Structure-Functional Analysis Of SARS Coronavirus Nonstructural Protein14

The N7-methylguanosine cap is the structural feature of the eukaryotic mRNA. The most viruses, including coronavirus, have evolved their own RNA capping modification which replicates in the cytoplasm of eukaryotic cells.In a previous study, we adopted a yeast genetic system to filter the cap-forming enzymes encoded by severe acute respiratory syndrome coronavirus (SARS), and found that nonstructural protein14of SARS coronavirus (nsp14) has an activity of (guanine-N7)-methyltransferase (N7-MTase) in vivo in yeast system and in vitro by employing purified protein and the RNA substrate. The nsp14of coronavirus has been proved to have an activity as3’-5’exonuclease enzyme by analyzing the mutation. By structurebased sequence alignment, the carboxy-terminal part of nspl4has conservative characteristics of cellular N7-MTase, and we also identified that the N7-methyltransferase enzyme activity is located in the C-terminal part of nsp14by mutation analysis. The active site of exoribonuclease enzyme is not required for N7-methyltransferase activity, but the exonuclease domain. The two functional domains co-located on the coronavirus nsp14may represent a novel RNA processing enzyme.A cap structure at the5’end of genomic RNA and subgenomic RNAs of coronavirus, which is generated through consecutive methylations by guanine-N7-methyltransferase and2’-O-methyltransferase encoding virally. The N7-MTase of coronavirus is very special for its physical linkage with an exonuclease domain on the coronavirus nsp14. In this study, deletions and site-directed mutations in the SARS coronavirus nspl4were used to analyze the structure-function relationships of N7-MTase. The results showed that the domain of ExoN is closely involved in the activity of the N7-MTase, unlike all other viruses, in which structural domains on the same polypeptide are separable from each other.12crucial critical amino acid residues of were identified to be necessary for the N7-MTase by the yeast genetic system and biochemical experiments in vitro. Two of them were located at the N terminus of the core ExoN domain, suggesting that the ExoN domain is an important role in the N7-methyltransferase activity. Most of the other10critical residues distributing throughout the N7-MTase domain located in the methylase S-adenosyl-L-methionine (SAM) binding sites and key structural elements of the MTase fold of nsp14. The sequence motif DxGxPxA (amino acids331to338) was considered to be the most important position of the SAM binding site. These results reveal the structure and function relationships of coronavirus nsp14N7-MTase.Mutation analysis of the replication system showed the activity of N7-methyltransferase was very important for the SARS virus replication/transcription, and can be an attractive drug target for anti-SARS-CoV treatment.