No abstract

Coronaviruses (CoVs) are an important cause of illness in humans and animals. Most human coronaviruses commonly cause relatively mild respiratory illnesses; however two zoonotic coronaviruses, SARS-CoV and MERS-CoV, can cause severe illness and death. Investigations over the past thirty-five years have illuminated many aspects of coronavirus replication. The focus of this review is the functional analysis of conserved RNA secondary structures in the 5’ and 3’ of the betacoronavirus genomes. The 5’ 350 nucleotides folds into a set of RNA secondary structures which are well conserved, and reverse genetic studies indicate that these structures play an important role in the discontinuous synthesis of subgenomic RNAs in the betacoronaviruses. These cis-acting elements extend 3’ of the 5’UTR into ORF1a. The 3’UTR is similarly conserved and contains all of the cis-acting sequences necessary for viral replication. Two competing conformations near the 5’ end of the 3’UTR have been shown to make up a potential molecular switch. There is some evidence that an association between the 3’ and 5’UTRs is necessary for subgenomic RNA synthesis, but the basis for this association is not yet clear. A number of host RNA proteins have been shown to bind to the 5’ and 3’ cis-acting regions, but the significance of these in viral replication is not clear. Two viral proteins have been identified as binding to the 5’ cis-acting region, nsp1 and N protein. A genetic interaction between nsp8 and nsp9 and the region of the 3’UTR that contains the putative molecular switch suggests that these two proteins bind to this region.

Since the first reports of a novel SARS-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how SARS-CoV-2 emerged in the human population. Recent debate has coalesced around two competing ideas: a “laboratory escape” scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.

Severe acute respiratory syndrome (SARS) is a life-threatening infectious disease which has been difficult to study and treat because of the lack of a readily available animal model. Intranasal infection of A/J mice with the coronavirus murine hepatitis virus strain 1 (MHV-1) produced pulmonary pathological features of SARS. All MHV-1-infected A/J mice developed progressive interstitial pneumonitis, including dense macrophage infiltrates, giant cells, and hyaline membranes, resulting in death of all animals. In contrast, other mouse strains developed only mild transitory disease. Infected A/J mice had significantly higher cytokine levels, particularly macrophage chemoattractant protein 1 (MCP-1/CCL-2), gamma interferon, and tumor necrosis factor alpha. Furthermore, FGL2/fibroleukin mRNA transcripts and protein and fibrin deposits were markedly increased in the lungs of infected A/J mice. These animals developed a less robust type I interferon response to MHV-1 infection than resistant C57BL/6J mice, and treatment with recombinant beta interferon improved survival. This study describes a potentially useful small animal model of human SARS, defines its pathogenesis, and suggests treatment strategies.

All coronaviruses (CoVs), including the causative agent of severe acute respiratory syndrome (SARS), encode a nucleocapsid (N) protein that harbors two independent RNA binding domains of known structure, but poorly characterized RNA binding properties. We show here that the N-terminal domain (NTD) of N protein from mouse hepatitis virus (MHV), a virus most closely related to SARSCoV, employs aromatic amino acid-nucleobase stacking interactions with a triple adenosine motif to mediate high-affinity binding to single-stranded RNAs containing the transcriptional regulatory sequence (TRS) or its complement (cTRS). Stoichiometric NTD fully unwinds a TRS-cTRS duplex that mimics a transiently formed transcription intermediate in viral subgenomic RNA synthesis. Mutation of the solvent-exposed Y127, positioned on the β-platform surface of our 1.75 Å structure, binds the TRS far less tightly and is severely crippled in its RNA unwinding activity. In contrast, the C-terminal domain (CTD) exhibits no RNA unwinding activity. Viruses harboring Y127A N mutation are strongly selected against and Y127A N does not support an accessory function in MHV replication. We propose that the helix melting activity of the coronavirus N protein NTD plays a critical accessory role in subgenomic RNA synthesis and other processes requiring RNA remodeling.