Study Of Antagonizing JAK-STAT Signal Pathway By Infectious Bronchitis Virus

There be four types of Coronavirus:α type,β type,γ type and δ type.At present,seven kinds of coronaviruses are known to infect and affect human health,of which three are highly pathogenic coronaviruses:MERS-CoV,SARS-CoV and SARSCoV-2,which broke out at the end of 2019 and is causing a global pandemic.Coronavirus is ubiquitous in animals,which not only has the risk of cross-species infection,but also causes serious harm to the aquaculture industry.Infectious bronchitis virus belongs to coronavirus family and γ-coronavirus genus.It was first discovered in 1930s.It mainly infects birds and has many serotypes.It causes serious damage to respiratory tract,kidney,fallopian tube and other organs of chickens,mainly causes the decline of egg laying rate and fallopian tube damage,and causes serious economic losses to the poultry industry.Innate immune response is the first line of defense against the virus,and type Ⅰinterferon(IFN)is a key component of this response.After virus infection,it activates pathogen recognition receptors in cells,induces the expression and secretion of interferon,then activates JAK-STAT signal pathway,induces the expression of hundreds of antiviral interferon-stimulating genes(IFN stimulated gene,ISGs),and protects against virus infection and replication.Like other viruses,IBV has evolved strategies that can evade and counteract the body’s antiviral innate immune response so that it can survive on its own.It has been reported that IBV can inhibit the phosphorylation and nuclear translocation of STAT1 in IFN-β-induced JAK-STAT signaling pathway in the late stage of infection,but the detailed mechanism is still unclear.In this study,IBV was used as the research object to screen the viral proteins that antagonize the JAK-STAT signal pathway of IBV,and to explore the functional conservation of viral proteins in coronaviruses.The main contents and results are as follows:In order to verify whether IBV antagonizes JAK-STAT signal pathway,Vero cells were infected with IBV and PEDV was used as positive control.The results showed that IBV infection did not activate JAK-STAT signal pathway,and could inhibit the phosphorylation of STAT1 and STAT2 and nuclear translocation of STAT1 induced by IFN-β in different degrees.It is speculated that the protein encoded by IBV itself is involved in antagonizing the JAK-STAT signal pathway.In order to explore whether the protein encoded by IBV is involved in antagonizing JAK-STAT signal pathway,23 proteins encoded by IBV were cloned and expressed.JAK-STAT signal pathway was stimulated by IFN-β and nuclear translocation of STAT1 was induced.The viral proteins antagonizing STAT nuclear translocation were screened by indirect immunofluorescence.The results showed that nsp2,nsp4,nsp6,nsp13,nsp15 and N proteins encoded by IBV inhibited the nuclear translocation of STAT1 induced by IFN-β,and it was also found that nsp2,nsp5,nsp6,nsp8,nsp13,nspl5,N and E proteins could also inhibit p-STAT1 nuclear translocation.Further Western blotting analysis showed that the selected viral protein did not inhibit the phosphorylation of STAT1 and STAT2,suggesting that the viral protein of IBV may antagonize the JAK-STAT signal pathway by inhibiting the nuclear translocation of STAT.To further verify the above results,we constructed expression plasmids for STAT1 and STAT2,and co-transfected the screened viral proteins with them.The study found that the viral protein nsp15 can down-regulate the expression of exogenous STAT1 and STAT2 proteins,and its nuclease activity and nsp15 Trimerization plays a key role in antagonizing the JAK-STAT signaling pathway.It has also been confirmed that IBV N protein is involved in inhibiting the nuclear translocation of STAT1 induced by IFN-β;the immunoprecipitation test initially confirmed that the N protein of IBV interacts with STAT1 and STAT2;further studies have found that the N proteins from different types of coronaviruses are all It can inhibit the nuclear translocation of STAT1,indicating that the N protein antagonizes the function of the JAK-STAT signaling pathway,which is universal in coronaviruses.In this study,by screening the IBV protein that antagonizes JAK-STAT signal pathway,it is found that the N protein of coronavirus is the antagonistic protein of JAKSTAT signal pathway.Follow-up studies will explore the mechanism of N protein inhibiting STAT1 nuclear translocation and provide theoretical support for the prevention and control of coronavirus.