key: cord-016652-x8t3lf1x authors: Matthews, David; Emmott, Edward; Hiscox, Julian title: Viruses and the Nucleolus date: 2011-05-23 journal: The Nucleolus DOI: 10.1007/978-1-4614-0514-6_14 sha: doc_id: 16652 cord_uid: x8t3lf1x The nucleolus is a dynamic sub-nuclear structure integral to the function of a eukaryotic cell. Some of its major roles involve ribosome subunit biogenesis, RNA processing, cell cycle control and responding to cellular stress, such as infection. Our understanding of the relationship between viruses and the nucleolus has moved from a phenomenological approach describing protein localisation to functional studies involving genetic analysis and proteomic approaches. These advances have provided fundamental insights as to how and why the nucleolus is targeted by many different viruses both to usurp normal functioning and to recruit nucleolar proteins to facilitate virus replication. This knowledge has been exploited for therapeutic strategies involving targeted inhibition of virus replication and live-attenuated recombinant vaccines. ; Lee et al. (2003) ; Lutz and Kedinger (1996) ; Tollefson et al. (2007) ; Matthews and Russell (1998) Asfaviridae Lower et al. (1995) Many of these proteins have been shown to interact with nucleolar proteins The reason why RNA viruses, and positive-strand RNA viruses in particular, interact with the nucleolus when the site of genome replication is in the cytoplasm is less intuitive. In this latter case, viral proteins that are normally required in the cytoplasm must transit through the nuclear pore complex both to and from the nucleus. This process is crucial for virus biology because if the viral proteins that are required for cytoplasmic functions such as RNA synthesis and encapsidation are sequestered in the nucleolus or nucleus, then progeny virus production will be affected as has been revealed by inhibitor and genetic studies (Lee et al. 2006; Tijms et al. 2002) . Viruses may interact with the nucleolus to usurp host cell functions and recruit nucleolar proteins to facilitate virus replication. Investigating the interactions between viruses and the nucleolus may facilitate the design of novel anti-viral therapies both in terms of recombinant vaccines (Pei et al. 2008 ) and molecular intervention (Rossi et al. 2007) , and also contribute to a more detailed understanding of the cell biology of the nucleolus. For many years our understanding of the interaction of viruses and the nucleolus was phenomenological and focused on identifying viral proteins that localised to this structure, their mechanisms of trafficking and potential interaction with nucleolar proteins (e.g. see Table 14 .1). However, recent research capitalising on advances in proteomics, viral genetics and cellular imaging techniques are beginning to increase our understanding of the mechanisms viruses use to subvert host cell nucleoli and facilitate virus biology . New data are now emerging that support the view that many viruses interact with the nucleus and nucleolus, particularly to facilitate virus replication. One of the best-studied viruses in terms of viral interactions with the nucleolus is HIV-1 and is described in detail in Chap. 17. Although HIV has clearly defined cytoplasmic and nuclear replication strategies, the virus has a positive-sense RNA genome in the sense that the viral capsid contains two copies of positive-sense RNA, but these are reverse transcribed in the cytoplasm and then trafficked to the nucleus, where ultimately the new genome is transcribed and trafficked back to the cytoplasm. Part of the reasoning for the interaction of HIV-1 with the nucleolus is the trafficking of intronless mRNA from the nucleus into the cytoplasm (Michienzi et al. 2000) . This is a property shared with herpes viruses and indicated that different viruses have evolved similar strategies involving subversion of nucleolar function for the benefit of virus biology (Boyne and Whitehouse 2006) . In the case of HIV-1, this knowledge has also led to the design and implementation of effective genetic therapies against the virus (Unwalla et al. 2008 ). A large number of viruses with DNA genomes have been shown to interact with nucleolus, and this perhaps is not surprising as most DNA viruses replicate in the nucleus. A genome-wide screen of three distinct herpesviruses, herpes simplex virus 1 (HSV-1), cytomegalovirus (CMV) and Epstein-Barr virus (EBV), has shown that at least 12 herpesvirus-encoded proteins specifically localise to the nucleolus (Salsman et al. 2008) , which are implicated in many aspects of the herpesvirus life cycle. Therefore, a number of proteomic studies are currently being undertaken to study changes, in a global context, within the nucleolar proteome during virus infections, and are discussed later (Lam et al. 2010) . Several different herpes virus proteins have been shown to cause the redistribution of nucleolar proteins and hence disruption of the nucleolus. These include herpes simplex virus 1, the major tegument structural protein VP22 (Lopez et al. 2008) , and the US11 (Xing et al. 2010) and UL24 proteins (Bertrand and Pearson 2008; Lymberopoulos and Pearson 2007) . Such disruption in many cases may have a direct effect on nucleolar function. A significant area of virus biology that has been investigated is the role of viral proteins that traffic through the nucleolus. For example, a number of HIV proteins that traffic through the nucleolus have been implicated in virus mRNA processing (Dundr et al. 1995) . Similar observations have also been made in herpesviruses Whitehouse 2006, 2009; Leenadevi and Dalziel 2009) . Initial studies utilising the prototype g-2 herpesvirus, herpes virus saimiri (HVS), demonstrated that the HVS nucleolar trafficking ORF57 protein induces nucleolar redistribution of the host cell human TREX proteins, which are involved in mRNA nuclear export (Boyne and Whitehouse 2006) . Intriguingly, ablating ORF57 nucleolar trafficking led to a failure of ORF57-mediated viral mRNA nuclear export (Boyne and Whitehouse 2006) . The precise role of this nucleolar sequestration is yet to be determined, but possible effects on viral mRNA/protein processing and viral ribonucleoprotein particle assembly are currently being investigated. This property may also be conserved in other ORF57 homologues as recent analysis has shown that the ORF57 protein from Kaposi's sarcoma associated herpesvirus (KSHV) also dynamically traffics through the nucleolus (Boyne et al. 2008b) . Moreover, on the rapid disorganisation of the nucleolus a reduction is observed in virus mRNA nuclear export (Boyne and Whitehouse 2009 ). The formation of an ORF57-mediated export competent ribonucleoprotein particle within the nucleolus may also have implications for the translation of viral mRNAs. For example, it has recently been demonstrated that the cellular nucleo-cytoplasmic shuttle protein, PYM, which is involved in translation enhancement, is redistributed to the nucleolus in the presence of the KSHV ORF57 protein (Boyne et al. 2010 ). This interaction effectively enhances the translation of the predominantly intronless transcripts made by KSHV, and draws parallels with potential translation enhancement of positive strand RNA virus genomes through their interaction with the nucleolus (discussed later). A second area of virus replication where nucleolar proteins are sequestered involves the replication of the virus DNA genome. For example, we (Matthews) and others have observed that nucleolar antigens upstream binding factor (UBF) and nucleophosmin (B23.1) are both sequestered into adenovirus DNA replication centres where they promote viral DNA replication (Hindley et al. 2007; Lawrence et al. 2006; Okuwaki et al. 2001) . Similarly, in HSV-1 infected cells, a number of nucleolar proteins including nucleolin and UBF are recruited into viral DNA replication centres . These are specific sites where replication and encapsidation of the HSV-1 genome occurs. Evidence suggests that sequestration of UBF is essential for viral DNA replication as overexpression of tagged version of UBF acts in a dominant-negative manner inhibiting virus DNA replication (Stow et al. 2009 ). Moreover, depletion of nucleolin results in reduced virus gene expression and infectious virion production (Calle et al. 2008; Sagou et al. 2010) . In addition to enhancing virus replication, nucleolar proteins are redistributed to alter cellular pathways during infection. For example, the nucleolar targeted HSV-1 US11 protein has been shown to interact with homeodomain-interacting protein kinase 2 (HIPK2), which plays a role in p53-mediated cellular apoptosis and hypoxic response (Calzado et al. 2009 ) and also participates in the regulation of the cell cycle (Calzado et al. 2007 ). This interaction alters the sub-cellular localisation of HIPK2 and protects against HIPK2-mediated cell cycle arrest (Giraud et al. 2004) . In contrast, the cellular protein, protein interacting with the carboxyl terminus-1 (PICT-1), can sequester the virally encoded apoptosis suppressor protein, KS-Bcl-2 protein, from the mitochondria into the nucleolus to down-regulate its anti-apoptotic activity (Kalt et al. 2010 ). This is a potential interesting interplay between two sub-cellular structures involved in the viral stress response (Olson 2009 ), and maybe more common and widespread. For example, bacterial infection has been shown to disrupt the nucleolus through regulating mitochondrial dysfunction (Dean et al. 2010). Although many RNA virus proteins have been shown to localise to the nucleolus, most attention has focused on viral capsid proteins. These are proteins that associate with the viral genome for encapsidation and assembly of new virus particles. These proteins may also modulate replication (and transcription, where appropriate) of the viral genome. Increasingly, capsid proteins have also been shown to have a number of roles in modulating host cell signalling pathways and functions. These capsid proteins are referred to as capsid, nucleoproteins or nucleocapsid proteins, depending on the virus. In many cases, they are phosphorylated (Chen et al. 2005) , which can modulate activity (Spencer et al. 2008) . Many examples of these proteins have been shown to localise to the nucleolus both when over-expressed and also in infected cells. These include proteins from positive-strand animal and plant RNA viruses, including the coronavirus nucleocapsid protein (Chen et al. 2002; Hiscox et al. 2001; Wurm et al. 2001) , the arterivirus nucleocapsid protein (Rowland et al. 1999) , the alphavirus capsid protein (Jakob 1994 ) and non-structural protein nsP2 (Rikkonen et al. 1992 (Rikkonen et al. , 1994 and the umbravirus ORF3 protein (Ryabov et al. 2004 ). Capsid proteins from negative-strand RNA viruses also localise to the nucleolus. These have strain dependent localisation of a number of different influenza virus proteins (Emmott et al. 2010c; Han et al. 2010; Melen et al. 2007; Volmer et al. 2010) . For many years this has followed a phenomenological pattern and viral capsid and RNA-binding proteins might simply localise to the nucleolus because they diffuse through the nuclear pore complex and associate with compartments in the nucleus that have high RNA contents -the nucleolus in particular because it is transcriptionally active. In this case, sub-cellular localisation to the nucleolus would have no physiological consequence for the virus or the cell. However, RNA virus replication is error prone and selection pressure might apply to such a fortuitous localisation (given the ~4,500+ nucleolar proteins and their diverse roles (Ahmad et al. 2009 )), with the concomitant effect that the virus could select for changes that ultimately disrupt nucleolar function and/or recruit nucleolar proteins to aid virus replication. There is a potential correlation between the nucleolar localisation of a viral protein and the loss of an essential nucleolar function. The molecular mechanisms responsible for this effect are unknown, but the displacement and re-localisation of nucleolar proteins by viral proteins could increase or decrease the nucleolar, nuclear and/or cytoplasmic pool of these proteins. Certainly, the accumulation of viral proteins in the nucleolus could potentially cause volume exclusion or crowding effects, which have been proposed to play a fundamental role in the formation of nuclear compartments including the nucleolus, and can be addressed by proteomic strategies. Therefore, disruption of nucleolar architecture and function might be common in virus-infected cells if viral proteins target the nucleolus or a stage of the virus lifecycle disrupts nucleolar proteins. For example, poliovirus infection results in the selective redistribution of nucleolin from the nucleolus to the cytoplasm (Waggoner and Sarnow 1998) and inactivation of UBF, which shuts off RNA polymerase I transcription in the host cell. The infection of cells with IBV has been shown to disrupt nucleolar architecture (Dove et al. 2006b ) and cause arrest of the cell cycle in the G2/M phase and failure of cytokinesis (Dove et al. 2006a) . The IBV and arterivirus nucleocapsid proteins associate with nucleolin and fibrillarin, respectively. Similarly, the HIV-1 Rev protein has been shown to localise to the DFC and GC and over-expression of Rev protein alters the nucleolar architecture and is associated with the accumulation of nucleophosmin (Dundr et al. 1995) . Many different virus proteins localise to the nucleolus (Table 14 .1). However, predicting viral (and cellular) nucleolar targeting signals has historically been problematic and only recently has bioinformatic software been developed to fascilitate this (Scott et al. 2011) . Nucleolar trafficking might be mediated by virtue of the fact that viral proteins that are trafficked to the nucleolus contain motifs that resemble host nucleolar targeting signals, that is, a form of molecular mimicry is used . The discovery of specific nucleolar trafficking signals in viral proteins has indicated a functional mechanism behind this observed localisation (Lee et al. 2003; Reed et al. 2006; . Analysis of the different nucleolar trafficking signals identified in viral proteins using dynamic live-cell imaging has certainly demonstrated that different proteins can confer differential trafficking rates and localisation patterns (Emmott et al. 2008) . This is very similar to cellular nucleolar proteins (Lechertier et al. 2007 ). In some virus proteins, both NLSs and nucleolar targeting signals act in concert to direct a protein to the nucleolus. The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein localises to the nucleolus and has been shown to contain two potential NLSs, a pat4 and a downstream pat7 motif (Rowland et al. 1999 . Analysis revealed that a 31 amino acid sequence incorporating the pat7 motif could direct the nucleocapsid protein to both the nucleus and nucleolus. The protein also contains a predicted NES, presumably to allow the protein to traffic back into the cytoplasm to contribute to viral function in this compartment. This is common with other similar related proteins. For example, in the avian coronavirus nucleocapsid protein an eight amino acid sequence is necessary and sufficient to target the protein to the nucleolus (Reed et al. 2006 ) and contains an NES (Reed et al. 2007 ). Intriguingly, genetic analysis (Lee et al. 2006) , dynamic livecell imaging (You et al. 2008 ) and use of trafficking inhibitors (Tijms et al. 2002) paint a picture of the requirement of these positive sense RNA virus capsid proteins localising to the nucleolus as soon as they are translated, prior to their involvement in virus replication or assembly. This may be related to subversion of host cell function, protein modification (e.g. phosphorylation) or recruitment of nucleolar proteins. Viral proteins might also traffic to the nucleolus through association with cellular nucleolar proteins . For example, the hepatitis delta antigen has been shown to contain a nucleolar targeting signal that also corresponded to a site that promoted binding to nucleolin (Lee et al. 1998) . Mutating this region prevented nucleolin binding to the delta antigen and nucleolar trafficking. By implication, this relates nucleolin binding to nucleolar trafficking (Lee et al. 1998) . Certainly, interaction with nucleophosmin and hepatitis delta antigens can modulate viral replication (Huang et al. 2001 ) and more recently combined proteomic-RNAi screens have revealed many other nucleolar proteins that can be associated with this viral protein (Cao et al. 2009 ). Trafficking and accumulation of viral proteins to and from the nucleolus, similar to cellular proteins, may also be cell cycle related. For example, the coronavirus nucleocapsid protein localises preferentially to the nucleolus in the G2 phase of the cell cycle (Cawood et al. 2007) , as does the human cytomegalovirus protein UL83 in the G1 phase (Arcangeletti et al. 2011) . Again these trafficking profiles may be related to the interaction with cellular nucleolar proteins (Emmott and Hiscox 2009). Semliki Forest virus non-structural protein nsP2 can localise to the nucleolus (Peranen et al. 1990; Rikkonen et al. 1992 Rikkonen et al. , 1994 and disruption of this localisation through a single amino acid change results in a reduction in neurovirulence (Fazakerley et al. 2002) . Such in vitro data has also been backed up by persuasive in vivo data. Mutation of the arterivirus nucleocapsid protein pat7 NLS motif in the context of a full-length clone revealed that this sequence could have a key role in virus pathogenesis in vivo, as animals infected with mutant viruses had shorter viraemia than wild-type viruses (Lee et al. 2006; Pei et al. 2008) . Interestingly, reversions occurred in the mutated nucleocapsid gene sequence and although the amino acid sequence of the pat7 motif was altered, its function was not; this new signal was defined as a pat8 motif (Lee et al. 2006) . The clear implications of this groundbreaking work is that disruption of nucleolar trafficking of a viral protein proves functional relevance and illustrates the potential of exploiting this knowledge for the generation of growth attenuated recombinant vaccines (Pei et al. 2008; Reed et al. 2006 Reed et al. , 2007 . Similarly, point mutations in the Japanese encephalitis virus (JEV) core protein that abolished nuclear and nucleolar localisation resulted in recombinant viruses with impaired replication in mammalian cells, compared to wild type virus (Mori et al. 2005; Tsuda et al. 2006) . Curiously, replication of recombinant viruses was not impaired in insect cells, illustrating this could potentially be related to differences in nucleolar architecture and proteomes between these cell types (Thiry and Lafontaine 2005) . The JEV core protein has been shown to interact with nucleophosmin and is translocated from the nucleolus to the cytoplasm. Flaviviruses in general (JEV, Dengue virus and West Nile virus) appear to have a part-nuclear stage to the synthesis of viral RNA and several components of the viral replicase together with newly synthesised RNA have been found in the nucleus of infected cells (Uchil et al. 2006) . One intriguing question that has yet to be elucidated is how such viral RNA traffics from the nucleus to the cytoplasm. Most cellular mRNAs are spliced and it is part of the splicing process that signals nuclear export. Certain DNA viruses, such as herpesvirus saimiri, produce intron-less mRNA and these viruses have evolved specific viral proteins (such as herpesvirus saimiri ORF57 (Boyne et al. 2008a) ), which interact with the cellular mRNA export machinery (e.g. the mRNA processing and export factor ALY) to traffic viral mRNA from the nucleus to the cytoplasm (Boyne et al. 2008b (Boyne et al. , 2010 Boyne and Whitehouse 2006) and a similar process might be required by RNA viruses. For example, tomato bushy stunt virus (TBSV) redistributes ALY from the nucleus to the cytoplasm, and this might be a way the virus mediates host cell protein synthesis (Uhrig et al. 2004 ). In plants RNA silencing, a host defence mechanism targets virus RNAs for degradation in a sequence-specific manner and viruses use several mechanisms to counteract this system (Canto et al. 2006) . TBSV encodes a protein, P19, which interferes with this pathway. However, ALY might transport P19 from the cytoplasm to the nucleus or nucleolus and disrupt its silencing suppression activity. Nucleolin has also been shown to be involved in the trafficking of herpes simplex virus type 1 nucleocapsids from the nucleus to the cytoplasm (Sagou et al. 2010) , drawing parallels with the involvement of nucleolar proteins in the movement of plant viruses (Kim et al. 2007a, b) . Different plant virus proteins involved in long-distance phloem-associated movement of virus particles or with roles in binding to the RNA virus genomes localise to the nucleolus and other sub-nuclear structures (Kim et al. 2007b; Ryabov et al. 2004 ). This may be mediated by association with nuclear proteins, as is the case with fibrillarin and the ORF3 protein of plant umbraviruses (Kim et al. 2007a) . Hijacking the nucleolus is not exclusive to plant viruses and may also occur with mammalian viruses. Similar to the plant rhabdovirus maize fine streak virus (MFSV), whose nucleocapsid and phosphoproteins localise to the nucleolus (Tsai et al. 2005) , the animal negative-stranded RNA virus Borna disease virus has been reported to use the nucleolus as a site for genome replication, and its RNA-binding protein has the appropriate trafficking signals for import to and export from the cytoplasm to the nucleus (Pyper et al. 1998 ). The hepatitis delta virus genome also has differential synthesis in the nucleus with RNA being transcribed in the nucleolus (Huang et al. 2001) ; this is similar to the potato spindle tuber viroid where RNAs of opposite polarity are sequestered in different nuclear compartments, with the positive-sense RNA being transported to the nucleolus. Again localisation to different sub-nuclear strcutures may have different roles in the virus life cycle (Li et al. 2006 ). An intriguing recent discovery has been made showing that adenoassociated virus (AAV) encodes an additional protein called assembly-activating protein (AAP) that localises to the nucleolus and promotes assembly of the viral capsid (Sonntag et al. 2010) . As a result of their limited genomes and coding capacities, recruitment of cellular proteins with defined functions in RNA metabolism would be a logical step to facilitate RNA virus infection. As nucleolar proteins have many crucial functions in cellular RNA biosynthesis, processing and translation, it comes as no surprise that nucleolar proteins are incorporated into the replication and/or translation complexes formed by RNA viruses. Given that some nucleolar proteins have many different functions, the same nucleolar protein might be used by a virus for different aspects of the replication pathway. Studies suggest that the human rhinovirus 3 C protease (3Cpro) pre-cursors, 3CD' and/or 3CD, localise in the nucleoli of infected cells early in infection and inhibit cellular RNA transcription via proteolytic mechanisms (Amineva et al. 2004 ). This general property is not restricted to human rhinovirus and in terms of the inhibition of cellular translation has also been described for encephalomyocarditis virus (Aminev et al. 2003a, b) , again suggesting roles in translational regulation. Given the many roles of the nucleolus in the life cycle of the cell, including as stress sensor (Boulon et al. 2010; Mayer and Grummt 2005) , it would seem reasonable that comprehensive unbiased analysis of the nucleolar proteome would yield interesting data, particularly, with providing clues as to what cellular nucleolar functions may be altered by virus infection and what mechanisms the nucleolus may use to respond to this. How the nucleolar proteome changes in response to virus-infection has been investigated using stable isotope labelling with amino acids in cell culture (SILAC) coupled to LC-MS/MS and bioinformatics (Fig. 14.1) . These studies, led by our laboratories, have analysed purified nucleoli and the nucleus, and have directly stemmed from the pioneering work of the Lamond laboratory in analysing purified nucleoli using quantitative proteomics (Andersen et al. 2005 (Munday et al. 2010) . Overall, our data indicates that only a small proportion of nucleolar proteins change in abundance in virus-infected cells, Fig. 14. 1 Diagram of a "classic" SILAC experiment. This technology allows high-throughput quantitative proteomics and has been readily applied to the nucleolus, especially when coupled with dynamic live-cell imaging (Andersen et al. 2005) . The ability to simultaneously compare up to three different conditions through selection of the appropriate isotope label has enabled the recent studies of how the nucleolar proteome changes in virus-infected cells (Emmott et al. 2010a; Emmott et al. 2010b; Emmott et al. 2010c; Hiscox et al. 2010; Lam et al. 2010) and these tend to be virus-specific. For example, in adenovirus infected cells just 7% of proteins identified show a twofold or greater change compared to almost a third of nucleolar antigens showing a greater than twofold change when cells are treated with ActD which inhibits rRNA synthesis (Lam et al. 2010) . What is notable is that direct comparison between the adenovirus data set and the ActD dataset shows no clear correlation Lam et al. 2010) , further supporting the case that adenovirus induces effects on the nucleolus distinct from that of a generalised, non-specific shut down of nucleolar function. This fits well with a previous observation that adenovirus infection does not affect rRNA synthesis even 36 h post-infection (Lawrence et al. 2006 ). These results were initially surprising given the number of different viral proteins that can localise to this structure and how they interact with nucleolar proteins. This suggests that the nucleolar proteome and architecture is resilient during early stages of infection but may become disrupted as more and more damage accumulates inside cells because of virus activity, as clearly evidenced in live-cell imaging experiments (Bertrand and Pearson 2008; Dove et al. 2006b; ). Coupling quantitative proteomic analysis of the nucleolus and deep sequencing throughout infection in time-course experiments of lytic, latent, acute and persistent viruses would reveal valuable insights into the response of the nucleolus to virus infection. Likewise, being able to move from studying cell culture-adapted laboratory strains into clinical isolates replicating in primary cells would yield more biologically relevant information, particularly with regard to the severity of disease and nucleolar changes. These technologies could also be applied to large-scale analysis of viral proteins that traffic to the nucleolus and the cellular nucleolar proteins that they associate with (e.g. using SILAC and EGFP-traps (Trinkle-Mulcahy et al. 2008)), thus generating and integrating interactome networks with the nucleolar proteome during infection. NOPdb: nucleolar proteome database-2008 update Encephalomyocarditis viral protein 2A localizes to nucleoli and inhibits cap-dependent mRNA translation Encephalomyocarditis virus (EMCV) proteins 2A and 3BCD localize to nuclei and inhibit cellular mRNA transcription but not rRNA transcription Rhinovirus 3 C protease precursors 3CD and 3CD' localize to the nuclei of infected cells Nucleolar proteome dynamics Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro Np9 protein of human endogenous retrovirus K interacts with ligand of numb protein X Functional role of pX open reading frame II of human T-lymphotropic virus type 1 in maintenance of viral loads in vivo A temporal study of the expression of the capsid, cytoplasmic inclusion and nuclear inclusion proteins of tobacco etch potyvirus in infected plants Visualization of the interaction between the precursors of VPg, the viral protein linked to the genome of turnip mosaic virus, and the translation eukaryotic initiation factor iso 4E in Planta The conserved N-terminal domain of herpes simplex virus 1 UL24 protein is sufficient to induce the spatial redistribution of nucleolin M148R and M149R are two virulence factors for myxoma virus pathogenesis in the European rabbit The nucleolus under stress Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA Nucleolar disruption impairs Kaposi's sarcoma-associated herpesvirus ORF57-mediated nuclear export of intronless viral mRNAs Herpesvirus saimiri ORF57: a post-transcriptional regulatory protein Recruitment of the complete hTREX complex is required for Kaposi's sarcoma-associated herpesvirus intronless mRNA nuclear export and virus replication Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs Nucleolin is required for an efficient herpes simplex virus type 1 infection HIPK2: a versatile switchboard regulating the transcription machinery and cell death From top to bottom: the two faces of HIPK2 for regulation of the hypoxic response Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient A single amino acid change in the nuclear localization sequence of the nsP2 protein affects the neurovirulence of semliki forest virus Analysis of the subcellular localization of the proteins Rep, Rep' and Cap of porcine circovirus type 1 Bovine immunodeficiency virus tat gene: cloning of two distinct cDNAs and identification, characterization, and immunolocalization of the tat gene products Human T-cell leukemia virus type I p30 nuclear/nucleolar retention is mediated through interactions with RNA and a constituent of the 60 S ribosomal subunit US11 of herpes simplex virus type 1 interacts with HIPK2 and antagonizes HIPK2-induced cell growth arrest Nuclear and nucleolar localization of an African swine fever virus protein, I14L, that is similar to the herpes simplex virus-encoded virulence factor ICP34.5 The nucleoprotein and the viral RNA of infectious salmon anemia virus (ISAV) are localized in the nucleolus of infected cells The bovine immunodeficiency virus rev protein: identification of a novel lentiviral bipartite nuclear localization signal harboring an atypical spacer sequence Cucumber mosaic virus 2b protein subcellular targets and interactions: their significance to RNA silencing suppressor activity Involvement of the nucleolus in replication of human viruses Identification of nucleolus localization signal of betanodavirus GGNNV protein alpha Characterization of the nuclear and nucleolar localization signals of bovine herpesvirus-1 infected cell protein 27 New regulatory mechanisms for the intracellular localization and trafficking of influenza A virus NS1 protein revealed by comparative analysis of A/PR/8/34 and A/Sydney/5/97 Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 VP22 tegument protein subcellular associations Nucleolar localization of potato leafroll virus capsid proteins Relationship between adenovirus DNA replication proteins and nucleolar proteins B23.1 and B23.2 Direct interaction between nucleolin and hepatitis C virus NS5B Brief review: the nucleolus -a gateway to viral infection? The interaction of animal cytoplasmic RNA viruses with the nucleus to facilitate replication RNA viruses: hijacking the dynamic nucleolus The coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus Nucleolar proteomics and viral infection The HBZ-SP1 isoform of human T-cell leukemia virus type I represses JunB activity by sequestration into nuclear bodies Nucleolar localization of mouse mammary tumor virus proteins in T-cell lymphomas Identification and characterization of the UL24 gene product of herpes simplex virus type 2 The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication Expression and processing of a small nucleolar RNA from the Epstein-Barr virus genome A novel, mouse mammary tumor virus encoded protein with Rev-like properties Nucleolar accumulation of Semliki Forest virus nucleocapsid C protein: influence of metabolic status, cytoskeleton and receptors GLTSCR2/PICT-1, a putative tumor suppressor gene product, induces the nucleolar targeting of the Kaposi's sarcoma-associated herpesvirus KS-Bcl-2 protein Interaction of a plant virus-encoded protein with the major nucleolar protein fibrillarin is required for systemic virus infection Cajal bodies and the nucleolus are required for a plant virus systemic infection Electron microscopy of ribonucleic acid in nuclear particulate aggregates of hepatitis D using nuclease-gold complexes Functional similarity of HIV-I rev and HTLV-I rex proteins: identification of a new nucleolar-targeting signal in rev protein Nucleo-cytoplasmic redistribution of the HTLV-I Rex protein: alterations by coexpression of the HTLV-I p21x protein Proteomics analysis of the nucleolus in adenovirus-infected cells Immunocytology shows the presence of tobacco etch virus P3 protein in nuclear inclusions Nucleolar protein upstream binding factor is sequestered into adenovirus DNA replication centres during infection without affecting RNA polymerase I location or ablating rRNA synthesis A B23-interacting sequence as a tool to visualize protein interactions in a cellular context The nucleolin binding activity of hepatitis delta antigen is associated with nucleolus targeting Adenovirus core protein VII contains distinct sequences that mediate targeting to the nucleus and nucleolus, and colocalization with human chromosomes Precursor of human adenovirus core polypeptide Mu targets the nucleolus and modulates the expression of E2 proteins Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication The alcelaphine herpesvirus-1 ORF 57 encodes a nuclear shuttling protein Functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein ICP4 with EAP, a nucleolar-ribosomal protein RNA-templated replication of hepatitis delta virus: genomic and antigenomic RNAs associate with different nuclear bodies Capsid protein of cucumber mosaic virus accumulates in the nuclei and at the periphery of the nucleoli in infected cells Nucleolar and nuclear localization properties of a herpesvirus bZIP oncoprotein, MEQ The major tegument structural protein VP22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection Identification of a Rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses HTDV/ HERV-K Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter Involvement of UL24 in herpes-simplex-virus-1-induced dispersal of nucleolin Relocalization of upstream binding factor to viral replication compartments is UL24 independent and follows the onset of herpes simplex virus 1 DNA synthesis Involvement of the UL24 protein in herpes simplex virus 1-induced dispersal of B23 and in nuclear egress The products of gene US11 of herpes simplex virus type 1 are DNA-binding and localize to the nucleoli of infected cells Stable expression of hepatitis delta virus antigen in a eukaryotic cell line Adenovirus core protein V is delivered by the invading virus to the nucleus of the infected cell and later in infection is associated with nucleoli Cellular stress and nucleolar function Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27 Nuclear and nucleolar targeting of influenza A virus NS1 protein: striking differences between different virus subtypes Karyophilic properties of Semliki Forest virus nucleocapsid protein Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA Localization and importance of the adenovirus E4orf4 protein during lytic infection The lactate dehydrogenase-elevating virus capsid protein is a nuclear-cytoplasmic protein The protein ICP0 of herpes simplex virus type 1 is targeted to nucleoli of infected cells Nuclear localization of Japanese encephalitis virus core protein enhances viral replication Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus Functional domain structure of human T-cell leukemia virus type 2 rex Nucleolar localization of human hepatitis B virus capsid protein Nucleolar targeting signal of human T-cell leukemia virus type I rex-encoded protein is essential for cytoplasmic accumulation of unspliced viral mRNA Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins Induction of apoptosis by viruses: what role does the nucleolus play? Inhibition of human immunodeficiency virus type 1 and type 2 Tat function by transdominant Tat protein localized to both the nucleus and cytoplasm Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins Functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2 The nucleolus is the site of Borna disease virus RNA transcription and replication The complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection Control of nuclear and nucleolar localization of nuclear inclusion protein a of picorna-like Potato virus A in Nicotiana species Proapoptotic effect of hepatitis C virus CORE protein in transiently transfected cells is enhanced by nuclear localization and is dependent on PKR activation Delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein Characterization of the nuclear export signal in the coronavirus infectious bronchitis virus nucleocapsid protein Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2 Nuclear targeting of Semliki Forest virus nsP2 Intracellular transport of the murine leukemia virus during acute infection of NIH 3T3 cells: nuclear import of nucleocapsid protein and integrase Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus Genetic therapies against HIV Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences The localisation of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus Structural and functional characterization of human immunodeficiency virus tat protein Human endogenous retrovirus HERV-K(HML-2) encodes a stable signal peptide with biological properties distinct from Rec Intracellular location of two groundnut rosette umbravirus proteins delivered by PVX and TMV vectors Identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance RNA movement protein Nucleolin is required for efficient nuclear egress of herpes simplex virus type 1 nucleocapsids Genome-wide screen of three herpesviruses for protein subcellular localization and alteration of PML nuclear bodies Identification of the caprine arthritis encephalitis virus Rev protein and its cis-acting Rev-responsive element The Rev protein of visna virus is localized to the nucleus of infected cells PNAC: a protein nucleolar association classifier Characterization of signals that dictate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of Tomato leaf curl Java virus associated with DNA beta satellite Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing A viral assembly factor promotes AAV2 capsid formation in the nucleolus Role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein Upstream-binding factor is sequestered into herpes simplex virus type 1 replication compartments Nucleolin associates with the human cytomegalovirus DNA polymerase accessory subunit UL44 and is necessary for efficient viral replication Reversible nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion Birth of a nucleolus: the evolution of nucleolar compartments Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus Identification of a new human adenovirus protein encoded by a novel late l-strand transcription unit Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes Complete genome sequence and in planta subcellular localization of maize fine streak virus proteins Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication Nuclear localization of flavivirus RNA synthesis in infected cells Relocalization of nuclear ALY proteins to the cytoplasm by the tomato bushy stunt virus P19 pathogenicity protein Use of a U16 snoRNA-containing ribozyme library to identify ribozyme targets in HIV-1 Avian reovirus sigmaA localizes to the nucleolus and enters the nucleus by a nonclassical energy-and carrier-independent pathway Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells Viral ribonucleoprotein complex formation and nucleolarcytoplasmic relocalization of nucleolin in poliovirus-infected cells Interactions of minute virus of mice and adenovirus with host nucleoli Intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus Proteins C and NS4B of the flavivirus Kunjin translocate independently into the nucleus Subcellular compartmentalization of adeno-associated virus type 2 assembly The N-terminal domain of PMTV TGB1 movement protein is required for nucleolar localization, microtubule association, and long-distance movement Localisation to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division Molecular anatomy of subcellular localization of HSV-1 tegument protein US11 in living cells Nucleolar localization of the UL3 protein of herpes simplex virus type 2 Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein A model for the dynamic nuclear/ nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging G0/G1 arrest and apoptosis induced by SARS-CoV 3b protein in transfected cells Intracellular localization of the UL31 protein of herpes simplex virus type 2 Acknowledgements DAM and JAH would like to acknowledge their co-workers and collaborators over the years for developing viral interactions with the nucleolus. DAM's research on the nucleolus is supported by the Wellcome Trust and JAH's by the BBSRC and a Leverhulme Trust Research Fellowship. EE is supported by a BBSRC Astbury DTG studentship.