Study On The Role Of CGAS/STING In Porcine Circovirus Type 2 Infection

Porcine Circovirus Type 2(PCV2)is a single-stranded circular DNA virus that is the key pathogen of porcine circovirus associated disease(PCVAD),which causes weaning piglet multi-system failure syndrome(PMWS)Molecules(Karuppannan and Opriessnig 2017).PCV2 infected pigs can be immunosuppressed,the natural immune response weakened,susceptible to a variety of pathogens,leading to a serious mixed pathogen infection,high mortality,to the pig industry caused huge economic losses.cGAS / STING plays an important role in the body's antiviral immune response and the secretion of some inflammatory factors,but it has not yet reported how cGAS,STING play an important role in the process of the PCV2 infection.In this study,PK-15 cells were used as the model cells and wild-type PK-15 cells were infected with the CRISPR-Cas9 lentivirus knockout system targeting the porcine cGAS gene and the sting gene and screened with the appropriate concentration of puromycin to obtain positive clones Cells were obtained from cGAS knockouted cells and sting knockouted cells.The wild-type PK-15 cells,cGAS knockouted cells and sting gene knockouted cells were infected with PCV2 virus with the same titer respectively.The changes of cytokines IFN-?,IL-6 and IL-1? and the changes of viral replicates were observed after PCV2 infection by Real-time PCR and ELISA Methods.The following experimental results were obtained:1.A cGAS knockouted cell and a sting knockouted cell were successfully constructed using the CRISPR-Cas9 gene knockout system.In the cGAS gene knockouted cells constructed at 376 sites of cGAS gene,the 277 th to 284 th base of the CDS region was deleted and 7 bases were absent,resulting in the subsequent sequence shift.The amino acids at positions 92-111 of the cGAS protein were mutated and terminated at the 111 th amino acid.In the sting gene knockouted PK-15 cell,which was located at the 196 locus of sting gene,The 194-198 base of the sting gene was deleted,and the subsequent sequence of frames was mutated,resulting in the initiation of multiple amino acid sequences from the 65 th amino acid.The results of western blotting showed that the expression of cGAS protein was not detected in knockouted cells targeting cGAS gene 376,and the expression of STING protein was not detected in knockouted cells located at 196 locus of sting gene.2.PCV2 infected wild-type PK-15 cells,cGAS knockouted PK-15 cells(376PK-15cGAS-/-)and sting knockouted PK-15 cells(196PK-15sting-/-).Results of Real-time PCR show that compared with wild-type PK-15 cells,the deletion of cGAS gene increased the secretion of IL-6 and IL-1? at 6 h ~ 24 h of PCV2 infection.In the early stage of PCV2 infection(6 h,12 h)IFN-? mRNA level was significantly increased,and the mRNA level of IFN-? was significantly increased at 24 h after infection.The deletion of sting gene resulted in a significant increase in IL-6 and IFN-? levels at 6 h ~ 24 h of PCV2 infection,and had no significant effect on IL-1? mRNA levels.The results of ELISA were consistent with the results of Real-time PCR.The effect of PCV2 on cGAS gene knockouted cells and sting gene knockouted cells was significantly lower than that in wild-type PK-15 cells.In this study,cGAS and STING were involved in PCV2 infection-induced immune response.cGAS protein may be involved in the regulation of inflammatory body formation and PCV2 infection in PCV2-infected PK-15 cells.While the STING protein is more biased to participate in the regulation of PCV2 infection caused by antiviral immune response and type I interferon production process.And both proteins are the upstream molecules that regulate PCV2-induced inflammatory responses,antiviral responses.cGAS and sting deletion will have an impact on PCV2 virus replication.