Regulation Of Cellular Antiviral Response By Protein Kinase A

Viral infection triggers antiviral innate immune responses of host cells.After detection of pathogen associated molecular patterns(PAMPs)by germline-coded pattern-recognition receptors(PRRs),host cells would initiate a series of signaling cascades,leading to the induction of a large number of downstream antiviral genes including type I interferons(IFNs),interferon-stimulating genes and inflammatory cytokines,which will ultimately contribute to clearance of the invaded viruses by triggering apoptosis of virus-infected cells,suppressing viral replication,as well as facilitating the adaptive immune response.Host cells have employed kinds of delicate regulatory mechanisms to timely terminate the expression of IFNs and downstream antiviral genes and avoid excessive immune responses at the late phase of viral infection,RNA virus-triggered induction of downstream antiviral genes is mainly mediated by the cytoplasmic RIG-I-like receptor(RLR)family members including RIG-I and MDA5,which recognize viral RNA in the cytosol upon viral infection.After RNA-binding and activation,RIG-I/MDA5 translocate to mitochondria and interact with the mitochondrial-localized adaptor VISA through their respective CARD domain.Then,VISA forms large prion-like polymers via its CARD domain and recruit several TRAFs,as well as kinases IKK and TBK1,which in turn phosphorylate and activate transcriptional factors NF-kB and IRF3 respectively,leading to the ultimate induction of inflammatory cytokines and type I IFNs.At the late phase of viral infection,the activated VISA undergoes K48-linked poly ubiquitination by different E3 ubiquitin ligases and subsequent proteasome-dependent degradation to avoid sustained activation of the signaling transduction.Multiple signaling molecules including RIG-I?cGAS?MITA?TBK1 and IRF3 have been previously reported to undergo phosphorylation upon viral infection,which play important regulatory roles on the timely termination of antiviral immune responses.However,whether phosphorylation also targets the critical adaptor VISA for turning-off its activation has not been reported so far.In order to figure out the potential kinases that could mediate phosphorylation of VISA and regulate its activity,we screened a pool of 352 kinases and identified two candidate kinases,namely PRKACA and PRKACB,two catalytic subunits of cAMP-dependent Protein Kinase A(PKA),which redundantly catalyze phosphorylation of VISA and suppress its activation,as well as negatively regulate RNA virus-induced expression of type I IFNs and cellular antiviral immune responses.Biochemical experiments showed that PKA phosphorylates Thr54 of VISA,which inhibited oligomerization of the CARD of VISA,as well as facilitated the recruitment of E3 ligase MARCH5 and the subsequent MARCH5-mediated K48-linked ubiquitination and degradation of VISA.Mice treated with H89,a PKA inhibitor,produced higher levels of serum cytokines including IFN-?4,IFN-? and IL-6 upon EMCV infection and were more resistant to EMCV infection-induced death.Interestingly,we observed that viral infection triggers accumulation of cellular cAMP and the increase of the activity of PKA,especially at the late phase.Exogenous added cAMP could significantly inhibit virus infection-induced expression of type I IFNs and downstream antiviral genes,which is consistent with the critical role of PKA in termination of antiviral immune response at the late phase of viral infection.Taken together,these results suggest that cAMP-dependent protein kinase A phosphorylates Thr54 of VISA and negatively regulated RNA virus-induced expression of type I interferons and downstream antiviral genes at the late phase of viral infection,so as to prevent sustained activation of signaling and avoid excessive immune responses.Our present study will contribute to the complete therapy design and new drug development for viral infection and autoimmune diseases.