Study On The Regulation And Mechanism Of STAG2 On PDCoV Infection And Replication

Porcine deltacoronavirus（PDCoV）is a member ofδ-coronavirus.Pigs of different ages will have different degrees of diarrhea after being infected with PDCoV.Piglets are often accompanied by symptoms of dehydration and vomiting.In addition to pigs,PDCoV can also infect humans as well as chickens,turkeys,mice,calves and other animals,bringing potential threats to the development of breeding industry and human public health and safety.Stromal antigen 2（STAG2）is one of the subunits of cohesin complex,which is widely found in vertebrate somatic cells.Some studies have shown that STAG2 gene deletion can significantly inhibit the replication of rotavirus,chikungunya virus and several influenza viruses in cells,but the interaction between STAG2 and coronavirus is rarely reported.Therefore,this experiment takes the STAG2 gene as the research object to explore whether it can regulate the replication of PDCoV,and clarify its molecular mechanism,so as to provide a new idea for the study of the interaction between PDCoV and host protein.In order to explore the effect of STAG2 on PDCoV replication and host cytokine expression,three groups of sg RNA targeting STAG2 gene were designed through sg RNA online design website and ligated with lenti CRISPR v2 vector to construct three groups of recombinant plasmids named p STAG2-sg1,p STAG2-sg2 and p STAG2-sg3 respectively.The constructed recombinant plasmid and lentivirus packaging plasmid were co-transfected into 293T cells,and the lentivirus was harvested at 48 h and 72 h after transfection.Porcine intestinal epithelial cells（IPEC-J2）were infected with lentivirus.A STAG2 gene knockout monoclonal cell line derived from p STAG2-sg1 group was obtained by puromycin screening and limited dilution method,which was named IPEC-J2-STAG2^-/-（KO）.The cell line was successfully constructed and had good genetic stability by sequencing and Western blot methods.In order to further explore the regulatory effect of STAG2 on PDCoV replication,IPEC-J2-STAG2^-/-and wild type IPEC-J2 cells（WT）were infected with PDCoV（MOI=0.1）.It was found that PDCoV replication in KO cells was significantly lower than that in KO cells by indirect immunofluorescence assay;the m RNA transcription level and protein expression of PDCoV N protein in the two kinds of cells at different time points were detected by RT-q PCR and Western blot methods.The results of RT-q PCR showed that compared with WT cells,with PDCoV infection after 12 h,24 h,36 h,48 h,the m RNA level of PDCoV N protein in KO cells was down-regulated by 71%,72%,70%and 31%.Western blot results showed that the expression of PDCoV N protein in KO cells was significantly lower than that in WT cells.In order to further verify the effect of STAG2 gene deficiency on PDCoV replication,two kinds of cells were infected with PDCoV（MOI=0.1）.The virus fluids of the offspring at 12 h,24 h and36 h after infection were collected,and the titers of PDCoV were detected by TCID₅₀.The results showed that the TCID₅₀titers of progeny viruses collected from KO and WT cells at 12 h,24 h and 36 h after infection with PDCoV were 10^-6.48and 10^-7.46,10^-6.47and 10^-7.47,10^-6.49and 10^-7.48respectively.The above results show that STAG2 gene deficiency can significantly inhibit the replication of PDCoV in IPEC-J2 cells.In order to further study the molecular mechanism of STAG2 gene deficiency inhibiting PDCoV replication,the expression of innate immune related factors in WT and KO cells was detected by RT-q PCR.The results showed that compared with WT cells,the m RNA transcription levels of IFN-β,IFN-λ1,IFN-λ3,ISG15,ISG54,ISG56,OAS1,OASL and STAT1 were up-regulated in KO cells.In order to explore whether a large number of expressed ISGs is produced through JAK-STAT signal pathway,the expression of phosphorylated STAT1 protein（p-STAT1）in WT and KO cells was detected by Western blot.The results showed that p-STAT1protein expression was detected in KO cells.In order to further explore whether the deletion of STAG2 gene affects the adsorption of the virus and then affects the replication of the virus,the adsorption of PDCoV in WT and KO cells was detected by RT-q PCR.The results showed that compared with WT cells,the m RNA transcription level of PDCoV N protein in KO cells was down-regulated by about 80%.In order to further verify whether the decrease of virus adsorption is related to interferon-induced transmembrane protein（IFITM）,the level of IFITM3 m RNA transcription in WT and KO cells was detected by RT-q PCR.The results showed that the IFITM3m RNA transcription level in KO cells was up-regulated by 1.05 times as compared with that in WT cells.These results suggest that the deletion of STAG2 gene can induce the production of inherent antiviral factors such as IFNs,and the JAK-STAT signal pathway is activated,which produces a large amount of ISGs and inhibits virus replication in host cells.At the same time,interferon can also induce the production of IFITM3,weaken the adsorption process of the virus,and then inhibit the replication of PDCoV.To sum up,this study successfully constructed a STAG2 gene knockout IPEC-J2 cell line by using CRISPR/Cas9 gene editing technique,which was used as a model to reveal for the first time that the deletion of STAG2 factor can regulate the expression of related cytokines to initiate innate immune response,thus inhibiting the adsorption and replication of PDCoV in IPEC-J2cells.This study provides a new insight for the study of the interaction between PDCoV and host protein,and also provides a new clue for further understanding the role of STAG2 in coronavirus infection.