SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored Plpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKK?-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKK?, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3- TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which Plpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.     

Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63

Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.     

抗病毒天然免疫通路中IRF-3调控新机制

干扰素调节因子-3(interferon regulatory factor-3,IRF-3)是IRF家族中重要 转录因子之一,在调控干扰素(interferon, IFN)基因表达和抗病毒天然免疫反应中具有重要作 用. 最新发现的MITA (mediator of IRF-3 activation, 又称STING/ERIS)蛋白是宿主抗病 毒天然免疫反应中的一种重要调节分子. 病毒侵染时,MITA与IRF-3相互作用,特异性激活 IRF-3,并募集TANK结合激酶1（TANK binding kinase 1, TBK1）与IFN通路中的线粒体抗 病毒信号蛋白MAVS(mitochondrial anti-viral signaling protein)形成复合物,且MITA可 被TBK1磷酸化,诱导Ⅰ型IFN及IFN刺激基因(interferon stimulate genes, ISG)的表达 ,诱发抗病毒天然免疫反应. 同时还发现,泛素连接酶RNF5（ring finger protein 5）可对MITA 发生泛素化修饰从而抑制其对IRF-3活化,实现对宿主抗病毒天然免疫反应负调节作用. 本 室研究发现,严重性急性呼吸系统综合症冠状病毒(severe acute respiratory syndrome co ronavirus, SARS-CoV）和人类新型冠状病毒（human coronavirus NL63, HCoV-NL63）的 木瓜样蛋白酶（papain-like protease, PLP）利用其特有的去泛素化酶（deubiquitinase, DUB）活性,通过宿主细胞泛素-蛋白酶体信号系统对IRF-3的泛素化等翻译后修饰进行调节 ,从而成为该种病毒逃逸机体抗病毒防御系统主要手段之一.     

Coronavirus membrane-associated papain-like proteases induce autophagy through interacting with Beclin1 to negatively regulate antiviral innate immunity

Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 (PLP2-TM) of coronaviruses acts as a novel autophagyinducing protein. Intriguingly, PLP2-TM induces incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2-TM interacts with the key autophagy regulators, LC3 and Beclin1, and promotes Beclin1 interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclin1 partially reverses PLP2-TM’s inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results suggested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclin1, which in turn modulates coronavirus replication and antiviral innate immunity.     

p53 Degradation by a Coronavirus Papain-like Protease Suppresses Type I Interferon Signaling

Infection by human coronaviruses is usually characterized by rampant viral replication and severe immunopathology in host cells. Recently, the coronavirus papain-like proteases (PLPs) have been identified as suppressors of the innate immune response. However, the molecular mechanism of this inhibition remains unclear. Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63 (HCoV-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53. Meanwhile, we identify IRF7 (interferon regulatory factor 7) as a bona fide target gene of p53 to mediate the p53-directed production of type I interferon and the innate immune response. By promoting p53 degradation, PLP2 inhibits the p53-mediated antiviral response and apoptosis to ensure viral growth in infected cells. Thus, our study reveals that coronavirus engages PLPs to escape from the innate antiviral response of the host by inhibiting p53-IRF7-IFNβ signaling.     

Message in a bottle: lessons learned from antagonism of STING signalling during RNA virus infection

STING has emerged in recent years as an important signalling adaptor in the activation of type I interferon responses during infection with DNA viruses and bacteria. An increasing body of evidence suggests that STING also modulates responses to RNA viruses, though the mechanisms remain less clear. In this review, we give a brief overview of the ways in which STING facilitates sensing of RNA viruses. These include modulation of RIG-I-dependent responses through STING's interaction with MAVS, and more speculative mechanisms involving the DNA sensor cGAS and sensing of membrane remodelling events. We then provide an in-depth literature review to summarise the known mechanisms by which RNA viruses of the families Flaviviridae and Coronaviridae evade sensing through STING. Our own work has shown that the NS2B/3 protease complex of the flavivirus dengue virus binds and cleaves STING, and that an inability to degrade murine STING may contribute to host restriction in this virus. We contrast this to the mechanism employed by the distantly related hepacivirus hepatitis C virus, in which STING is bound and inactivated by the NS4B protein. Finally, we discuss STING antagonism in the coronaviruses SARS coronavirus and human coronavirus NL63, which disrupt K63-linked polyubiquitination and dimerisation of STING (both of which are required for STING-mediated activation of IRF-3) via their papain-like proteases. We draw parallels with less-well characterised mechanisms of STING antagonism in related viruses, and place our current knowledge in the context of species tropism restrictions that potentially affect the emergence of new human pathogens.     

Rac1 and PAK1 are upstream of IKK-epsilon and TBK-1 in the viral activation of interferon regulatory factor-3

The anti-viral type I interferon (IFN) response is initiated by the immediate induction of IFN beta, which is mainly controlled by the IFN-regulatory factor-3 (IRF-3). The signaling pathways mediating viral IRF-3 activation are only poorly defined. We show that the Rho GTPase Rac1 is activated upon virus infection and controls IRF-3 phosphorylation and activity. Inhibition of Rac1 leads to reduced IFN beta promoter activity and to enhanced virus production. As a downstream mediator of Rac signaling towards IRF-3, we have identified the kinase p21-activated kinase (PAK1). Furthermore, both Rac1 and PAK1 regulate the recently described IRF-3 activators, I kappa B kinase- and TANK-binding kinase-1, establishing a first canonical virus-induced IRF-3 activating pathway.     

STING Negatively Regulates Double-Stranded DNA-Activated JAK1-STAT1 Signaling via SHP-1/2 in B Cells

Recognition of cytosolic DNA initiates a series of innate immune responses by inducing IFN-I production and subsequent triggering JAK1-STAT1 signaling which plays critical roles in the pathogenesis of infection, inflammation and autoimmune diseases through promoting B cell activation and antibody responses. The stimulator of interferon genes protein (STING) has been demonstrated to be a critical hub of type I IFN induction in cytosolic DNA-sensing pathways. However, it still remains unknown whether cytosolic DNA can directly activate the JAK1-STAT1 signaling or not. And the role of STING is also unclear in this response. In the present study, we found that dsDNA directly triggered the JAK1-STAT1 signaling by inducing phosphorylation of the Lyn kinase. Moreover, this response is not dependent on type I IFN receptors. Interestingly, STING could inhibit dsDNA-triggered activation of JAK1-STAT1 signaling by inducing SHP-1 and SHP-2 phosphorylation. In addition, compared with normal B cells, the expression of STING was significantly lower and the phosphorylation level of JAK1 was significantly higher in B cells from MRL/lpr lupus-prone mice, highlighting the close association between STING low-expression and JAK1-STAT1 signaling activation in B cells in autoimmune diseases. Our data provide a molecular insight into the novel role of STING in dsDNA-mediated inflammatory disorders.     

Arenavirus nucleoprotein targets interferon regulatory factor-activating kinase IKKε

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.     

Binding of bacterial secondary messenger molecule c di-GMP is a STING operation

Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell. Amongst the spillage, the secondary messenger molecule, cyclic dimeric guanosine monophosphate (c di-GMP), was recently shown to be bound by stimulator of interferon genes (STING). Binding of c di-GMP by STING activates the Tank Binding Kinase (TBK1) mediated signaling cascades that galvanize the body' defenses for elimination of the pathogen. In addition to c di-GMP, STING has also been shown to function in innate immune responses against pathogen associated molecular patterns (PAMPs) originating from the DNA or RNA of pathogens. The pivotal role of STING in host defense is exemplified by the fact that STING^-/- mice die upon infection by HSV-1. Thus, STING plays an essential role in innate immune responses against pathogens. This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the immune defenses against invading microbes. Similarly, STING could be targeted for mitigating the inflammatory responses augmented by the innate immune system. This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in delineating the mechanism of STING-mediated responses.