Characterization Of The Guanine-N7Methyltransferase Activity Of Coronavirus Nsp14on Nucleotide GTP

Coronaviruses are frequently associated with respiratory and enteric diseases in humans, livestock, and companion animals. Coronaviruses possess a nearly30kb positive-stranded RNA genome, and the two large open reading frames (ORFs) la and lb, located at the5’-two-thirds of the genome, encode the proteins making the replication and transcription complex (RTC).Most eukaryotic viruses that replicate in the cytoplasm, including coronaviruses, have evolved strategies to cap their RNAs. Previous studies have shown that nonstructural protein (nsp)13functions as RNA helicase and5’-triphosphatase, and we and others identified nsp14as an exoribonuclease and cap (guanine-N7)-methyltransferase (N7-MTase), and nsp16as a2’-0methyltransferase (2’-O-MTase).In our previous work, nsp14of severe acute respiratory syndrome coronavirus (SARS-CoV) was identified as N7-MTase. In this study, we found that GTP, dGTP as well as cap analogues GpppG, GpppA and m7GpppG could be methylated by SARS-CoV nsp14. In contrast, the nsp14could not modify ATP, CTP, UTP, dATP, dCTP, dUTP or cap analogue m7GpppA. Critical residues of nsp14essential 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 nsp14of other coronaviruses. Moreover, the accumulation of m7GTP or presence of protein nsp14could interfere with protein translation of cellular mRNAs. In summary, we provided direct evidence that coronavirus nsp14could methylate free GTP. These results confirm and extend our previous observations on the cap N7-MTase activity of SARS-CoV nspl4. As the N7-methylated GTP is inhibitory to mRNA translation, the present finding provides a clue for further research on the molecular mechanisms of coronavirus replication and pathogenesis.