Following the infection of cells with positive strand RNA viruses dramatic rearrangements of intracellular membranes can be observed which are generally double membrane vesicles (DMV) and convoluted membranes (CMs) attached to a cellular membrane. These structures contain viral dsRNA intermediates and thus prevent pattern recognition receptors from recognizing dsRNA and thus prevent the induction of an antiviral response. In addition, DMVs sequester viral and cellular enzymes necessary for viral replication as well as providing a scaffold for viral particle assembly and/or means for the newly synthesized viral RNA to enter the cytoplasm.
Coronaviridae are no exception. In the case of two prototype Coronaviridae, the murine Coronavirus (Mouse Hepatitis Virus, MHV) and human SARS-associated Coronavirus, SARS-CoV, these replication-transcription complexes (RTCs) -another terminology for DMV- consist of the non-structural proteins (nsp) required for RNA synthesis, RNA-dependent RNA Polymerase (RdRp/nsp12), as well as the membrane spanning nsp3, 4, and 6 proteins, although nsp3 and 4 are sufficient for inducing the DMVs and the formation of DMVs is not dependent on active RNA synthesis.
Whereas nsp12 contains the enzymatic activity for RNA synthesis, the remaining nsps anchor the DMV to the ER membrane. In this context it is interesting to note that most fusion proteins of these nsps derived from MHV or SARS-CoV do not retain the ability of nsp3 and 4 to anchor DMVs to the ER and with the exception of nsps 2 and 6 fail to recruit nsps required for viral replication such as the viral RNA Helicase. The notable exception are fusion proteins in which the large luminal loop between the first and second transmembrane domain of nsp4 derives from the same species as the nsp3, a notion which was confirmed by truncation analysis of nsp4, whereas the luminal loop of nsp3 may have a stabilizing function and be involved in recruitment larger nsps via nsp6.
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| Non structural proteins involved in Coronavirus replication are encoded within the PP1ab |
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| Localisation of nsp3, nsp4 and nsp4 of MHV and SARS-CoV |
In both SARS-CoV and MHV infected cells, dsRNA intermediates -and thus active viral RNA synthesis- co-localising with RdRp/nsp12 with are localized in the interior of the DMV at early timepoints post infection. As the infection progresses however the co-localisation of dsRNA and newly synthesized viral RNA becomes less apparent suggesting that viral RNA transcription ceases. In the case of MHV, it has been demonstrated that the RTCs translocate to the assembly site of new viral particles at 8-16 h p.i. thus obliviating the need of active RNA transcription. At this timepoint the DMVs are not only devoid of dsRNA but also of RNA helicases.
Replication structures of Arteriviridae: similarities to Coronaviridae
Similar to the Coronaviridae, following infection of cells with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) or Equine Arterivirus (EAV) -two of the most commonly studied Arteriviridae- the replicative proteins (with the notable exception of nsp1 and nsp2TF) are assembled in virus induced membrane structures consisting of modified intracellular membranes in the perinuclear region of the infected cell. As it is the case for Coronavirus infected cells, these DMVs are associated with viral replication and transcription and thus contain viral proteins and dsRNA intermediates as well as viral positive and negative sense ssRNA. In the case of the Arteriviridae, the scaffold of the RTCs seems to consist of the putative membrane spanning proteins nsp2, nsp3 and nsp5; similar to the Coronaviridae, these are expressed as part of the orf1a gene which is processed by autoprocessing using viral proteases. In addition, both the nsp2 and nsp3 are sufficient to induce the formation of DMVs (as is the case for the Coronavirus nsp3 and 4) and DMVs can form structures of modified ER which are interconnected by their outer membrane as revealed in tomographic studies. Although host proteins implicated in the initiation of arteriviral RNA synthesis binding either to the 3’ end of the genome or the anti-genome of PRRSV or EAV have been postulated to localize to DMVs in the addition of viral proteins the precise composition of these has not been identified.
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| Hypothetical model of how Arteri- and Coronavirus nsps induce the formation of LC3-I and LC3-II positive particles by stimulating autophagy |
As in the case of COVID-19 and other members of the Coronaviridae, the replication of EAV and PRRSV is sensitive to inhibitors from members of the Cyclophilin family, probably Cyclophilin A. Furthermore, similar to IBV and other Coronaviruses, microtubule-associated protein 1 light chain 3 (LC3) and ER degradation-enhancing α-mannosidase-like1 (EDEM1) proteins associate with DMVs at 16 h p.i. (in the case of MHV) and the depletion of LC3 reduces EAV replication. These results suggest that in both Coronavirus and Arterivirus infected cells autophagy may play a role late in infection similar to Rotavirus infected cells. So far it remains to be seen which viral protein -or combination of of viral proteins- is responsible for the recruitment of the components of the autophagy pathway and to which extent autophagy inhibitors are preventing the spread of viral particles and CPE. In the opinion of the author, the most likely pathway inducing the recruitment of LC3 to DMVs is by inducing ER stress by inhibition of mTOR via the arteriviral nsp2 and 3 (and Coronavirus nsp3 and 4) proteins - in other words the question which remains to be answered is, if the formation of DMVs and the induction of autophagosome like structures influence viral assembly and if the stabilization of these structures has any effect. Also it remains to be seen if the recruitment of ER chaperones such as EDEM1 by the murine Coronavirus and EAV is required for the correct folding of viral proteins. In this context it is important to note that viral replication itself does not require the presence of autophagy related protein (ATG) 7 and thus the lipidiated form of LC3 (LC3II). It would be of interest to investigate if in ATG7 depleted cells infected with EAV, SARS-CoV or MHV, EDEM1 is stabilized and if the formation of viral particles is affected. Finally, is the recruitment of EDEM1 necessary for the detection and degradation of viral glycoproteins such as the Coronavirus S protein that are misfolded akin to the cellular unfold protein response ? Furthermore, it might be possible that RTC are predominating LC3-II positive whereas the assembly particles containing the coronaviral S, E, M and N proteins as well as EDEM1 are predominantly LC3-I.
The application of Cyclophilin A therefore might lead to an increase in misfolded viral and cellular proteins that are recognised by EDEM1 and degraded in autophagosomes. If the infection of cells with Corona-or Arteriviridae interferes with the ability of cells to respond to aggregated misfolded proteins, this might explain why COVID-19 infected cells are sensitive to Cyclosporin A. Who is up to the challenge?
Further Reading
Hagemeijer MC, Vonk AM, Monastyrska I, Rottier PJ, & de Haan CA (2012). Visualizing coronavirus RNA synthesis in time by using click chemistry. Journal of virology, 86 (10), 5808-16 PMID: 22438542
Knoops K, Kikkert M, Worm SH, Zevenhoven-Dobbe JC, van der Meer Y, Koster AJ, Mommaas AM, & Snijder EJ (2008). SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS biology, 6 (9) PMID: 18798692
van den Worm SH, Knoops K, Zevenhoven-Dobbe JC, Beugeling C, van der Meer Y, Mommaas AM, & Snijder EJ (2011). Development and RNA-synthesizing activity of coronavirus replication structures in the absence of protein synthesis. Journal of virology, 85 (11), 5669-73 PMID: 21430047
Angelini MM, Akhlaghpour M, Neuman BW, & Buchmeier MJ (2019). Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles. mBio, 4 (4) PMID: 23943763
Hagemeijer, M., Monastyrska, I., Griffith, J., van der Sluijs, P., Voortman, J., van Bergen en Henegouwen, P., Vonk, A., Rottier, P., Reggiori, F., & de Haan, C. (2019). Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4 Virology, 458-459, 125-135 DOI: 10.1016/j.virol.2019.04.027
Nal, B. (2005). Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E Journal of General Virology, 86 (5), 1423-1434 DOI: 10.1099/vir.0.80671-0
Bost AG, Prentice E, & Denison MR (2001). Mouse hepatitis virus replicase protein complexes are translocated to sites of M protein accumulation in the ERGIC at late times of infection. Virology, 285 (1), 21-9 PMID: 11414802
Lontok E, Corse E, & Machamer CE (2004). Intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site. Journal of virology, 78 (11), 5913-22 PMID: 15140989
Snijder, E., Kikkert, M., & Fang, Y. (2019). Arterivirus molecular biology and pathogenesis Journal of General Virology, 94 (Pt_10), 2141-2163 DOI: 10.1099/vir.0.056341-0
Monastyrska I, Ulasli M, Rottier PJ, Guan JL, Reggiori F, & de Haan CA (2019). An autophagy-independent role for LC3 in equine arteritis virus replication. Autophagy, 9 (2), 164-74 PMID: 23182945
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
de Wilde AH, Raj VS, Oudshoorn D, Bestebroer TM, van Nieuwkoop S, Limpens RW, Posthuma CC, van der Meer Y, Bárcena M, Haagmans BL, Snijder EJ, & van den Hoogen BG (2019). MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment. The Journal of general virology, 94 (Pt 8), 1749-60 PMID: 23620378
Ciechomska IA, Gabrusiewicz K, Szczepankiewicz AA, & Kaminska B (2019). Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine a-induced cell death. Oncogene, 32 (12), 1518-29 PMID: 22580614
Reggiori F, de Haan CA, & Molinari M (2011). Unconventional use of LC3 by coronaviruses through the alleged subversion of the ERAD tuning pathway. Viruses, 3 (9), 1610-23 PMID: 21994798
Reggiori, F., Monastyrska, I., Verheije, M., Calì, T., Ulasli, M., Bianchi, S., Bernasconi, R., de Haan, C., & 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), 500-508 DOI: 10.1016/j.chom.2010.05.013
de Haan CA, Molinari M, & Reggiori F (2010). Autophagy-independent LC3 function in vesicular traffic. Autophagy, 6 (7), 994-6 PMID: 20814233 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
Arnoldi F, De Lorenzo G, Mano M, Schraner EM, Wild P, Eichwald C, & Burrone OR (2019). Rotavirus increases levels of lipidated LC3 supporting accumulation of infectious progeny virus without inducing autophagosome formation. PloS one, 9 (4) PMID: 24736649
Shenkman M, Groisman B, Ron E, Avezov E, Hendershot LM, & Lederkremer GZ (2019). A shared endoplasmic reticulum-associated degradation pathway involving the EDEM1 protein for glycosylated and nonglycosylated proteins. The Journal of biological chemistry, 288 (4), 2167-78 PMID: 23233672
Park S, Jang I, Zuber C, Lee Y, Cho JW, Matsuo I, Ito Y, & Roth J (2019). ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase. Histochemistry and cell biology PMID: 24664425
Zuber C, Cormier JH, Guhl B, Santimaria R, Hebert DN, & Roth J (2007). EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites. Proceedings of the National Academy of Sciences of the United States of America, 104 (11), 4407-12 PMID: 17360537
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