Unveiling The Mechanism Of The Innate Immune Responses For Group â…  Coronaviruses HcoV-229E

After infection, the first obstacles viruses have to face is a powerful host immune response. Based on antigenicity, genome organization and sequence homology, coronaviruses are divided into three groups. Previous studies showed that group II coronaviruses, exemplified by SARS-CoV and MHV, do not induce or induce delayed and weak innate immune responses in infected cells. The underlying reason for this phenomenon is not fully understood. Two major mechanisms were proposed. One is by evading the recognition of host innate immune RNA sensors. Both SARS and MHV replicate inside double membrane vesicles (DMVs), the viral RNA is probably masked in this compartment. The other mechanism is by viral coding proteins that strongly antagonize host innate immunity. Like non-structural protein1, papain-like protease and nucleocapsid protein of SARS-CoV and MHV, etc.In the present study, We investigated the innate immune responses in different cells infected by group Ⅰ coronaviruses HCoV-229E, HCoV-NL63and TGEV. We discovered some unique and common features for group I coronaviruses but drastic different to that for group Ⅱ coronaviruses. Immunoblotting analysis showed that the IFN-β transcriptional factor IFR-3was dimerized and phosphorylated in all three coronaviruses infected cells. Real-time quantitative PCR showed that IFN-β gene transcription was steadily detected in HCoV-229E infected cells. ELISA test detected released IFN-β protein in HCoV-229E and TGEV infected MRC-5and A549cells, but only very weak level of IFN-β protein detected in HCoV-NL63infected LLC-MK2cells. However, suppression of group I coronaviruses replication by pretreatment with type I interferon was only observed in primary fibroblast cells but not in epithelial and carcinoma cells. By transmission electron microscope assay we also found double membrane vesicles (DMVs) in HCoV-229E-infected MRC-5cells and HCoV-NL63-infected LLC-Mk2cells. As the special characters of HCoV-229E from other coronaviruses, we constructed all its genes into expressing plasmid pcDNA. We also constructed293cell lines stably transfected with IFN-β or ISG15promoter-driven luciferase reporters. We transfect these plasmids into cells and all the viral coding proteins were detected by western blotting. We then transfected the stable cell lines by recombined plasmids, and activated the innate immunity by infected with NDV or co-transfected with these plasmids and poly I:C. We found that the IFN-β and ISG15promoters were both stimulated by NDV or poly I:C but only nspl exhibited an antagonist activity against the host innate immunity. ELISA test also revealed that the level of released IFN-β protein in nsp1transfected cells was less than cells transfected with control plasmid or other viral gene plasmids. Another work of us showed that nsp1proteins of group Ⅰ coronaviruses share structural and functional similarities with SARS-CoV nsp1. Nsp1of different coronaviruses might use the same mechanism to antagonize all host protein synthetise but not just target at innate immunity. This indicating that the nspl protein is actually a virulence factor for facilitating viral spread, but is not a major determinant in coronavirus pathogenicity.These results indicate that replicate in DMVs or nsp1proteins may not be the key factor used by coronavirus to evade host innate immunity. However, the unique character of the human coronavirus229E, which has low pathogenicity but could stimulate strong innate immune responses, may promote it to become a perfect vaccine vector of other higher pathogenic coronaviruses.