The recent emergence of a novel coronavirus (2019‐nCoV), which is causing an outbreak of unusual viral pneumonia in patients in Wuhan, a central city in China, is another warning of the risk of CoVs posed to public health. In this minireview, we provide a brief introduction of the general features of CoVs and describe diseases caused by different CoVs in humans and animals. This review will help understand the biology and potential risk of CoVs that exist in richness in wildlife such as bats.

The N7-methylguanosine (m7G) cap is the defining structural feature of eukaryotic mRNAs. Most eukaryotic viruses that replicate in the cytoplasm, including coronaviruses, have evolved strategies to cap their RNAs. In this report, we used a yeast genetic system to functionally screen for the cap-forming enzymes encoded by severe acute respiratory syndrome (SARS) coronavirus and identified the nonstructural protein (nsp) 14 of SARS coronavirus as a (guanine-N7)-methyltransferase (N7-MTase) in vivo in yeast cells and in vitro using purified enzymes and RNA substrates. Interestingly, coronavirus nsp14 was previously characterized as a 3′-to-5′ exoribonuclease, and by mutational analysis, we mapped the N7-MTase domain to the carboxy-terminal part of nsp14 that shows features conserved with cellular N7-MTase in structure-based sequence alignment. The exoribonuclease active site was dispensable but the exoribonuclease domain was required for N7-MTase activity. Such combination of the 2 functional domains in coronavirus nsp14 suggests that it may represent a novel form of RNA-processing enzymes. Mutational analysis in a replicon system showed that the N7-MTase activity was important for SARS virus replication/transcription and can thus be used as an attractive drug target to develop antivirals for control of coronaviruses including the deadly SARS virus. Furthermore, the observation that the N7-MTase of RNA life could function in lieu of that in DNA life provides interesting evolutionary insight and practical possibilities in antiviral drug screening.

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.

RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans , while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but not in RNAi-depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle and provide further evidence to support that RNAi-mediated antiviral response exists in mammalian cells.