Search

Highlights • We found SARS-CoV nsp14 could methylate GTP and dGTP. • Critical residues of nsp14 essential for the activity on GTP were identified. • m7GTP or nsp14 could interfere with protein translation., 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.

Highlights ► We identify the region of aa 65–107 of nsp10 as the interaction domain with nsp16 in vivo. ► We identify the region of aa 42–120 of nsp10 as the functional domain for activating nsp16 in vitro. ► We design and synthesize two inhibitory peptides K12 and K29 that could inhibit the MTase activity of nsp10/nsp16 complex., 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.