the crystal structure of the nsp16/nsp10/SAM complex, and the structural analysis revealed that the details of the inter-molecular interactions and indicated that nsp10 may stabilize the SAM-binding pocket and extend the capped RNA-binding groove. The interaction interface of nsp16/nsp10 is unique for coronaviruses and thus may provide an attractive target for developing specific antiviral drugs for control of coronaviruses including the deadly SARS coronavirus.Keywords
This publication has 67 references indexed in Scilit:
- Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5Nature Immunology, 2011
- 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family membersNature, 2010
- Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*Online Journal of Public Health Informatics, 2010
- Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferaseProceedings of the National Academy of Sciences of the United States of America, 2009
- Flavivirus methyltransferase: A novel antiviral targetAntiviral Research, 2008
- Structural insights into the mechanism and evolution of the vaccinia virus mRNA cap N7 methyl-transferaseThe EMBO Journal, 2007
- On macromolecular refinement at subatomic resolution with interatomic scatterersActa crystallographica. Section D, Structural biology, 2007
- Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 ReplicationJournal of Virology, 2007
- Analysis of Murine Hepatitis Virus Strain A59 Temperature-Sensitive Mutant TS-LA6 Suggests that nsp10 Plays a Critical Role in Polyprotein ProcessingJournal of Virology, 2007
- The CCP4 suite: programs for protein crystallographyActa crystallographica. Section D, Structural biology, 1994