N Proteins Of Novel Coronavirus GDS04 And HKU2 Inhibit Type Ⅰ Interferon Pathway

In 2017,outbreak of a new type of alpha coronavirus was reported in a farm in Guangdong,China.The virus infects pigs and causes diarrhea.The mortality was very high in piglets.The virus was later identified as a coronavirus and was named as PEAV-GDS04(also known as SeACoV).Full-genomic phyligenetic analysis showed that PEAV-GDS04 coronavirus and bat coronavirus HKU2 are closely related and may be transmitted by HKU2 across species.Coronavirus nucleocapsid protein(N)is a highly expressed protein in viral infected cells.It is known that the N proteins from some coronaviruses have the ability to regulate host immune response,enhance viral infection and pathogenesis.N protein is also an important biomarker for diagnosis of coronavirus.So,the purposes of the current research were to first obtain anti-N polyclonal antibody by expressing and purification of the N protein of GDS04 in E.coli cells and second,to investigate anti-interferon(IFN)activity of the N protein in mammalian cells.The N genes of porcine coronavirus GDS04 and bat coronavirus HKU2 were cloned into prokaryotic and eukaryotic expression vectors via molecular methods.The N protein expression level in transfected cells was examined by Western-blot.Dual luciferase reporters were used to investigated the anti-IFN or anti-NF-kB activity of the N proteins.The effect of the N protein on downstream ISG expression was examined by qRT-PCR and dual luciferase reporter experiments.Expression of N protein in E.coli was induced by IPTG,and purified by nickel column.Mice were immunized with an adjuvant emulsified recombinant N protein and sera were collected for analysis.The results showed that the N proteins of GDS04 and HKU2 viruses were highly expressed in 293 T,Hela and A549 cells.The expressed N protein inhibited the type I interferon production in these cells.In particular,the N protein of GDS04 and HKU2 inhibited the production of IFN-β and its downstream target ISG54.However,the N protein had no effect on the NF-κB signaling pathway.The prokaryotic expression vector of GDS04 was successfully constructed,and the corresponding recombinant protein was expressed and purified.Mice polyclonal antibody was prepared by immunizing mice with purified N protein.The obtained anti-N mouse antiserum had a high titer for N protein and was used for ELSIA and Wester-blot detection of N protein expression of GDS04 and HKU2.Conclusion: this preliminarily study revealed that the N proteins of the novel α-coronavirus GDS04 and related bat HKU2 coronavirus have the role in regulating host immune response by inhibiting host type I interferon pathway.The anti-n polyclonal antibody can be used for detection of N protein by Western protein and ELISA.