Regulation Of IRF3Signaling Pathway By The Papain-like Protease Of The SARS Coronavirus

The innate immune system is the first line of defense against invading viruses.Host cells sense pathogen-associated molecular patterns (PAMPs) by widelyexpressed pattern-recognition receptors (PRRs), which trigger the induction of type Iinterferon (IFNs) and proinflammatory cytokines. Human coronaviruses (CoV)such as severe acute respiratory syndrome coronavirus (SARS-CoV) and NL63-CoVhave evolved strategies to escape from the innate immune system. It was reportedthat SARS-CoV encodes at least eight proteins acting as innate immune antagonists,including the nonstructural protein1(NSP1), NSP7, NSP15, the open reading frame3b (ORF3b), ORF6, papain-like protease (PLpro), membrane protein (M) andnucleocapsid (N). Most of them have been shown to target IFN regulatory factor3(IRF3) to prevent the activation of the IFN system. However, the regulationmechanism of IRF-3activation by PLpro is poorly understoodPLpro is a catalytic domain of SARS NSP3, which is also a deubiquitinase(DUB). A transmembrane (TM) form of PLpro (PLpro-TM) was used in the studyto dissect the possible negative regulation mechanism of IRF-3activation by PLpro,as NSP3predominantly resides in the endoplasmic reticulum(ER) region of a cell.We found that (1) PLpro-TM inhibits RIG-I and other signal proteins mediatedactivation of IRF3signaling pathway by dual luciferase activity assay;(2) PLpro-TMdisrupts the interaction between TRIM25(RING-finger E3ubiquitin ligase) andRIG-I, and deubiquitinates the Lys63-linked ubiquitin moiety from the N-terminalCARDs of RIG-I, which have little effects on the activation of IRF3;(3) PLpro-TMblocks the signal transduction from MAVS to TRAF3and STING. However, thissignaling disruption does not effectively inhibit the activation of IRF3;(4) PLpro-TMremoves the lysine-63-linked polyubiquitin chains from TRAF3, resulting in itsdissociation from the downstream signaling complex containing TANK-bindingkinase1(TBK1) and IκB kinase epsilon (IKK);(5) PLpro-TM inhibits dimerizationand phosphorylation of IRF3induced by overexpression of stimulator of interferongenes (STING), perhaps mainly because PLpro-TM disrupt the interaction betweenSTING and IRF3, which keeps TBK1from phosphorylating IRF3. Also perhaps thisinhibition has something to do with the interaction between PLpro-TM and the TM of STING, which may inhibit membrane trafficking of STING. Accordingly, our datareveal a novel regulation mechanism of antiviral immune response of PLpro throughdisrupting TRAF3and STING mediated IRF3pathway, which would be helpful fordevelopment of novel anti-coronavirus therapy.