Virology tidbits: March 2015

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 p14^Arfmediated 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 p21^Waf1 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 p21^Waf1 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 p21^Waf1 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
MHV or IBV N

Delocalisation of Fibrillarin in various cell lines expressing
SARS-CoV N

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
pathways

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.

Virology tidbits

Virology tidbits

Tuesday, 31 March 2015

Viruses and the Nucleolus: Coronavirus N, Cytokinesis and Autophagy