Coronavirus nsp, ER stress, UPR, and ERAD

As discussed before, the essential step in the intracellular life cycle of many positive strand ssRNA viruses is the generation of double membrane vesicles (DMVs) that in most cases (but not all) contain the viral replication and transcription complexes (RTC) and hence serve as the replication platform. In the case of both the Nidovirales and the Flaviviridae these are formed by subverting the membrane of the ER in the absence of conventional ER and secretory pathway markers. Additionally,  non-structural proteins (nsps or NS) do not contain classic sequences such as the KDEL sequence that direct these proteins to the ER. The analysis of the DMVs/RTCs derived from members of both viral families -in particular Mouse Hepatitis Virus (MHV), Equine Arterivirus (EAV), and Japanese Encephalitis Virus (JEV)- however revealed that the DMVs not only contain viral RNA and proteins but also cellular proteins, more specifically components that form the ER-associated degradation (ERAD) vesicle thereby segregating the ERAD factors EDEM-1, OS-9, and SEL-1 from the ER lumen in addition to displaying the non-lipidiated form of LC3, LC3-I, on their cytoplasmic surface. In non-infected cells, the ER is the site of maturation for membrane and secretory proteins. Unfolded or misfolded proteins are normally dislocated across the ER membrane followed by proteasomal and endolysosomal degradation in a process collectively known as ERAD which involves the recruitment of ERAD associated proteins and the subsequent formation of EDEMosomes which form mature autophagosomes and autolysosomes; alternatively, proteins are targeted for proteasomal degradation. Any viral protein therefore which binds EDEM-1, OS-9, and /or SEL-1 therefore would eventually be degraded unless this pathway is inhibited by viral proteins. In the case of the Coronaviral nsp-6 protein, inhibits the maturation of endosomes into lysosomes by preventing the binding and activation of mTORC-1 to the surface of lysosomes through an unknown mechanism as described elsewhere. It would be expected that inhibiting this pathway induces the accumulation of unfolded or misfolded proteins and subsequently induction of apoptosis. In order to counteract the negative effect of accumulated unfolded or misfolded proteins and subsequent ER stress the host cell however induces a unfolded protein response (UPR), a signaling pathway that starts with the activation of three ER stress transducers, (a) PKR- like ER protein kinase (PERK), (b) Activating Transcriptional Factor- 6 (ATF6) and (c) Inositol-requiring Protein-1 (IRE1), which not only increases the expression of genes encoding for components of the ERAD pathway but also induces apoptosis and upregulates the expression of cytokines such as Interferon-β and Interleukin-6 (which of course can be counteracted by CoV). In terms of infection of cells with a Coronavirus such as the avian Infectious Bronchitis Virus (IBV), the infection induces the PERK and IRE1 dependent pathways (as well as PKR, but this pathway is induced by dsRNA and not via ER stress).

Overview of Coronavirus induced UPR via ER stress by orf8ab, nsp-3/-4/-6, and structural S/M/E proteins

As shown in the figure, EDEM1 expression is induced via the splicing of XBP1 whereas the induction of PERK induces apoptosis and the expression of pro-inflammatory cytokines. Whilst the induction of these pathways following the infection with MHV, IBV, or SARS-CoV has been well established it is not clear if the expression of nsp-6, -3, or -4 ( or a combination of these in the absence or presence of other components of the viral RTCs) is sufficient for the induction of the UPR. In infected cells, the maturation of the viral E, M, and S protein involves glycosylation that might increase ER stress in addition to the formation of DMVs. Also, in cells expressing SARS-CoV orf3a the UPR is induced in a PERK dependent manner. Further evidence that components of the ERAD pathway are required for Coronavirus replication and that ERAD tuning is subverted by Coronaviral proteins stems from observations that silencing of EDEM-1, SEL1L, and LC3 impairs the replication of MHV.

Obvious candidates for viral proteins which are responsible for recruiting components of the ERAD pathway are non-structural proteins which are known to localize to the ER and induce the formation of DMVs - in other words, nsp-3,-4, and -6. In principal, these proteins could be recruited in two ways, either by sequestering EDEM-1 and OS-9 following the insertion into the ER or being sequestered to the ER by EDEM-1 and/OS-9. In both cases, EDEM-1/OS-9 might recognize the carboxyterminal helical domain of the respective nsps’. 

Induction of ERAD pathway via nsps-3/-4/-6: localisation and glycosylation recruits EDEM-1/OS-9 (see next figure for legend)

The author of this post favors a model in which cytosolic ERAD recognizes the carboxyterminal helical domain and translocates nsp-3/-4/-6 to the ER where the protein is inserted into the membrane where (accumulated) cytosolic nsps’ are recognised as part of t ERAD-C pathway. Since the nsp’s in question are not marked for entry into the ER, the cell tags them for degradation via the formation of EDEMosomes. This step might be necessary since none of the nsps in question has an intrinsic KDEL sequence which would be recognised by the Signal Recognition Particle (SRP) and thus allow the formation of the RTC. Following the insertion into the membrane, EDEM-1/OS-9 then recruits SEL1L and subsequently LC3-I, forming EDEMosomes, a step which might be preceded by N-glycosylation of the nsps' in question. Targeting nsp-3/-4/-6 by ERAD-C and localizing to the ER might therefore be a requisite for N-glycosylation. Since the ERAD-C pathway is best characterized in S. Cerevisiae,  investigating this possibility might however be problematic unless coronaviral proteins are glycosylated oat the same sites as in mammalian cells. Also, naturally studies using the replicon systems available would not possible.  

Induction of ERAD pathway via nsps-3/-4/-6: ERAD-C localizes nsps followed by glycosylation  and recruitment of LC3-I by SEL1L

In addition, it might be possible that the recruitment of nsp-6 sequesters PtdIns thus inducing the recruitment of factors necessary for the formation of the omegasome. The induction of PtdInsP3 by recruiting the cellular Vp34 kinase complex might also counteract the induction of apoptosis by prolonged ER stress and thus favour viral replication.

Finally, it should be noted that so far omegasomes have shown not to contain EDEM-1/OS-9 so it might be possible that nsp-6 either does not recruit components of the ERAD pathway, preferentially induces the formation of omegasomes via PtdIns, or that EDEMosomes are degraded (in contrast to omegasomes). Since in MHV infected cells the degradation of EDEMosomes is blocked it is possible that nsp-6 induced vesicles contain EDEM-1 or that nsp-6 by itself does not recruit EDEM-1/OS-9. This however is open for investigation and I hope that somebody is up to challenge.

The basic question of course is why any virus would express proteins that upon localisation to the ER induce a response which is potentially lethal to the host cell? The UPR not only induces apoptosis or the expression of proteins such EDEM-1 that have the potential to degrade viral components but also increases the expression of ER chaperones. This protein family -with BiP, Calnexin, ERp57, and PDI the most abundant members- have key roles in the folding of cellular membrane proteins and therefore may be also used for correct folding of viral proteins located in the envelope of viruses or having other structural finctions. Since these proteins are generally only required relatively late in the replication cycle, viruses need to develop strategies to avoid early apoptosis. One example is African Swine Fever Virus (ASFV) that triggers an UPR and prevents early apoptosis by inducing the ATF6 pathway. The SARS-CoV orf8ab protein in a similar way not only localizes to the ER lumen, but induces UPR by activation of ATF6.  If however the mere expression of nsp-3/-4/-6 causes UPR and subsequently ATF6 - this remains to be seen.   

Further reading

Bernasconi R, & Molinari M (2011). ERAD and ERAD tuning: disposal of cargo and of ERAD regulators from the mammalian ER. Current opinion in cell biology, 23 (2), 176-83 PMID: 21075612

Verchot, J. (2014). The ER quality control and ER associated degradation machineries are vital for viral pathogenesis Frontiers in Plant Science, 5 DOI: 10.3389/fpls.2014.00066 

Noack J, Bernasconi R, & Molinari M (2014). How viruses hijack the ERAD tuning machinery. Journal of virology PMID: 24990995 

Fung TS, & Liu DX (2014). Coronavirus infection, ER stress, apoptosis and innate immunity. Frontiers in microbiology, 5 PMID: 24987391 

Minakshi R, Padhan K, Rani M, Khan N, Ahmad F, & Jameel S (2009). The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor. PloS one, 4 (12) PMID: 20020050 

Reggiori F, Monastyrska I, Verheije MH, Calì T, Ulasli M, Bianchi S, Bernasconi R, de Haan CA, & Molinari M (2010). Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication. Cell host & microbe, 7 (6), 500-8 PMID: 20542253

Hagemeijer MC, Ulasli M, Vonk AM, Reggiori F, Rottier PJ, & de Haan CA (2011). Mobility and interactions of coronavirus nonstructural protein 4. Journal of virology, 85 (9), 4572-7 PMID: 21345958 

Bernasconi R, Noack J, & Molinari M (2012). Unconventional roles of nonlipidated LC3 in ERAD tuning and coronavirus infection. Autophagy, 8 (10), 1534-6 PMID: 22895348 

Bernasconi R, Galli C, Noack J, Bianchi S, de Haan CA, Reggiori F, & Molinari M (2012). Role of the SEL1L:LC3-I complex as an ERAD tuning receptor in the mammalian ER. Molecular cell, 46 (6), 809-19 PMID: 22633958 

Ast T, Aviram N, Chuartzman SG, & Schuldiner M (2014). A cytosolic degradation pathway, prERAD, monitors pre-inserted secretory pathway proteins. Journal of cell science PMID: 24849653

Sun S, Shi G, Han X, Francisco AB, Ji Y, Mendonça N, Liu X, Locasale JW, Simpson KW, Duhamel GE, Kersten S, Yates JR 3rd, Long Q, & Qi L (2014). Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival. Proceedings of the National Academy of Sciences of the United States of America, 111 (5) PMID: 24453213 
 
Baliji, S., Cammer, S., Sobral, B., & Baker, S. (2009). Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by Using Bioinformatics and Molecular Approaches Journal of Virology, 83 (13), 6957-6962 DOI: 10.1128/JVI.00254-09

Clementz MA, Kanjanahaluethai A, O'Brien TE, & Baker SC (2008). Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles. Virology, 375 (1), 118-29 PMID: 18295294 

Gadlage MJ, Sparks JS, Beachboard DC, Cox RG, Doyle JD, Stobart CC, & Denison MR (2010). Murine hepatitis virus nonstructural protein 4 regulates virus-induced membrane modifications and replication complex function. Journal of virology, 84 (1), 280-90 PMID: 19846526 

Kanjanahaluethai A, Chen Z, Jukneliene D, & Baker SC (2007). Membrane topology of murine coronavirus replicase nonstructural protein 3. Virology, 361 (2), 391-401 PMID: 17222884 


Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, Griffiths G, & Ktistakis NT (2008). Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. The Journal of cell biology, 182 (4), 685-701 PMID: 18725538 

Thakur PC, Davison JM, Stuckenholz C, Lu L, & Bahary N (2014). Dysregulated phosphatidylinositol signaling promotes endoplasmic-reticulum-stress-mediated intestinal mucosal injury and inflammation in zebrafish. Disease models & mechanisms, 7 (1), 93-106 PMID: 24135483 

Galindo, I., Hernáez, B., Muñoz-Moreno, R., Cuesta-Geijo, M., Dalmau-Mena, I., & Alonso, C. (2012). The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection Cell Death and Disease, 3 (7) DOI: 10.1038/cddis.2012.81 

Sung SC, Chao CY, Jeng KS, Yang JY, & Lai MM (2009). The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6. Virology, 387 (2), 402-13 PMID: 19304306