Virology tidbits

Virology tidbits

Thursday, 11 September 2014

SARS-CoV: formation of the RTC and mitophagy; role of p6 and orf9b

Science by it s very nature is always in flux, articles being reviewed and published, answering questions raised before and contributing to the vast trove of knowledge.
Consequently, some articles published in the recent weeks and months which caught the attention of the author of this blog after the very fact that posts have been published shed some light on issues raised; others of whom the author was aware did not make the cut in previous contributions. This post is therefore an update of an area already well covered  -the ER stress response induced by Coronaviruses and the formation of RTCs. Here, the role of the bat and SARS-CoV derived p6 protein in the induction of the RTC will be discussed, proposing a model where the induction of the ER stress response will allow the formation of RTC by inducing autophagy. Whilst the nature of the RTC and the role of the CoV non-structural proteins as well the potential role of these proteins in the ER stress response have been discussed in detail before, the role of p6 has been neglected. Also, a recent paper highlighting the protective role of the IRE1α induction by Infectious Bronchitis Virus (IBV) will be included thus allowing the introduction of a model where both the non-structural and the structural protein induce a ER stress response which ultimately induces the formation of the RTC, not only antagonising apoptosis but also supporting viral replication by inducing the formation of autophagosomes-like vesicles. I need to mention however that this model should not be seen as a “universal model” since some of the proteins are not encoded in the genomes of all CoVs, and indeed may serve SARS-CoV and bat derived SARS-like CoV better than IBV or TGEV. Nevertheless, it raises the question if altering the genome of the latter viruses by including these additional proteins alters the severity of these viruses. One of such proteins is p6 and creating a recombinant MHV strain expressing SARS-CoV derived p6 indeed increased viral replication and the severity of the disease in infected mice. Before I introduce the model therefore, we shall take a detour and examine some of the properties associated with p6 which make this protein interesting. Also the role of the SARS-CoV orf9b protein in the induction of mitophagy will be discussed. 


                 SARS-CoV and bat derived SARS-like CoV p6

Both in infected and transfected cells, the p6 protein of SARS-CoV and bat derived SARS-like CoV localises predominantly to the ER and Golgi, where it interacts with Karyopherin alpha 2 (KPNA2). KPNA2 in turn binds to Karyopherin beta 1 (KPNB1) and prevents the nuclear import of STAT1, thus blocking the type I interferon response. In addition to binding KPNA2, p6 co-localises with SARS-CoV orf9b, orf 8a/b and nsp3, interactions which may contribute to the formation of vesicles that either target mitochondria and the MAVS/TRAF3/TRAF6 signalosome or enhance viral replication. p6 itself is a relatively small protein, which originally has been identified as a β-interferon antagonist and a protein that stimulates DNA synthesis whilst inhibit the expression of co-transfected plasmids. In transfected cells, the expression of p6 induces the formation of DMV like structures derived from the ER and induces ER stress and co-localises with nsp-3 and nsp8 (a primer independent, non-canonical, RNA Polymerase) in infected cells. As discussed below, p6 therefore may be part of the viral RTC and induces or supports the formation of RTCs.

                orf9b and the MAVS/TRAF3/TRAF6 signalosome

The SARS-CoV orf9b protein has been shown to localise to the mitochondria where it sequesters the Mitochondrial antiviral-signaling protein (MAVS) into punctae and subsequently leading to the ubiquitination and degradation of MAVS, which is accompanied by a decrease in TRAF-3 and -6. In addition, the expression of orf9b induces ATG5 dependent formation of the autophagosome as well as inhibiting mitochondrial fission via degradation of Dynamin related protein1 (DRP1). As discussed previously for Measles Virus and Chikungunya Virus, the activation of antiviral signalling via MDA5 or RIG-1 can be inhibited by inducing mitophagy via formation of the MAM complex. In the case of SARS-CoV this may be achieved by binding of orf9b to p6 instead of cellular proteins; indeed it may be possible that the interaction of p6 with orf9b tethers mitochondria to the ER in order to allow the formation of the autophagosome and subsequent mitophagy in a p62/SQSTM1 and NIX dependent manner, similar to the perinuclear sequestering of mitochondria and mitophagy in HCV infected cells. Since orf9b has been reported to localise to the ER as well, alternatively the protein might not be a genuine mitochondrial protein but an adaptor protein that binds to MAVS and facilitates the recruitment of ubiquitin ligases to the tethered mitochondria. So far however there is no evidence that this is the case, but once the domain necessary for interaction with MAVS has been identified this should be clarified.  Also, it might be possible that orf9b binds to MAM and facilitates  the recruitment of mitochondria which contain orf9b - in other words, forming dimers independent of MAVS mediated interaction with MAM. 

SARS-CoV orf9b: potential mechanisms of mitochondrial recruitment
prior mitophagy

Interestingly the levels of LC3-II  in cells expressing orf9b are not significantly increased; as pointed out before however, it might be possible that the process involves LC3-C instead of the more common LC3-B isoform, mediated by NDP52. 

                   p6, nsp-3/-4/-6 and the formation of viral RTC

As discussed before, the viral nsp-3, -4, and -6 proteins induce the formation of autophagosome like vesicles which are not subject to degradation via the autolysosome probably due to the inhibition of mTORC1 by nsp-6, but are important for providing a scaffold for the formation of the RTC.

The structure of p6 is predicted to be an amphipathic α-helix with the N terminal hydrophobic domain located within the ER membrane and the C-terminal domain in the cytoplasm; the protein therefore forms a hairpin structure rather than being a transmembrane  protein. Deletion analysis of the N terminal domain showed that this region is responsible for the induction of membranous vesicles and reticular structures akin to the structures observed in infected cells. Although these structures are induced in the absence of other proteins, the localisation of p6 overlaps with nsp-3, suggesting that both proteins might interact (or not?), as well with  nsp-8, a non-canonical viral RNA Polymerase. If however, p6 is required for the formation of the RTC is doubtful since the absence of p6 in viruses other than SARS-CoV does not prevent the formation of RTC. p6 might however enhance viral replication (1) by recruiting a second viral RdRP (nsp-8) to nsp-12 as well as nsp-7 and thus to the RTC, (2) due to the ability to inhibit STAT1 dependent and MAVS dependent antiviral signalling pathways and (3) increase the formation of membrane vesicles which form the scaffold for RTCs.

nsp-3/-4/-6 and p6: formation RTC and LC3C-II positive vesicles

Since SARS-CoV p6 induces the formation of membrane vesicles, it is probably not surprising that p6 induces ER stress and subsequently apoptosis. Indeed, p6  induces the expression of GRP94 in JNK dependent manner as well as Caspase-3 dependent apoptosis. 

In conclusion, the viral orf9b protein might have a function akin to the interaction of Measles Virus C protein with IRGM, namely in clustering mitochondria by binding to orf9b -similar to  HCV NS4B/5A- by inducing the formation of membranous vesicles. If however these functions are associated with mitophagy and autophagy remains to be seen. p6 however can be associated with increased viral replication, probably because of recruitment of nsp-8 as well as the inhibition of STAT1. The p6 protein on the other hand, might increase viral replication by forming a complex of nsp-12 together with the non-canonical nsp-8 Polymerase in addition to enhancing the formation of DMVs. 

If the formation of the RTC induces apoptosis via induction of the ER stress response, then how do infected cells survive long enough for the virus to replicate? As we have seen in previous posts, the ER stress response also induces the expression of chaperones as well autophagy related genes and activates Beclin-1 by phosphorylating Bcl-2 - processes that inhibit apoptosis at least temporarily. Also, the three branches of the ER stress response are activated successively. Recent evidence from obtained in Vero and H1299 infected with IBV suggest that the activation of IRE1α and subsequent splicing of XBP1 protects cells from premature apoptosis by upregulating EDEM1, ERdj4, and p58IPK, as well as phosphorylating Akt. Finally, does the upregulation of autophagy related genes following the infection of cells with SARS -if it occurs- contribute to orf9b mediated mitophagy?

Coronavirus and the ER stress response: pathway overview

Further reading

Zhong Y, Tan YW, & Liu DX (2012). Recent progress in studies of arterivirus- and coronavirus-host interactions. Viruses, 4 (6), 980-1010 PMID: 22816036 

Shi CS, Qi HY, Boularan C, Huang NN, Abu-Asab M, Shelhamer JH, & Kehrl JH (2014). SARS-Coronavirus Open Reading Frame-9b Suppresses Innate Immunity by Targeting Mitochondria and the MAVS/TRAF3/TRAF6 Signalosome. Journal of immunology (Baltimore, Md. : 1950), 193 (6), 3080-9 PMID: 25135833 

Tangudu C, Olivares H, Netland J, Perlman S, & Gallagher T (2007). Severe acute respiratory syndrome coronavirus protein 6 accelerates murine coronavirus infections. Journal of virology, 81 (3), 1220-9 PMID: 17108045 

Hussain S, & Gallagher T (2010). SARS-coronavirus protein 6 conformations required to impede protein import into the nucleus. Virus research, 153 (2), 299-304 PMID: 20800627 

Otera H, Ishihara N, & Mihara K (2013). New insights into the function and regulation of mitochondrial fission. Biochimica et biophysica acta, 1833 (5), 1256-68 PMID: 23434681 

Kumar P, Gunalan V, Liu B, Chow VT, Druce J, Birch C, Catton M, Fielding BC, Tan YJ, & Lal SK (2007). The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein. Virology, 366 (2), 293-303 PMID: 17532020 

Ye Z, Wong CK, Li P, & Xie Y (2008). A SARS-CoV protein, ORF-6, induces caspase-3 mediated, ER stress and JNK-dependent apoptosis. Biochimica et biophysica acta, 1780 (12), 1383-7 PMID: 18708124 

Subissi L, Imbert I, Ferron F, Collet A, Coutard B, Decroly E, & Canard B (2014). SARS-CoV ORF1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets. Antiviral research, 101, 122-30 PMID: 24269475 

Fung TS, Liao Y, & Liu DX (2014). The ER stress sensor IRE1α protects cells from apoptosis induced by coronavirus infectious bronchitis virus. Journal of virology PMID: 25142592 

Guo W, Ding J, Zhang A, Dai W, Liu S, Diao Z, Wang L, Han X, & Liu W (2014). The inhibitory effect of quercetin on asymmetric dimethylarginine-induced apoptosis is mediated by the endoplasmic reticulum stress pathway in glomerular endothelial cells. International journal of molecular sciences, 15 (1), 484-503 PMID: 24451129 

Cruz JL, Sola I, Becares M, Alberca B, Plana J, Enjuanes L, & Zuñiga S (2011). Coronavirus gene 7 counteracts host defenses and modulates virus virulence. PLoS pathogens, 7 (6) PMID: 21695242

Neuman BW, Angelini MM, & Buchmeier MJ (2014). Does form meet function in the coronavirus replicative organelle? Trends in microbiology PMID: 25037114 

Angelini MM, Akhlaghpour M, Neuman BW, & Buchmeier MJ (2013). Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles. mBio, 4 (4) PMID: 23943763 

Subissi L, Posthuma CC, Collet A, Zevenhoven-Dobbe JC, Gorbalenya AE, Decroly E, Snijder EJ, Canard B, & Imbert I (2014). One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proceedings of the National Academy of Sciences of the United States of America PMID: 25197083

Moshynskyy I, Viswanathan S, Vasilenko N, Lobanov V, Petric M, Babiuk LA, & Zakhartchouk AN (2007). Intracellular localization of the SARS coronavirus protein 9b: evidence of active export from the nucleus. Virus research, 127 (1), 116-21 PMID: 17448558 

Sharma K, Åkerström S, Sharma AK, Chow VT, Teow S, Abrenica B, Booth SA, Booth TF, Mirazimi A, & Lal SK (2011). SARS-CoV 9b protein diffuses into nucleus, undergoes active Crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus. PloS one, 6 (5) PMID: 21637748

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