Virology tidbits

Monday, 15 September 2014

SARS-CoV Nucleocapsid protein and Mitochondria: a personal story

The coronavirus N protein is located inside the virus particle where it binds to the viral RNAas well as the viral M protein to form the viral ribonucleoparticle (RNP), thus being a multifunctional protein that os also possibly involved in the translation of viral subgenomic (sgRNA), in the transcription of the viral genomic RNA and in the replication of the viral genome and possible encapsidation. All processes involve RNA-protein interactions between the N protein and viral sgRNAs, specifically between N and the leader sequence. The general physical properties of the nucleocapsid protein are similar in all coronaviruses although sequence identity is low. The polypeptide ranges from 377 to 455 amino acids in length, corresponding to a size of about 50 kDa. N protein derived from various Coronavirus’ has been shown to be phosphorylated by cellular kinase, namely ATR, and phosphorylation of N has been implicated in regulating its localisation to the site of viral replication, the ER-Golgi Intermediate Compartment (ERGIC), although its impact on viral replication has been controversial.

Sequence analysis of MHV N protein (52 kDa) exhibits a three-domain structure, with a central RNA binding domain (designated as domain II) flanked by two domains (domains I and III), with spacer sequences of lower identity between them.The three-domain structure of N is shared among the Coronaviridae as shown by amino acid sequence analysis. The function of domains I and III remain elusive, although he nucleolar localisation has been mapped to be with this region in addition to a Nuclear export sequence (NES). Amino acid analysis of the C-terminal domain of domain III of IBV revealed a potential binding site for ribosomal proteins in addition to a nucleolar localisation signal sequence (NuLs or NoLs).

In the case of SARS-CoV, the open reading frame encoding the N gene -orf9- encodes for a second gene, orf9b, which is expressed by a ribosomal frameshift. The orf9b protein is 98 AA in length with a NES within the C terminal end of the protein, and although the protein is mainly localised in the cytoplasm of the cell, nuclear localisation has been reported as well. As described in a previous post, orf9b has been recently implicated in mediating suppressing innate immunity by targeting mitochondrial antiviral signalling and causing mitochondrial elongation by degradation of Dynamic related protein 1 (Drp1) via the proteasome (in addition to possible mitophagy). Nuclear orf9b has also been reported to trigger the nuclear translocation of activated Caspase-3, thus triggering DNA fragmentation and apoptosis. In the case of full length N, the expression of SARS-CoV N in COS-1 cells has been shown to induce apoptosis via the mitochondrial pathways. In particular the latter results beg the question to which extent do the individual domains of SARS-CoV N contribute to the observed effect. Whilst I do not have the complete answer, I did some research by myself prior to the publication of the result published on full length N and the alternative orf9b protein.

Apart from the widtype (wt) SARS N construct, I created expression plasmids either laking the N-terminal 45 AA (designated as SARS N Δ3, since there were two other mutants which not important in this context) and a a mutant lacking the first 150 AA (designated SARS N Δ4). The expression of both the wt and mutant plasmids revealed that SARS N localises both to the cytoplasm and nucleus with a strong nucleolar localisation as reported for the N proteins derived from MHV, IBV, and TG, as evidenced with the co-localisation of a nucleolar marker protein, Fibrillarin.

SARS N mutants used

Counterstaining A549 cells with Mitotracker revealed however that the Mitochondria exhibit a distribution pattern which does not reflect a reticular pattern but the appearance of “ballooning”structures, which is even more pronounced in cells expressing SARS N Δ3 but not in cells expressing EYFP-SARS N Δ4. Furthermore, A549 cells expressing SARS N Δ3, exhibit a decrease in these structures, suggesting that the expression of SARS N (a) induces mitochondrial fusion and (b) that the presence of the N-terminal 45 AA somehow prevents the degradation of mitochondria.

To further test if the expression of SARSN induces depolarisation of the mitochondrial membrane a flow cytometry based JC-1 assay was performed which indicated that both in Africa Green Monkey (Vero) and human (A549) cells, SARS N WT does not induce depolarisation of the mitochondria; in contrast the expression of SARS N Δ3 induces mitochondrial depolarisation in A549 cells, whereas the expression of pCi-SARS N Δ4 does not.

Since the fusion of mitochondria is known to induce mitochondrial mass, a 10-N-nonyl acridine orange (NAO) based assay was performed, which in short measures the amount of cardiolipin by cardiolipin-induced dimerisation of NAO and thus allows to determine changes in the mitochondrial mass. Indeed, expression of SARS N Δ3 but not SARS N WT increases NAO fluorescence, whereas the expression of pCi-SARS N Δ4 does not.

Finally, does the expression of SARS N WT induce the activation of Caspase-3, which is activated as a result of mitochondrial fusion and depolarisation. Both indirect immunofluorescence analysis of the distribution of activated Caspase-3a and the cleavage of a fluorogenic substrate ((DEVD)₂-Rhodamine 110) suggest that active Caspase-3 is located within the nucleus as well as the cytoplasm. It should be noted that within the cytoplasm the pattern of active Caspase-3 is reticular rather than uniform (more pronounced in cells transfected with SARS N Δ3 than SARS N WT); if this reflects an association with mitochondria however is not clear and was not tested. Interestingly, A549 and feline CRFK cells transfected with both SARS N wt and SARS N Δ3 undergo apoptosis whereas Vero cells do not, suggesting that host cell factors contribute to the induction of SARS N induced apoptosis.

At that time we did not test if the expression of SARS N wt and its mutants does induce mitophagy or not. Also, we did not test for the induction of reactive oxygen species nor did we perform analysis for the ER stress response. Based on the available data however it might be possible that the SARS N Δ3 mutant might accumulate in the ER. In hindsight, both the JC-1 and the NAO assay should have performed on isolated mitochondria as well, in addition to studies involving Mitofusin 2.

What appears however is that the full length N as opposed to the alternative orf9b protein does not induce mitochondrial elongation but mitochondrial fission. One question which I hope is answered is if the expression of orf9b is regulated during the infection cycle in cells infected with SARS, and if this “switch” -it it occurs- is regulated by the interaction of viral and cellular proteins with the viral RNA.

On a more personal note, I found these results intriguing. Back then not much was known about the SARS N protein and using the tools I had gave me some results which I am still intrigued by.

Further reading

Surjit M, Kumar R, Mishra RN, Reddy MK, Chow VT, & Lal SK (2005). The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation. Journal of virology, 79 (17), 11476-86 PMID: 16103198

Calvo E, Escors D, López JA, González JM, Alvarez A, Arza E, & Enjuanes L (2005). Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells. The Journal of general virology, 86 (Pt 8), 2255-67 PMID: 16033973

White TC, Yi Z, & Hogue BG (2007). Identification of mouse hepatitis coronavirus A59 nucleocapsid protein phosphorylation sites. Virus research, 126 (1-2), 139-48 PMID: 17367888

Spencer KA, Dee M, Britton P, & Hiscox JA (2008). Role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein. Virology, 370 (2), 373-81 PMID: 17931676

Peng TY, Lee KR, & Tarn WY (2008). Phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization. The FEBS journal, 275 (16), 4152-63 PMID: 18631359

Fang S, Xu L, Huang M, Qisheng Li F, & Liu DX (2013). Identification of two ATR-dependent phosphorylation sites on coronavirus nucleocapsid protein with nonessential functions in viral replication and infectivity in cultured cells. Virology, 444 (1-2), 225-32 PMID: 23849791

Reed ML, Howell G, Harrison SM, Spencer KA, & Hiscox JA (2007). Characterization of the nuclear export signal in the coronavirus infectious bronchitis virus nucleocapsid protein. Journal of virology, 81 (8), 4298-304 PMID: 17202223

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

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

Zhang L, Wei L, Jiang D, Wang J, Cong X, & Fei R (2007). SARS-CoV nucleocapsid protein induced apoptosis of COS-1 mediated by the mitochondrial pathway. Artificial cells, blood substitutes, and immobilization biotechnology, 35 (2), 237-53 PMID: 17453707

Christensen ME, Jansen ES, Sanchez W, & Waterhouse NJ (2013). Flow cytometry based assays for the measurement of apoptosis-associated mitochondrial membrane depolarisation and cytochrome c release. Methods (San Diego, Calif.), 61 (2), 138-45 PMID: 23545197

Wlodkowic D, Skommer J, & Darzynkiewicz Z (2012). Cytometry of apoptosis. Historical perspective and new advances. Experimental oncology, 34 (3), 255-62 PMID: 23070010

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 p6

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