The Mechanism Of Porcine Circovirus Type 2 Rep Protein Escapes Type Ⅰ Interferon-mediated Antiviral Response

Innate immune system is the host’s first line to defense against pathogen invasion,in which type Ⅰ interferon-mediated antiviral response plays an important role in host’s antiviral processes.When host cells are invaded by pathogenic microorganisms,the host cells recognize the pathogens’ pathogen-associated molecular patterns(PAMPs)through the cell’s pattern recognition receptors(PRRs),thereby activating the receptor-related signaling pathways to induce the production of type Ⅰ interferon through the signal cascade amplification mechanism.IFNAR1 and IFNAR2 on the cell surface recognize and bind with secreted type Ⅰ interferon to activate JAK1 and TYK2.Subsequently,STAT1 and STAT2 are recruited by receptors and then phosphorylated by activated tyrosine kinases,inducing heterodimer formation of activated STAT1 and STAT2.The heterodimers interact with IRF9 to further form the heterotrimeric complex ISGF3,which is transferred into the nucleus and binds to the ISRE promoter to trigger the transcription and expression of a large number of ISGs,finally help the host establish antiviral status.At the same time,many viruses have evolved multiple strategies to escape the type Ⅰ interferon-mediated antiviral response.Previous studies have shown that when the JAK-STAT signaling pathway is inhibited by its specific inhibitor AG490,it can promote PCV2 proliferation in PK-15 cells.However,it is not clear whether PCV2 can escape the antiviral response,and the specific mechanism has not been reported.Based on the above information,we screened the main encoded proteins and explored their roles in the JAK-STAT signaling pathway to clarify the molecular mechanism between the pathogenicity of PCV2 and the host’s innate immunity.In the study,we first infected PK-15 cells with PCV2 and added IFN-α stimulation,and then confirmed that PCV2 can significantly inhibit the type Ⅰ interferon-mediated antiviral response by qPCR method.Next,we constructed six PCV2-encoding protein expression plasmids.Then six PCV2-encoding proteins were transfected into 293T cells and stimulated with IFN-α.The double luciferase reporter system and real-time fluorescence quantitative PCR assay results showed that PCV2 Rep significantly inhibited the ISRE promoter activity induced by IFN-α(P<0.001)and the mRNA levels of ISG15,ISG54 and ISG56(P<0.01).To further explore the mechanism of action,the expression plasmids of Rep were transfected into 293T cells and PK-15 cells and stimulated with IFN-α.Westen Blotting showed that PCV2 Rep did not influence the expression and phosphorylation level of JAKs,STATs and IRF9 in the JAK-STAT pathway.The expression plasmids of Rep and ISGF3 complex component(STAT1,STAT2,IRF9)cotransfected into 293T cells.The real-time fluorescence quantitative PCR assay results showed that Rep inhibited the mRNA expression levels of ISG54 and ISG56 induced by ISGF3(P<0.05),indating that PCV2 Rep targeted at ISGF3 or its downstream to inhibit the production of ISGs.The expression plasmids of Rep,STAT1,STAT2 and IRF9 cotransfected into 293T cells and stimulated with IFN-α.Co-IP results showed that Rep did not influence the formation of the ISGF3 complex.Then the expression plasmids of Rep transfected into 293T cells and stimulated with IFN-α.The experiment of nuclear and cytoplasmic extraction showed that Rep inhibited ISGF3 complex translocation from the cytoplasm to the nucleus.These results indicated that PCV2 Rep blocked the process of ISGF3 complex entering into the nuclear,thereby inhibiting the downstream signaling pathway.The nuclear transport of ISGF3 complex is mainly mediated by the Karyopherin alpha-1(KPNA1)which is a nuclear transport protein.The expression plasmids of Rep and KPNA1 cotransfected into 293T cells and stimulated with IFN-α.Co-IP results showed that Rep interacted with KPNA1 and blocked the binding of p-STAT1 with KPNA1.These results showed that PCV2 Rep blocked ISGF3 complex entering into the nucleus by targeting at KPNA1,thereby inhibiting the production of ISGs.Nuclear transport proteins mediate the nuclear transport of proteins which have nuclear localization sequences(NLS).A NLS sequence 8RSGPQPHKRWVFTLNNPSEDERKKIR33 in PCV2 Rep was found and predicted that it had high nuclear entry activity using the cNLS Mapper software.In order to observe whether Rep NLS is a key sequence that affects the binding between p-STAT1 and KPNA1.The RepΔNLS expression plasmid lacking NLS was constructed based on the Rep expression plasmid.The expression plasmids of Rep or RepΔNLS,KPNA1,STAT1 cotransfected into 293T cells and stimulated with IFN-α.Co-IP results showed that RepΔNLS restored the interaction between p-STAT1 and KPNA1 compared to Rep.In addition,the expression plasmids of Rep or RepΔNLS transfected into 293T cells and stimulated with IFN-α.The real-time fluorescence quantitative PCR assay results showed that RepΔNLS significantly increased the expression of ISG54 and ISG56 mRNA compared with Rep(P<0.05),indicating NLS was the key sequence to inhibit the interaction of p-STAT1 with KPNA1 and the production of ISGs.In conclusion,PCV2 Rep inhibited the binding of KPNA1 and p-STAT1 through interacting with KPNA1,thereby inhibiting the ISGF3 complex into the nucleus and the production of ISGs induced by IFN-α,and NLS played an important role in the process of Rep inhibiting the production of antiviral proteins.