The Physiological Function And Molecular Mechanism Studies On Ns12.9 Accessory Protein Of Human Coronavirus OC43(HCoV-OC43)

Coronaviruses(CoVs) are large, enveloped ssRNA viruses that are widely distributed among human, mammals and birds, causing principally respiratory or gastrointestinal illness but in some cases hepatitis or neurologic diseases. It is conceivable that coronaviruses are pathogenic viruses of both medical and veterinary importance. Although human coronaviruses(HCoVs) have been identified in 1960 s, HCoVs become the subject of much interest in the past decade until the global pandemic of highly pathogenic coronavirus as the causative agent of severe acute respiratory syndrome(SARS) in 2003. Interspersed among the set of canonical structural genes, such as replicase, S, E, M, and N, CoVs genomes contain a series of accessory genes, in a wide range of configurations. CoVs accessory genes exhibit individual specificity with numbers varying from one(e.g., HCoV-NL63) to eight(e.g., SARS-CoV). Strikingly, an accessory gene between the S and E gene loci, we designate it as SNE, is contained in all CoVs, suggesting that it may play important and conserved roles during CoVs infection. In our previous study, we found that this accessory protein of SARS-CoV(SARS-3a) acts as a viroporin and regulates the virus production via an unknown mechanism.Viroporins are small hydrophobic proteins that are encoded by a wide range of RNA and DNA viruses, such as influenza A virus matrix protein 2(M2), HIV-1 viral protein U(Vpu) and JC polyomavirus agnoprotein. Viroporins share several features, including membrane localization, self-oligomerization and ion channel formation. Viroporins insert into the host cell membrane to form hydrophilic pores that disrupt a number of physiological properties of the cell to potentially aid in several diverse steps of the viral life cycle. Given viroporins are essential for viral life cycle, they are attractive targets for antiviral therapy, and several compounds that block their ion channel activity are tested in clinical trial. Moreover, viroporin-defective viruses may be a good candidate for the development of live attenuated vaccines.HCoV-OC43 was isolated from a patient who had symptoms of the common cold in the 1960 s. Besides being a major causative agent of the common cold, HCoV-OC43 infection results in pneumonia or even death in the immunocompromised patients and elderly, which underscores the importance of further study on HCoV-OC3 pathogenesis. The SNE locus of HCoV-OC43 encodes a potential protein with a molecular weight of 12.9 kDa, and this protein is named ns12.9. However, its function during viral infection remains unknown. Here, we engineered a recombinant mutant virus lacking the ns12.9 protein(HCoV-OC43-Δns12.9) to characterize the contributions of ns12.9 in HCoV-OC43 replication. The ns12.9 accessory protein is a transmembrane protein and forms ion channels in both Xenopus oocytes and yeast through homo-oligomerization, suggesting that ns12.9 is a newly recognized viroporin. HCoV-OC43-Δns12.9 presented at least 10-fold reduction of viral titer in vitro and in vivo, indicating that the ns12.9 viroporin plays a significant role in the production of infectious virions. Systematic dissection of single-cycle replication demonstrated that ns12.9 viroporin had no measurable effect on virus entry, subgenomic messenger RNA(sgmRNA) synthesis and protein expression. Further characterization revealed that HCoV-OC43-Δns12.9 was less efficient in virion morphogenesis than recombinant wild-type virus(HCoV-OC43-WT). Moreover, reduced viral replication, inflammatory response and virulence in HCoV-OC43-Δns12.9-infected mice were observed compared with HCoV-OC43-WT-infected mice, suggesting that the ns12.9 viroporin is involved in virion morphogenesis and the pathogenesis of HCoV-OC43 infection. Importantly, homologous SNE proteins and heterologous viroporins with ion channel activity could compensate the the mutant virus in replication, confirming that these SNE proteins exert a conserved viroporin function that is required for virion morphogenesis via the same mechanism during the viral infection.Taken together, we clarified the physiological function and molecular mechanism of ns12.9 accessory protein during HcoV-OC43 infection. Our findings contribute to a deeper understanding of the viral life cycle and pathogenesis of CoVs, and provide a solid theoretical foundation and experimental basis for the development of effective antiviral drugs and attenuated vaccine to treat or prevent CoVs infection.