Virology tidbits

Virology tidbits

Tuesday, 31 March 2015

Viruses and the Nucleolus: Coronavirus N, Cytokinesis and Autophagy

The nucleolus is a subnuclear structure not surrounded by a membrane, which is disassembled during mitosis and reformed during late telophase via the interaction of nucleolar proteins with loci of ribosomal DNA. At this point, the inhibition of CDK1 leads to the resumption of RNA Polymerase I (RNA Pol I) dependent transcription of rDNA, resulting in the expression of the pre-rRNA (18S, 5.8S, and 28S, with internal and external transcribed spacers) and the recruitment of the processing machinery, forming the prenucleolar bodies (PNBs) and subsequently processing factors as nucleoli mature.
During interphase -in particular during late G1 and early S phase- multiple nucleoli can be observed within the nucleus whereas in middle/late S phase these commonly fuse to become one or two nucleoli. Since large nucleoli are associated with (high) rates of cell proliferation due to an increase in RNA Pol I dependent transcription of rDNA, nucleolar size can be linked to cancer. This is also reflected by changes in the localisation pattern of nucleolar proteins such as Fibrillarin, a factor involved in the processing of newly synthesised pre-rRNA, whose localisation changes upon treatment with Actinomycin D. Since Actinomycin D inhibits RNA Pol I activity, Actinomycin D treated cells exhibit a weaker signal for Fibrillarin similar to cells treated with ALLN or the small molecule inhibitor 10058F4, indicating that Fibrillarin is associated with sites of active RNA Pol I transcription and that the perinuclear region is disassembled following the inhibition of RNA Pol I.

Domains and marker proteins of a prototype nucleolus

In general, the nucleolus can be subdivided into three different regions, the granular component (GC), the Fibrillar Centre (FC), and the Dense Fibrillar Centre (DFC), the latter being the “core” formed around the nucleolar organizing region (NOR) containing the 18S, 5.8S, and 28S rDNA loci which in the human genome are localised on the p- arms of the five acrocentric chromosomes.  Transcription by RNA Pol I occurs at the interface of the FC and DFC following the assembly of rDNA transcription factories in a c-Myc dependent manner. Traditionally, the nucleolus has been associated sole with the biogenesis of ribosomes and while this is certainly one of the main functions, in recent years multiple pathways -including apoptosis, autophagy, and DNA repair- have been identified which depend on nucleolar integrity. Nucleolar disruption induced by various stressors including DNA damage and inhibition of Pol I dependent transcription induces the activation of p53 via p14Arf mediated disruption of the MDM2/p53 complex - and subsequent induction of p53 responsive genes- as well as inducing non-canonical autophagy in B23/Nucleophosmin (NPM) dependent manner but independent of nucleophagy. Since (activated) p53 not only induces the expression of proapoptotic genes such PUMA and NOXA or the Cyclin E inhibitor p21Waf1 but also DRAM-1 and Sestrin-2, it might be possible that the induction of p53 following nucleolar stress induces autophagy in a DRAM-1 dependent pathway. Since the expression of p21Waf1 has also been linked to oncogene induced apoptosis and oncogene induced senescence (OIS), inhibition of the nucleolar stress response pathway by Akt and mTORC1 mediated phosphorylation of PRAS40 has been demonstrated to promote the inhibition of p53 by stabilising a complex consisting of the ribosomal protein L11, HDM2 and p53, thus preventing the induction of OIS by preventing the expression of p21Waf1 as well as inhibiting autophagy. 

Degradation of p53 in unstressed nucleoli by HDM2

Stabilisation of p53 following nucleolar stress induces apoptosis, senescence,
and autophagy via p14 which can be inhibited by nuclear PRAS40

Viral proteins and the nucleolus: Coronavirus Nucleocapsid protein as a case study

Viral proteins localise to the nucleolus either via nucleolar localisation sequences (NoLS) that are usually part of a NLS, via binding of nucleolar shuttle proteins (such as Coilin or Argonaute 4), or by binding nucleolar proteins.

Table: Examples of viral proteins localising to the nucleolus

A number of viral proteins have been shown to localise to the nucleolus or to interact with nucleolar proteins, included -but not limited to- viral proteins derived from HIV (Rev, Gag, Tat), Adenovirus (V, VI, and the viral genome), Herpesvirus Saimiri and KSHV (ORF57), Influenza A Virus (NS1), and both the major and minor Capsid proteins from Potato Leafroll virus (PLRV), indicating that the nucleolar localisation of viral proteins is a common feature. In addition, the nucleolar architecture is altered in cells infected with Human Cytomegalovirus (HCMV) as such that Nucleolin expression is not only induced by HCMV but also relocalised to the nucleolar periphery in close proximity to the viral replication centres thus maintaining the architecture of the replication centres.
In the case of plant viruses, it has been demonstrated that the recruitment of nucleolar proteins is required for the viral replication, viral movement, and the assembly of viral RNP particles. The ORF3 long distance movement protein localises to the nucleolus via binding to nuclear (and highly dynamic) Cajal Body (CB) as indicated by the reorganisation of CB into structures that fuse with the nucleolus, leading to the recruitment of Fibrillarin. Fibrillarin in turn is required for the assembly of infectious viral particles in the cytoplasm, which is similar to PLRV. Interestingly, both the Herpesvirus Saimiri  and KSHV ORF57 proteins induce the nucleolar redistribution of the human TREX (transcription/export) proteins that are involved in the export of mRNA, thus inducing the export of viral mRNA suggesting that the nucleolar localisation of viral proteins in general is essential for viral replication.

In the case of the Coronaviridae, the nucleocapsid (N) protein derived from a variety of different members, including IBV, MHV, TGEV, PEDV, and SARS-CoV, has been shown to localise to the nucleolus as indicated by co-localisation with nucleolar proteins, in particular Nucleolin and Fibrillarin. Since the N protein from both MHV and IBV does not co-localise with B23, it can be assumed that the N protein does localise within the DFC or the FC of the nucleolus.

CoV N protein localises to the nucleolus in LLCPK (TGEV) or Vero  (MHV, IBV, SARS-CoV) cells
and induces incomplete cytokinesis

Relocalisation of Fibrillarin in Vero cells expressing

Delocalisation of Fibrillarin in various cell lines expressing

The expression of N derived from IBV, MHV, TGEV, and SARS-CoV has also been shown to induce aberrant cytokinesis. If this feature however is related to the nucleolar localisation of N or not is at present not known. One possible scenario might be that the nucleolar localisation of N causes nucleolar stress as indicated by the relocalisation of Fibrillarin within the nucleolus. The induction of nucleolar stress induces the activation of p53 that can induce autophagy via DRAM-1 and/or Sestrin-2 and thus might promote the degradation of proteins required for the degradation of the midbody such as active RHOA. So far however this has not been demonstrated.
The activation of p53 however might also the induction of the intrinsic apoptotic pathway and the expression of SARS N has been demonstrated to induce the cleavage of Caspase-3 in the absence of apoptosis. Since activated Caspase-3 cleaves Beclin-1, Atg4D and Atg5 –thus inhibiting the formation of the autophagosome- only a subset of cells expressing N might exhibit an increase in autophagy. Apoptosis itself might be antagonized by binding PARP-1; indeed, PRRSV N protein has been shown to bind PARP-1 although the functional consequences are not known.

The expression of IBV (top) or MHV N sequesters B23
and induces incomplete cytokinesis

Model: induction of nucleolar stress induces multiple competing

Alternatively the sequestration of B23 by the coronaviral N protein might prevent the phosphorylation of B23 by Polo-like Kinase (Plk)-1 and thus induce mitotic defects including cytokinesis failure.

It would be interesting to investigate if the defects of cytokinesis can be alleviated by the expression of other Coronavirus proteins, in particular those whose expression inhibits the degradation of autophagosomes, namely nsp-6 and nsp-3/-4, or in the presence of autophagy inhibitors such Vps34 inhibitors or Chloroquine. Since only a subset of N expressing cells are undergoing aberrant cytokinesis and not all cells  expressing N exhibiting a cytokinesis defect also exhibit nucleolar localisation of N, aberrant cytokinesis might also be dependent on other factors. Clearly, further experiments are warranted to elucidate the mechanism.

Further reading

Olson MO, Hingorani K, & Szebeni A (2002). Conventional and nonconventional roles of the nucleolus. International review of cytology, 219, 199-266 PMID: 12211630

Leung, A., & Lamond, A. (2003). The Dynamics of the Nucleolus Critical Reviews in Eukaryotic Gene Expression, 13 (1), 39-54 DOI: 10.1615/CritRevEukaryotGeneExpr.v13.i1.40 

Shaw P, & Brown J (2012). Nucleoli: composition, function, and dynamics. Plant physiology, 158 (1), 44-51 PMID: 22082506 

Salvetti A, & Greco A (2014). Viruses and the nucleolus: the fatal attraction. Biochimica et biophysica acta, 1842 (6), 840-7 PMID: 24378568 
Grob A, Colleran C, & McStay B (2014). Construction of synthetic nucleoli in human cells reveals how a major functional nuclear domain is formed and propagated through cell division. Genes & development, 28 (3), 220-30 PMID: 24449107 

Havel JJ, Li Z, Cheng D, Peng J, & Fu H (2015). Nuclear PRAS40 couples the Akt/mTORC1 signaling axis to the RPL11-HDM2-p53 nucleolar stress response pathway. Oncogene, 34 (12), 1487-98 PMID: 24704832
Greco A (2009). Involvement of the nucleolus in replication of human viruses. Reviews in medical virology, 19 (4), 201-14 PMID: 19399920 

Hiscox JA (2007). RNA viruses: hijacking the dynamic nucleolus. Nature reviews. Microbiology, 5 (2), 119-27 PMID: 17224921 

Dang CV, & Lee WM (1989). Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins. The Journal of biological chemistry, 264 (30), 18019-23 PMID: 2553699 

Lochmann TL, Bann DV, Ryan EP, Beyer AR, Mao A, Cochrane A, & Parent LJ (2013). NC-mediated nucleolar localization of retroviral gag proteins. Virus research, 171 (2), 304-18 PMID: 23036987 

Melén K, Kinnunen L, Fagerlund R, Ikonen N, Twu KY, Krug RM, & Julkunen I (2007). Nuclear and nucleolar targeting of influenza A virus NS1 protein: striking differences between different virus subtypes. Journal of virology, 81 (11), 5995-6006 PMID: 17376915 

Taylor A, Jackson BR, Noerenberg M, Hughes DJ, Boyne JR, Verow M, Harris M, & Whitehouse A (2011). Mutation of a C-terminal motif affects Kaposi's sarcoma-associated herpesvirus ORF57 RNA binding, nuclear trafficking, and multimerization. Journal of virology, 85 (15), 7881-91 PMID: 21593148 

Haupt S, Stroganova T, Ryabov E, Kim SH, Fraser G, Duncan G, Mayo MA, Barker H, & Taliansky M (2005). Nucleolar localization of potato leafroll virus capsid proteins. The Journal of general virology, 86 (Pt 10), 2891-6 PMID: 16186245 

Shaw J, Love AJ, Makarova SS, Kalinina NO, Harrison BD, & Taliansky ME (2014). Coilin, the signature protein of Cajal bodies, differentially modulates the interactions of plants with viruses in widely different taxa. Nucleus (Austin, Tex.), 5 (1), 85-94 PMID: 24637832 

González I, Martínez L, Rakitina DV, Lewsey MG, Atencio FA, Llave C, Kalinina NO, Carr JP, Palukaitis P, & Canto T (2010). Cucumber mosaic virus 2b protein subcellular targets and interactions: their significance to RNA silencing suppressor activity. Molecular plant-microbe interactions : MPMI, 23 (3), 294-303 PMID: 20121451 

Boyne JR, & Whitehouse A (2006). Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA. Proceedings of the National Academy of Sciences of the United States of America, 103 (41), 15190-5 PMID: 17005724 

Boyne JR, Jackson BR, Taylor A, Macnab SA, & Whitehouse A (2010). Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs. The EMBO journal, 29 (11), 1851-64 PMID: 20436455 

Strang BL, Boulant S, Kirchhausen T, & Coen DM (2012). Host cell nucleolin is required to maintain the architecture of human cytomegalovirus replication compartments. mBio, 3 (1) PMID: 22318319 Callé A, Ugrinova I, Epstein AL, Bouvet P, Diaz JJ, & Greco A (2008). Nucleolin is required for an efficient herpes simplex virus type 1 infection. Journal of virology, 82 (10), 4762-73 PMID: 18321972 

Shi D, Lv M, Chen J, Shi H, Zhang S, Zhang X, & Feng L (2014). Molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus. Viruses, 6 (3), 1253-73 PMID: 24632575 

Chen H, Wurm T, Britton P, Brooks G, & Hiscox JA (2002). Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell. Journal of virology, 76 (10), 5233-50 PMID: 11967337 

Wurm T, Chen H, Hodgson T, Britton P, Brooks G, & Hiscox JA (2001). Localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division. Journal of virology, 75 (19), 9345-56 PMID: 11533198 

Hiscox JA, Wurm T, Wilson L, Britton P, Cavanagh D, & Brooks G (2001). The coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus. Journal of virology, 75 (1), 506-12 PMID: 11119619 

Kim JS, Ro SH, Kim M, Park HW, Semple IA, Park H, Cho US, Wang W, Guan KL, Karin M, & Lee JH (2015). Sestrin2 inhibits mTORC1 through modulation of GATOR complexes. Scientific reports, 5 PMID: 25819761 

Belaid A, Cerezo M, Chargui A, Corcelle-Termeau E, Pedeutour F, Giuliano S, Ilie M, Rubera I, Tauc M, Barale S, Bertolotto C, Brest P, Vouret-Craviari V, Klionsky DJ, Carle GF, Hofman P, & Mograbi B (2013). Autophagy plays a critical role in the degradation of active RHOA, the control of cell cytokinesis, and genomic stability. Cancer research, 73 (14), 4311-22 PMID: 23704209 

Pohl C (2009). Dual control of cytokinesis by the ubiquitin and autophagy pathways. Autophagy, 5 (4), 561-2 PMID: 19242118 

Pohl C, & Jentsch S (2009). Midbody ring disposal by autophagy is a post-abscission event of cytokinesis. Nature cell biology, 11 (1), 65-70 PMID: 19079246 

Zhang H, Shi X, Paddon H, Hampong M, Dai W, & Pelech S (2004). B23/nucleophosmin serine 4 phosphorylation mediates mitotic functions of polo-like kinase 1. The Journal of biological chemistry, 279 (34), 35726-34 PMID: 15190079 

Li Z, Shi K, Guan L, Jiang Q, Yang Y, & Xu C (2013). Activation of p53 by sodium selenite switched human leukemia NB4 cells from autophagy to apoptosis. Oncology research, 21 (6), 325-31 PMID: 25198662 

Liu L, Lear Z, Hughes DJ, Wu W, Zhou EM, Whitehouse A, Chen H, & Hiscox JA (2015). Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology. Veterinary microbiology, 176 (1-2), 109-19 PMID: 25614100

Cho DH, Jo YK, Hwang JJ, Lee YM, Roh SA, & Kim JC (2009). Caspase-mediated cleavage of ATG6/Beclin-1 links apoptosis to autophagy in HeLa cells. Cancer letters, 274 (1), 95-100 PMID: 18842334

Zhu Y, Zhao L, Liu L, Gao P, Tian W, Wang X, Jin H, Xu H, & Chen Q (2010). Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis. Protein & cell, 1 (5), 468-77 PMID: 21203962

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