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