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 5= cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. T he 5= ends of eukaryotic cellular mRNAs and most viral mRNAs possess a cap structure, which plays important roles in mRNA splicing, intracellular RNA transport, RNA stability, and translation initiation (1). Host and viral RNA molecules lacking the 5= cap structure are rapidly degraded in the cytoplasm (2). The cap-0 structure of mRNA is cotranscriptionally formed through sequential enzymatic reactions, including RNA triphosphatase (TPase), RNA guanylyltransferase (GTase), and RNA (guanine-N7)-methyltransferase (N7-MTase) (1). In higher eukaryotes and some viruses, cap-0 structure m7GpppN-RNA is further methylated at the ribose 2=-O position of the nascent mRNA by a ribose 2=-O-methyltransferase (2=-O-MTase) to form a cap-1 structure (m7GpppNm) and cap-2 structure (m7GpppNmNm). Both N7-MTase and 2=-O-MTase can catalyze the transfer of the methyl group from the methyl donor S-adenosyl-L-methionine (SAM or AdoMet) to RNA substrate and generate S-adenosyl-L-homocysteine (SAH or AdoHcy) as a by-product. The functions of viral RNA cap structure include the following: (i) the guanosine cap core structure protects the 5=-triphosphate from activating the host innate immune response (3, 4); (ii) the N7 methylation is essential for viral replication through the enhancement of viral RNA translation (5); and (iii) the 2=-O methylation functions to

Eukaryotic cellular mRNAs and many viral mRNAs contain a modified 5=-terminal "cap" structure that is essential for efficient splicing, nuclear export, translation, and stability of the mRNAs (1, 2). The cap structures are usually formed by three sequential enzymatic reactions: (i) the 5=-triphosphate end of the nascent mRNA is hydrolyzed to a diphosphate by RNA triphosphatase (TPase); (ii) a GMP residue derived from GTP is transferred to the diphosphate mRNA by RNA guanylyltransferase (GTase) via a two-step reaction; and (iii) the guanosine cap is methylated by guanine-N7-methyltransferase (N7-MTase) at the N7 position to generate a cap-0 structure (m 7 GpppN) in the presence of the methyl group donor S-adenosyl-

Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV.

Most eukaryotic viruses that replicate in the cytoplasm, including coronaviruses, have evolved strategies to cap their RNAs. In our previous work, the nonstructural protein (nsp) 14 of severe acute respiratory syndrome coronavirus (SARS-CoV) was identified as a cap (guanine-N7)-methyltransferase (N7-MTase). In this study, we found that GTP, dGTP as well as cap analogs GpppG, GpppA and m7GpppG could be methylated by SARS-CoV nsp14. In contrast, the nsp14 could not modify ATP, CTP, UTP, dATP, dCTP, dUTP or cap analog m7GpppA. Critical residues of nsp14 essential for the methyltransferase activity on GTP were identified, which include F73, R84, W86, R310, D331, G333, P335, Y368, C414, and C416. We further showed that the methyltransferase activity of GTP was universal for nsp14 of other coronaviruses. Moreover, the accumulation of m7GTP or presence of protein nsp14 could interfere with protein translation of cellular mRNAs. Altogether, the results revealed a new enzymatic activity of coronavirus nsp14.