Regulatory Roles And Mechanisms Of GC1qR And HDAC6 In The PCV2 Inhibition Of Type Ⅰ Interferon System Activation

Porcine circovirus type 2（PCV2）,as one of the most widespread pathogens in the world,has brought huge economic losses to the pig industry of our country.The pathogenicity of PCV2 infection alone is weak,however,PCV2 infection impairs the immune function of the host,resulting in increased susceptibility to various pathogens and increased mortality in PCV2-infected pigs.Therefore,to clarify the pathogenic mechanism of PCV2 infection,the primary task is to study the molecular mechanism of PCV2 infection-induced immune dysfunction,which also provides the best way for the effective prevention and control of PCVAD.Type I interferon is an important part of the innate immune system,and the expression products of interferon-stimulated genes（ISGs）are key innate immune molecules for the host to resist various viral infections and inhibit viral replication and spread.Current studies have confirmed that the cGAS-STING signaling pathway is the main pathway to recognize pathogenic DNA and induce the production of type I interferon,and interferon induces the expression of ISGs and exerts antiviral effect through the JAK-STAT signaling pathway.Our previous study found that PCV2 infection could not activate the cGAS-STING pathway to induce IFN-βproduction as effectively as other DNA viruses,meanwhile,PCV2infection could further inhibit the the cGAS-STING pathway activated by other DNA viruses.cGAS is an important molecule for intracellular DNA recognition and sensing,how PCV2regulates the function of cGAS and whether PCV2 inhibits the antiviral response induced by type I interferon is still unclear.In addition,PCV2 promotes its own replication by inducing autophagy in the host cells.gC1qR,an interacting molecule of PCV2 Cap,plays a key role in regulating autophagy,virus replication and mediating the formation of immune suppression.HDAC6 plays a key role in regulating autophagy,specific protein degradation,and RIG-I and TLR-mediated type I interferon production.However,how gC1qR and HDAC6 regulate cGAS-STING pathway activation and type I interferon response have not been reported.In this study,we studied and clarified the molecular mechanism of PCV2 inhibits the activation of cGAS-STING signaling pathway and the response of type I interferon induced by other DNA viruses,as well as the functional role of gC1qR/HDAC6 in this process.Furthermore,the molecular mechanism that PCV2 infection promotes susceptibility to other DNA viruses was elucidated from the level of innate immunity.The results will help us to reveal the molecular mechanism of PCV2 inducing host immune dysfunction comprehensively,and provide theoretical guidance for prevention and purification of PCV2.These results were as follows:1.PCV2 inhibits the activation of type I IFN signaling to facilitate the infection of other DNA viruses by reducing cGAMP productionEpidemiological investigation showed that the infection rate and pathogenic rate of porcine parvovirus（PPV）and porcine pseudorabies virus（PRV）were significantly increased in PCV2 positive pigs（P<0.01）;the PPV and PRV loads in native PCV2 positive pigs are higher than that in native PCV2 negative pigs（P<0.01）;PCV2 inhibited the secretion of IFN-βinduced by PPV or PRV infection（P<0.01）and promoted PPV and PRV replication（P<0.01）;PCV2 inhibits the transcription of Ifn-β,Ifit1,and Cxcl10 in piglet lungs and PK-15cells induced by PPV or PRV（P<0.01）;The inhibitory effects on ISD/PRV/PPV induced IFN-βinduction by PCV2 is dose and time-dependent,yet independent of type I interferon receptor;phosphorylated TBK1,phosphorylated IRF3,and intranuclear IRF3 are remarkably decreased in PCV2-infected cells（P<0.01）.2.PCV2 infection enhances the K48-linked poly-ubiquitination of porcine cGAS at K389,and activates HDAC6 to mediate the degradation of cGASThe cGAS levels were significantly decreased in PCV2-infected cells;the lysosomal acidification inhibitor chloroquine（CQ）could effectively prevented the reduction of cGAS protein in PCV2-infected cells,but the proteasome inhibitor MG132 did not;cGAS proteins were co-localized with LC3 in PCV2-infected cells;PCV2 infection enhances the K48-linked poly-ubiquitination of porcine cGAS at K389 and facilitates the interaction of cGAS with p62;the K48-linked ubiquitination of K389R cGAS mutant was abrogated in PCV2-infected cells,PCV2 infection failed to promote the interaction of K389R cGAS mutants with p62;PCV2infection activates HDAC6 to enhance the deacetylase activity,facilitates the interaction between K48-ubiquitinated cGAS and p62,whereas K389R mutated cGAS were not able to interacted with HDAC6 and p62;Knockdown of HDAC6 markedly decreased the degradation of cGAS,did not affect the K48-ubiquitination levels of cGAS in PCV2-infected cells;in HDAC6^-/-PK-15 cells,the interaction of ubiquitinated cGAS with p62 significantly decreased,which could be reversed by wild-type HDAC6 reconstitution,but not by a mutant HDAC6that loss of its ubiquitin-binding domain;in HDAC6^-/-PK-15 cells,rescued with mutated porcine HDAC6（D538A/D608A/D609A,named as HDAC6-3M）,which abolished the deacetylation activity of HDAC6 or inhibition of deacetylase activity of HDAC6 by its specific inhibitor（Tubastatin A,Tub A）could significantly decreased the interaction of ubiquitinated cGAS with p62 in PCV2-infected cells;PKCδis a key regulator in promoting the activation of HDAC6 in PCV2 infected cells.3.PCV2 infection can phosphorylate porcine cGAS via gC1qR-mediated Akt signaling to silence the catalytic activity of cGAS,promotes polyubiquitination of cGAS,and leads to autophagic degradation of cGAS in PCV2-infected cellsThere was no difference for cGAMP and IFN-βm RNA levels between CQ/3-MA-treated PCV2-infected cells,transfected with mutation of K389 site of porcine cGAS or used HDAC6-specific inhibitors could increase the levels of cGAS in PCV2-infected cells,but these interventions were not sufficient to weaken the inhibitory effects of PCV2 on cGAMP induction and IFN-βm RNA levels;PCV2 promotes the phosphorylation of cGAS at S278,which directly silences the catalytic activity of cGAS,phosphomimetic S278D mutant was not able to catalyze the substrate to produce cGAMP,but the cGAMP production was not found to be reduced in PCV2-infected cGAS^-/-PK-15 cells that rescued with porcine cGAS mutant（S278A）relative to the wild type cGAS;PCV2 promotes the phosphorylation of cGAS at S278via activation of Akt signaling,which directly silences the catalytic activity of cGAS.Subsequently,phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS resulting in a markedly increased cGAS degradation in PCV2infection-induced autophagic cells;the production of cGAMP and IFN-βm RNA in the cells infected with rAd-Cap were significantly lower than that in the cells infected with rAd-Rep or rAd-Blank;the phosphorylation of cGAS and HDAC6 activation were not detected in gC1qR^-/-cells,the gC1qR binding activity deficient mutant strain PCV2Rm A-infected cells showed significantly higher cGAMP production levels than PCV2-infected cells after secondary stimulation（P<0.01）,but IFN-βproduction levels in PCV2-infected cells were still significantly lower than PCV2Rm A Infected cells（P<0.01）;the phosphorylation level of porcine cGAS at S278 was lower in PCV2Rm A-infected cells than that in PCV2-infected cells,poly-ubiquitination and degradation of porcine cGAS were also decreased in PCV2Rm A-infected cells relative to that in PCV2-infected cells.Meanwhile,the level of Ac-tubulin(the natural active substrate of HDAC6 increased in PCV2Rm A-infected cells compared with PCV2-infected cells;more IFN-βwere induced in PCV2Rm A-infected cells than wild-type PCV2 infection upon PRV or PPV challenge;In PCV2Rm A infected cells,the levels of PPV and PRV were significantly lower than that in wild-type PCV2-infected cells;Histopathological examination showed that PRV infection induced lighter histological lesions in the brain and lung of PCV2Rm A-infected pigs than that in the PCV2-infected pigs.4.PCV2 infection inhibits the activation of type I interferon signaling via Capsid protein and host gC1qRPCV2 inhibited the transcription of IFN-stimulated genes（ISGs）transcription after IFN-αstimulation both in vivo and in vitro（P<0.05）;the ISRE promoter activity in PCV2-infected cells were significant reduced upon IFN-α-treatment;PCV2 significantly inhibited the phosphorylation of STAT1 and STAT2,the formation of ISGF3 complex,and the nuclear entry of phosphorylated STAT1 and STAT2 in a dose-dependent manner;the transcriptional factors of ISGF3 binding to the ISRE after IFN-αtreatment was also significantly inhibited in PCV2-infected cells;expressions both of PCV2 Rep and Cap could significantly inhibited IFN-mediated ISRE activation;PCV2 Cap inhibits STAT1/STAT2 phosphorylation and nuclear translocation induced by IFN,while Rep only inhibits the nuclear translocation of phosphorylated STAT1 and STAT2;both of PCV2 Rep and Cap significantly inhibited IFN-mediated ISRE activation;PCV2 Cap protein significantly inhibited the activation of ISRE in the early phase of infection,and PCV2 Rep can further inhibit the activation of ISRE in PK-15 cells induced by IFN-α;both endogenous STAT1 or STAT2 did not interact with PCV2 Cap;gC1qR^-/-PK-15 cells could remarkably attenuate the inhibitory of PCV2 on the IFN-α-induced Ifit1,Cxcl10,and Mx1 transcription（P<0.01）;compare to PCV2-infected wild type PK-15cells,the phosphorylation of STAT1 and STAT2 are significantly increased in gC1qR^-/-PK-15cells.the phosphorylated STAT1 and STAT2 are also increased in the nucleus in gC1qR^-/-PK-15 cells compared with wild type PK-15 cells.In summary,this study focused on the host innate immune response represented by the type I interferon system,and deeply analyzed the molecular mechanism of how PCV2 inhibits the activation of cGAS-STING signaling pathway induced by other DNA viruses and how PCV2 inhibits the type I interferon response.In this study,we clarified that the molecular mechanism of the negative regulation of cGAS phosphorylation induced by gC1qR/AKT in the early stage of PCV2 infection and the molecular mechanism of ubiquitination and degradation of cGAS by gC1qR/PKCδ/HDAC6/autophagy in the later stage of infection,we elucidate the interconnectedness and cooperative mode of two post-translational modifications,cGAS phosphorylation and ubiquitination,in inhibiting the activation of the cGAS-STING signaling pathway in PCV2-infected cells for the first time,we also clarified that the role and mechanism of PCV2 Cap and host gC1qR protein in inhibiting the response to type I interferon.The above two results clarified the molecular mechanism by which PCV2 infection inhibits the production and response of type I interferon,and revealed the reason why PCV2 infection caused susceptibility to other DNA viruses in innate immunity.It is further confirmed that gC1qR is an important regulator of PCV2 inhibiting antiviral innate immunity.Taken together,our findings uncover a systemic mechanism by which PCV2 infection inhibits type I interferon systems,which helps us to provide a basis for further research on the role and mechanism of PCV2 infection on host immune regulation and to develop a new PCV2 attenuated vaccines around gC1qR.