The antiviral response following the infection of cells with positive RNA viruses involves the expression of genes encoding Interferon-α and -β (IFN-α /-β), both of which induce the expression of a number of Interferon stimulated genes (ISGs) in a an autocrine and paracrine manner. The activation of the expression of IFN-α and -β itself however depends on a cytoplasmic signaling pathway which involves the recognition of viral ssRNA and dsRNA intermediates by cellular pattern recognition receptors (PRRs), with retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) the best characterised PRRs relevant for RNA viruses. Indeed, as a result of the Coronavirus infection, viral dsRNA intermediates binds both to MDA5 and PACT, following the recruitment of RIG-1 by the latter. This complex then associates with mitochondrial antiviral signaling protein (MAVS)/IFN-β promoter stimulator 1 (IPS-1) at the mitochondrial membrane. The localisation of RIG-1 in close proximity of mitochondria allows for the recruitment of stimulator of interferon genes (STING), which by itself is not a PRR but requires RIG-1 for its activation. Following the recruitment of STING in a RIG-1 dependent but MDA-5 independent manner, STING forms dimers with STING localises in the ER membrane, thus forming the mitochondria-associated membrane (MAM) complex as described in pervious post.
Dimerised STING subsequently translocates to perinuclear punctate structures where the dimer interacts with tank-binding kinase 1 (TBK1) and Interferon regulatory factor- 3 (IRF-3), which is preceded by K-63 ubiquitination and phosphorylation of STING. Phosphorylation of IRF-3 allows the translocation of IRF-3 into the nucleus where the expression of IFN-α and -β as well as other cytokines is induced. In addition to IRF-3, polyubiquitinated and phosphorylated STING also induces NF-kB signaling, but the precise mechanism is still debated. The significance of STING for antiviral signaling and inhibiting the replication of RNA viruses is highlighted by the observation that the replication of both negative and positive sense RNA viruses is enhanced in STING deficient cells and STING expression is increased in cells infected with several RNA viruses.
STING can also be activated by another cytoplasmic PPR, cyclic GMP-AMP synthase (cGAS). In contrast to MDA-5 or RIG-1, cGAS however does not detect viral ssRNA nor Poly(I:C) (but maybe dsRNA), yet it seems to be a restriction factor for all positive strand RNA viruses’ tested, including Equine Arterivirus (EAV) (no Coronavirus was tested), independent of of RIG-1. In this scenario, viral dsRNA intermediates recognised by cGAS activate cGAMP and activated cGAMP translocates to the ER where the it binds to STING, followed by the translocation of STING dimers to perinuclear punctae as described above. Alternatively, cGAS might be activated by the viral replication centers formed during the replication of positive strand RNA viruses. Indeed, the formation of Herpesvirus derived virus-like particles and cationic liposomes induce the translocation of STING to perinuclear punctae and the expression of IFN in a PI-3-K dependent manner. So far however the precise mechanism and the importance of this pathway in ablating the replication of positive strand RNA viruses is still under investigation. It might be possible that this pathway plays a role later in the infection following the onset of transcription of the viral RNA and the formation of RTCs.
|cGAS and STING|
Coronavirus PLP, PLPro, and N: taking the STING out of antiviral signaling?
Both the coronaviral PLP and PLPro proteases are encoded within the nsp-3 gene (in contrast to 3CLpro which is encoded by nsp-5) and partially are responsible for cleaving the viral orf1a polyprotein. In terms of antiviral signaling elicited by STING, it has been demonstrated that SARS-CoV PLPro as well as the PLP derived from HCoV-NL63 and the (neurotropic) murine Coronavirus, Mouse hepatitis Virus (MHV)-A59, antagonise STING induced induction of IFN following treatment of cells with Poly (I:C) in the absence of other viral proteins.
In the case of PLpro, a truncated version of nsp-3 encoding only the PLpro transmembrane domain is sufficient to inactivate STING mediated activation of IRF-3.
As outlined above, STING signalling involves the K-63 ubiquitination of STING prior to its association with TBK-1 and IRF-3. Since PLpro contains a deubiquitinase (DUB) domain similar to cellular DUBs, it has been proposed that PLpro deubquitinates STING, TBK-1, RIG-1, and IRF-3 as well as deISGylating cellular proteins. Although these properties have been confirmed both in vitro and in cell lines, the treatment with chemical inhibitors targeting the DUB activity as well as expressing PLpro mutants with mutations within the catalytic domain of PLpro did not antagonise the induction of type-I IFN following Poly (I:C) treatment completely. Subsequent studies showed that PLpro sequesters STING at the ER and thus prevents the formation of dimers independent of the catalytic domain. If PLpro however also facilities the degradation of STING -either via the Proteasome or the Autophagy pathway- has not been shown. Hypothetical, induction of autophagy might be possible in the absence of the DUB via binding of p62/SQSTM1 to the Ubiquitin-Like domain (UBL) of PLpro. In this scenario, not only would STING being degraded but the induction of autophagy itself via the ATG5/ATG12 complex can inhibit RIG-1 and MAVS signaling. Alternatively, sequestering of STING by the viral PLpro might prevent STING from being ubiquitinylated.
|STING binds Mitochondria via RIG-1|
|Interference of PLpro with STING mediated signaling|
In the case of alpha- and betacoronaviruses, nsp-3 encodes for two PLPs. In the case of HCoV NL-63 and Porcine Epidemic Diarrhoea Virus (PEDV) , the viral PLP2 has a DUB domain similar to SARS-CoV PLpro, which akin to PLpro antagonises STING mediated nuclear translocation of IRF-3. Furthermore, the catalytic domain of PLP2 is dispensable for STING inhibition and both PLP2 and full-length nsp-3 of HCoV-NL63 co-localise with STING in a pattern reminiscent of the ER. Akin to PLpro, PLP2 also prevents dimerisation of STING and TBK-1 as well as K63 polyubiquitination via the DUB domain although the DUB is not required for STING inactivation. Interestingly, in the case of PEDV PLP2, the ability of inhibiting STING is dependent on the C-terminal transmembrane domain, suggesting that this domain might be required for correct folding of the protein or for sequestering STING.
In short, both PLP2 and PLpro inactivate STING by a similar mechanism that might involve sequestering STING at the ER. Does the expression of either PLP or PLpro degrade STING? This remains to be investigated. Is the DUB required for the inactivation or merely involved in targeting ubiquitinated STING? Again, this remains to be investigated. Sequestering STING at the ER is not however limited to CoV. Viral proteins from Dengue Virus, Yellow Fever Virus and Hepatitis C Virus have all been postulated to bind STING. The arteriviral PLP2 suppresses antiviral signaling via the DUB domain; it remains to seen however if this is mediated by deubiquitinating STING. Unlike CoV PLP2 or PLpro however the catalytic site needs to be intact.
Finally, what about MERS-CoV? Recently published results suggest that MERS-CoV PLpro reduces the levels of both ISGylated and ubiquitinylated proteins and inhibiting antiviral MAVS signaling, suggesting sequestration of STING.
Last but not least, PEDV N protein has been shown to bind TBK-1 and thus block induction of IFN expression. Coronaviruses therefore block antiviral signaling at multiple stages of the replication cycle although nsp-3 might be a central part.
|A STING in antiviral signaling by CoV proteins|
Does the expression of PLP2 and/or or PLpro prevent preventing mitophagy by antagonizing Parkin mediated ubiquitination of mitochondria following the induction of ER stress or induced by SARS-CoV orf9b (assuming that SARS-CoV orf9b induces mitophagy)? Does the induction of autophagy antagonise the inactivation of STING? Does the co-expression of nsp-6 prevent mitophagy in the absence of PLP2/PLpro ? If so, then the clearance of damaged mitochondria might be affected in cells infected with CoV. In the context of inhibiting apoptosis induced by the expression of viral genes this however might advantageous for viral replication.
Another time, another place, somebody might just be curious as I am. So, who is up to the challenge?
Another time, another place, somebody might just be curious as I am. So, who is up to the challenge?
|PLpro and Mitophagy: Impact of clearance of damaged mitochondria|
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