Coronavirus PLpro, PLP2, and N: taking a STING out of antiviral signalling

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.

STING

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 mediated signaling pathway

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, PL^Pro,  and N: taking the STING out of antiviral signaling?

Both the coronaviral PLP and PL^Pro proteases are encoded within the nsp-3 gene (in contrast to 3CL^pro 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 PL^Pro   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.

 

PLP/PLpro of SARS-CoV, HCoV-NL63 and MHV-A59

In the case of PL^pro, a truncated version of nsp-3 encoding only the PL^pro  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 PL^pro contains a deubiquitinase (DUB) domain similar to cellular DUBs, it has been proposed that PL^pro 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 PL^pro mutants with mutations within the catalytic domain of PL^pro 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 PL^pro 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 PL^pro. 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 PL^pro, which akin to PL^pro 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 PL^pro, 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 PL^pro inactivate STING by a similar mechanism that might involve sequestering STING at the ER. Does the expression of either PLP or PL^pro 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 PL^pro however the catalytic site needs to be intact.

Finally, what about MERS-CoV? Recently published results suggest that MERS-CoV PL^pro 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 PL^pro   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/PL^pro ? 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?

PLpro and Mitophagy: Impact of clearance of damaged mitochondria

Further reading

Kok KH, Lui PY, Ng MH, Siu KL, Au SW, & Jin DY (2011). The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response. Cell host & microbe, 9 (4), 299-309 PMID: 21501829

Cornelissen T, Haddad D, Wauters F, Van Humbeeck C, Mandemakers W, Koentjoro B, Sue C, Gevaert K, De Strooper B, Verstreken P, & Vandenberghe W (2014). The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy. Human molecular genetics, 23 (19), 5227-42 PMID: 24852371

Sureshbabu A, & Bhandari V (2013). Targeting mitochondrial dysfunction in lung diseases: emphasis on mitophagy. Frontiers in physiology, 4 PMID: 24421769

Oh JE, & Lee HK (2014). Pattern recognition receptors and autophagy. Frontiers in immunology, 5 PMID: 25009542 Ablasser A, & Hornung V (2013). DNA sensing unchained. Cell research, 23 (5), 585-7 PMID: 23419517

van Kasteren PB, Bailey-Elkin BA, James TW, Ninaber DK, Beugeling C, Khajehpour M, Snijder EJ, Mark BL, & Kikkert M (2013). Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells. Proceedings of the National Academy of Sciences of the United States of America, 110 (9) PMID: 23401522

Ding Z, Fang L, Jing H, Zeng S, Wang D, Liu L, Zhang H, Luo R, Chen H, & Xiao S (2014). Porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between IRF3 and TBK1. Journal of virology, 88 (16), 8936-45 PMID: 24872591

Zheng D, Chen G, Guo B, Cheng G, & Tang H (2008). PLP2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type I interferon production. Cell research, 18 (11), 1105-13 PMID: 18957937

Yang X, Chen X, Bian G, Tu J, Xing Y, Wang Y, & Chen Z (2014). Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease. The Journal of general virology, 95 (Pt 3), 614-26 PMID: 24362959

Wang G, Chen G, Zheng D, Cheng G, & Tang H (2011). PLP2 of mouse hepatitis virus A59 (MHV-A59) targets TBK1 to negatively regulate cellular type I interferon signaling pathway. PloS one, 6 (2) PMID: 21364999

Clementz MA, Chen Z, Banach BS, Wang Y, Sun L, Ratia K, Baez-Santos YM, Wang J, Takayama J, Ghosh AK, Li K, Mesecar AD, & Baker SC (2010). Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases. Journal of virology, 84 (9), 4619-29 PMID: 20181693

Báez-Santos YM, Mielech AM, Deng X, Baker S, & Mesecar AD (2014). Catalytic Function and Substrate Specificity of the PLpro Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Journal of virology PMID: 25142582

Xing Y, Chen J, Tu J, Zhang B, Chen X, Shi H, Baker SC, Feng L, & Chen Z (2013). The papain-like protease of porcine epidemic diarrhea virus negatively regulates type I interferon pathway by acting as a viral deubiquitinase. The Journal of general virology, 94 (Pt 7), 1554-67 PMID: 23596270

Sun L, Xing Y, Chen X, Zheng Y, Yang Y, Nichols DB, Clementz MA, Banach BS, Li K, Baker SC, & Chen Z (2012). Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STING-mediated signaling. PloS one, 7 (2) PMID: 22312431

Sun Z, Li Y, Ransburgh R, Snijder EJ, & Fang Y (2012). Nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15. Journal of virology, 86 (7), 3839-50 PMID: 22258253

Ishikawa H, & Barber GN (2008). STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature, 455 (7213), 674-8 PMID: 18724357

Ouyang S, Song X, Wang Y, Ru H, Shaw N, Jiang Y, Niu F, Zhu Y, Qiu W, Parvatiyar K, Li Y, Zhang R, Cheng G, & Liu ZJ (2012). Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-GMP binding. Immunity, 36 (6), 1073-86 PMID: 22579474

Saitoh T, Fujita N, Hayashi T, Takahara K, Satoh T, Lee H, Matsunaga K, Kageyama S, Omori H, Noda T, Yamamoto N, Kawai T, Ishii K, Takeuchi O, Yoshimori T, & Akira S (2009). Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proceedings of the National Academy of Sciences of the United States of America, 106 (49), 20842-6 PMID: 19926846

Maringer, K., & Fernandez-Sesma, A. (2014). Message in a bottle: lessons learned from antagonism of STING signalling during RNA virus infection Cytokine & Growth Factor Reviews DOI: 10.1016/j.cytogfr.2014.08.004

Mielech AM, Chen Y, Mesecar AD, & Baker SC (2014). Nidovirus papain-like proteases: Multifunctional enzymes with protease, deubiquitinating and deISGylating activities. Virus research PMID: 24512893

Mielech AM, Kilianski A, Baez-Santos YM, Mesecar AD, & Baker SC (2014). MERS-CoV papain-like protease has deISGylating and deubiquitinating activities. Virology, 450-451, 64-70 PMID: 24503068

Xiong, H., Wang, D., Chen, L., Choo, Y., Ma, H., Tang, C., Xia, K., Jiang, W., Ronai, Z., Zhuang, X., & Zhang, Z. (2009). Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation Journal of Clinical Investigation, 119 (3), 650-660 DOI: 10.1172/JCI37617