Title: The nucleolar interface of <scp>RNA</scp> viruses
Abstract: Cellular MicrobiologyVolume 17, Issue 8 p. 1108-1120 MicroreviewFree Access The nucleolar interface of RNA viruses Stephen M. Rawlinson, Stephen M. Rawlinson Viral Pathogenesis Laboratory, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, AustraliaSearch for more papers by this authorGregory W. Moseley, Corresponding Author Gregory W. Moseley Viral Pathogenesis Laboratory, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, AustraliaFor correspondence. E-mail [email protected]; Tel. (+61) 83442288; Fax (+61) 93481421.Search for more papers by this author Stephen M. Rawlinson, Stephen M. Rawlinson Viral Pathogenesis Laboratory, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, AustraliaSearch for more papers by this authorGregory W. Moseley, Corresponding Author Gregory W. Moseley Viral Pathogenesis Laboratory, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, AustraliaFor correspondence. E-mail [email protected]; Tel. (+61) 83442288; Fax (+61) 93481421.Search for more papers by this author First published: 03 June 2015 https://doi.org/10.1111/cmi.12465Citations: 12AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Summary In recent years, understanding of the nucleolus has undergone a renaissance. Once considered primarily as the sites of ribosome biogenesis, nucleoli are now understood to be highly dynamic, multifunctional structures that participate in a plethora of cellular functions including regulation of the cell cycle, signal recognition particle assembly, apoptosis and stress responses. Although the molecular/mechanistic details of many of these functions remain only partially resolved, it is becoming increasingly apparent that nucleoli are also common targets of almost all types of viruses, potentially allowing viruses to manipulate cellular responses and the intracellular environment to facilitate replication and propagation. Importantly, a number of recent studies have moved beyond early descriptive observations to identify key roles for nucleolar interactions in the viral life cycle and pathogenesis. While it is perhaps unsurprising that many viruses that replicate within the nucleus also form interactions with nucleoli, the roles of nucleoli in the biology of cytoplasmic viruses is less intuitive. Nevertheless, a number of positive-stranded RNA viruses that replicate exclusively in the cytoplasm are known to express proteins that enter the nucleus and target nucleoli, and recent data have indicated similar processes in several cytoplasmic negative-sense RNA viruses. Here, we review this emerging aspect of the virus–host interface with a focus on examples where virus–nucleolus interactions have been linked to specific functional outcomes/mechanistic processes in infection and on the nucleolar interfaces formed by viruses that replicate exclusively in the cytoplasm. Introduction The nucleolus is the most prominent subnuclear structure and was first described in detail by cytologists over 170 years ago (Wagner, 1835; Montgomery, 1898). However, the function of the nucleolus remained unclear until the 1960s when it was firmly established as the site of ribosome biogenesis (Pederson, 2011). Until the 1990s, almost all studies of nucleoli focused on elucidating their structure and function as the cellular ribosome factory, and this role became an established dogma in modern biology. In the intervening years, our understanding of the nucleolus has fundamentally changed, with the entrenched idea of a largely static ribosome factory giving way to the current model of a highly dynamic, multifunctional structure (Boisvert et al., 2007). It has also become increasingly clear that the nucleolus represents a major player in the interface between viruses and host cells, with examples from almost all viral classes shown to form associations with nucleoli (Matthews et al., 2011). While much of the relevant data have been of a largely descriptive/phenomenological in nature, the emerging picture of a multifunctional nucleolus has brought new potential significance to these observations. This work has indicated the existence of two broad classes of virus–nucleolar interfaces, which are not necessarily mutually exclusive, whereby (i) viruses express proteins that target nucleoli to modify nucleolar/cellular functions and (ii) viruses recruit/exploit nucleoli and/or nucleolar components to mediate direct roles in the viral life cycle. Of particular interest in this respect are interactions formed by proteins expressed by RNA viruses that replicate their genomes outside the host cell nucleus, such that the role(s) of the nucleus/nucleolus is not immediately apparent. For such viruses, the formation of molecular interfaces with nucleoli is suggestive of highly specific modulatory mechanisms. This review will discuss these aspects of the virus–nucleolar interaction, with a particular focus on examples that go beyond the descriptive to highlight the two ‘classes’ of interface and on emerging evidence of the broad significance of nucleoli in infection by cytoplasmic viruses. Structure and function of the nucleolus Ribosomes are essential to all cellular life, and yet nucleoli are found only in eukaryotes. It has been suggested that this relates to the far greater complexity of ribosome biogenesis in eukaryotes than in prokaryotes, and consequently requires an organizing structure/platform (Pederson, 2011; Farley et al., 2015). Eukaryotic ribosomes are 40% larger than prokaryotes and require hundreds of additional RNA modifications and many additional rRNA and protein interactions (Hernandez-Verdun, 2011). As a result, ribosome biogenesis requires exquisite regulation to efficiently meet the needs of the cell, particularly under conditions of growth/mitosis when protein production is significantly increased (Smetana and Busch, 1974; Hernandez-Verdun, 2011). Multiple copies of rDNA are utilized for the generation of rRNA, and nucleoli form around rDNA clusters in chromosomes, called nucleolar organizing regions. This appears to enable rapid, efficient and highly regulated generation of ribosomes via a coordinated structural scaffold that concentrates ribosomal components, regulatory factors, interactions and reactions. This scaffold organization is most evident in the substructure of nucleoli, which are formed as membraneless bodies that, depending on cell type, are subdivided into three distinct structural/functional regions: the fibrillar centre (FC), which is surrounded by the dense fibrillar component (DFC) and the outer granular component (GC) (Hernandez-Verdun, 2011). These subcompartments appear to form an ‘assembly-line’ for the ribosome factory, with transcription occurring at the border of the FC and DFC and continuing through the DFC for completion in the GC (Fig. 1). Figure 1Open in figure viewerPowerPoint The nucleolar interface of cytoplasmic RNA viruses. The life cycle of +ssRNA (e.g. flaviviruses, picornaviruses) and −ssRNA viruses (e.g. paramyxoviruses, rhabdoviruses) is shown schematically; +ssRNA genomes are translated directly in the cytoplasm by cellular machinery, commonly generating a single polyprotein that is cleaved co- and post-translationally into individual viral proteins necessary for replication and release; −ssRNA genomes require co-delivery with a viral RNA-dependent RNA polymerase, which mediates primary transcription to generate +ve sense mRNA encoding viral protein. Most +RNA and −RNA viruses replicate exclusively in the cytoplasm but many also form interfaces with nucleoli (see expanded nucleolus) by (i) targeting viral proteins to nucleoli (blue/green arrows), usually to modulate nucleolar functions (e.g. host cell transcription, cell cycle progression) or (ii) recruiting/exploiting nucleolar proteins outside of the nucleolar compartment (red arrows) to directly facilitate key steps in the viral life cycle, with reports of roles at all stages of the viral infectious cycle. Most available data relate to +ssRNA viruses, but recent studies indicate that in spite of clear differences in replication, −ssRNA viruses also target the nucleolus with significance to infection through mechanisms/functions that are currently not determined (?). Specific examples of virus–nucleolar interfaces are shown, with nucleolar/host components in red and +ssRNA and −ssRNA virus components in blue and green respectively. The lower images of nucleoli indicate the nucleolar substructure, which typically comprises three subcompartments, the FC (white), surrounded by the DFC (light blue) and outer GC (yellow) important to the organization of the complex functions of the nucleolus. Details of specific viral interactions are discussed in the text and Table 1. The nucleolus may have evolved to meet the demands for ribosomal production in eukaryotic cells, but it is perhaps to be expected that other functions would evolve around this structure, exploiting its capacity to organize complex interactions. The finding that signal recognition particle assembly occurs in the nucleolus but that no components of ribosome biogenesis are utilized for this process provided the first clear indications of distinct cellular roles for the nucleolus (Jacobson and Pederson, 1998), leading to the development of the ‘plurifunctional nucleolus’ hypothesis (Pederson, 1998). The advent of new high-throughput technologies including quantitative proteomics techniques, such as stable isotope labelling with amino acids in cell culture, coupled with high-yield/purity enrichment of nucleoli, has now enabled unprecedented insights into the dynamic and complex nature of the nucleolus (Andersen et al., 2002; 2005; Pendle et al., 2005), with more than 6000 proteins shown to localize to nucleoli, ranging from the highly stable/resident [e.g. nucleolin, B23/nucleophosmin/NPM1 (B23), fibrillarin] to the transient (e.g. p53, p68, von-Hippel Lindau) (Mekhail et al., 2004; Andersen et al., 2005). Importantly, only 30% of the constituent proteins of the nucleolar proteome are known to be involved in ribosome biogenesis, with a large proportion having roles in processes such as cell cycle regulation, mRNA processing and DNA replication or repair, or having no established function (Ahmad et al., 2009) suggestive of additional cellular roles as yet unidentified. One corollary of this molecular complexity is that unravelling the precise functions of nucleoli has become increasingly difficult, with proteomic data far outpacing biological understanding. However, it appears that from an origin in ribosome biogenesis, the nucleolus has evolved as a multifunctional ‘meeting point’ to integrate a diverse array of interactions. In spite of the fact that the nucleolus is not membrane bound, nucleolar protein targeting appears to be highly specific, involving binding to nucleolar components such as rDNA, RNA or resident proteins (Emmott and Hiscox, 2009); this contrasts with targeting of organelles, such as mitochondria and the nucleus, which generally involves specific membrane translocation mechanisms (Rassow and Pfanner, 2000; Cautain et al., 2015). Many nucleolar proteins contain Arg/Lys-rich regions that act as nucleolar localization/targeting sequences (NoLSs), although a clear NoLS motif(s) has not been defined possibly due to the range of interactors that mediate nucleolar localization/retention. NoLSs are often intimately associated with nuclear localization sequences (NLSs) that are similarly rich in basic residues and mediate active import of proteins through the nuclear pore complex (NPC) embedded in the nuclear membrane (Cautain et al., 2015); this is likely to enable coordinated/efficient delivery to the nucleus and subsequent targeting to nucleoli. Viral targeting of the nucleolus Given the diverse functions of the nucleolus, it is not surprising that many viruses that replicate in the nucleus [including many DNA viruses, retroviruses and some nuclear-replicating negative-stranded RNA (−ssRNA) viruses] interact with the readily accessible nucleolus. Interactions have been described to enable the exploitation of nucleolar proteins to facilitate specific processes in viral replication or of nucleoli as structural platforms for virus assembly (Hiscox, 2007; Sonntag et al., 2010; Salvetti and Greco, 2014). Nucleolar targeting by nuclear viruses has been the subjects of several reviews (Hiscox, 2007; Greco, 2009; Hiscox et al., 2010; Wang et al., 2010a; Ni et al., 2012; Salvetti and Greco, 2014) and a number of known/hypothesized functions are summarized in Table 1. Table 1. Selected virus–nucleolar interactions with related functional significance/insights Family Replication site Virus Viral factor Host protein involved/targeted Functional data References RNA, positive-stranded Flaviviridae Cytoplasm WNV Capsid MDM2 Capsid activates p53-mediated apoptosis Yang et al. (2008) DDX56 Role in virus assembly Xu et al. (2011); Xu and Hobman (2012) JEV Capsid n.d. Nuclear/nucleolar targeting linked to pathogenesis in mice Mori et al. (2005) B23 B23 important for viral replication Tsuda et al. (2006) HCV Core B23, YY1 Activates transcription of B23 Mai et al. (2006) SL1, TBP Regulates transcriptional pathways; promotes cell growth and proliferation Kao et al. (2004) PKR, p53 Core protein induces apoptosis Otsuka et al. (2000); Realdon et al. (2004) HCV IRES Nucleolin Role in IRES-mediated translation initiation Yu et al. (2005) NS5B Nucleolin Interaction of NS5B and nucleolin critical for replication Hirano et al. (2003); Shimakami et al. (2006); Kusakawa et al. (2007) Picornaviridae Cytoplasm PV 3'NCR Nucleolin Nucleolin stimulates viral IRES-mediated translation Waggoner and Sarnow (1998); Izumi et al. (2001) 3Cpro UBF, SL1 Shutdown of RNA Pol I transcription Weidman et al. (2003) EMCV 2A, 3BCD B23 Shutdown of RNA Pol II transcription, and cap-dependent translation Aminev et al. (2003a,b) RV16 3CDpro OCT-1 Shutdown of cellular transcription Amineva et al. (2004) EV71 VP1 Nucleolin Facilitates virus entry Su et al. (2015) Togaviridae Cytoplasm M1 n.d. p21 Induces S-phase arrest and apoptosis Hu et al. (2009) Coronaviridae Cytoplasm PPRSV N Fibrillarin Nuclear/nucleolar localization linked to replication/pathogenesis Yoo et al. (2003); Lee and Gu (2010) IBV N n.d. Cell cycle-dependent nucleolar localization; affects nucleolar morphology Dove et al. (2006); Cawood et al. (2007) RNA, negative-stranded Paramyxoviridae Cytoplasm HPIV-3 F Nucleolin Cell surface-expressed nucleolin required for efficient entry into lung cells Bose et al. (2004) Hendra, Nipah, others M Several unconfirmed M protein of a number of paramyxoviruses localizes to the nucleolus; proteomic data indicate binding to several nucleolar proteins Pentecost et al. (2015) NDV M B23 M localizes to nucleolus early during infection; B23 redistributed to nucleoplasm late in infection; B23 is important for replication and cytopathic effect Peeples et al. (1992); Duan et al. (2014) RSV G Nucleolin Nucleolin acts as cellular receptor for entry Tayyari et al. (2011) Rhabdoviridae Cytoplasm RV P3 Nucleolin Nucleolin expression required for virus production Oksayan et al. (2015) Nucleus BDV N, M n.d. Site of RNA transcription and replication Pyper et al. (1998) Orthomyxoviridae Nucleus IAV NS1 Nucleolin, Fibrillarin Inhibits p53 activity and apoptosis; nucleolar targeting differs between viral subtypes Melen et al. (2007; 2012); Wang et al. (2010b; 2012) PB2, HA, NP, M1, NS1, NS2 Several – strain specific (e.g. ADAR1) Infection induced significant changes to nucleolar proteome Emmott et al. (2010a) DNA viruses Herpesviridae Nucleus HVS ORF57 hTREX Nucleolus required for viral mRNA nuclear export Boyne and Whitehouse (2006) PRV UL54 n.d. Mutation of the NoLS affects viral gene expression, DNA synthesis and viral production Li et al. (2011) HCMV UL44, UL84 Nucleolin Nucleolin required to maintain the architecture of replication compartments Strang et al. (2010; 2012); Bender et al. (2014) EBV EBNA-1 Nucleolin Nucleolin binding to EBNA-1 important for EBNA-1 role in EBV episome binding, maintenance and transcription Chen et al. (2014) Papillomaviridae Nucleus HPV E6, E7 B23, CDKN2A, UBF1 E6/E7 up-regulates B23, important for proliferation and inhibition of differentiation; E7 stimulates UBF-1-mediated rDNA gene transcription McCloskey et al. (2010); Dichamp et al. (2014) Parvoviridae Nucleus AAV2 AAP, VP3 n.d. Viral capsid assembly occurs in the nucleolus Sonntag et al. (2010) Retroviruses Retroviridae Nucleus/cytoplasm HIV-1 Tat, Rev hRIP Trafficking of intronless viral mRNA from the nucleus occurs via the nucleolus Michienzi et al. (2000); Sanchez-Velar et al. (2004) AAV2, adeno-associated virus 2; BDV, Borna disease virus; EBV, Epstein–Barr virus; EMCV, encephalomyocarditis virus; EV71, enterovirus 71; HCMV, human cytomegalovirus; HPV, human papillomavirus; HVS, herpes virus saimiri; IBV, avian infectious bronchitis virus; n.d., not determined; PRV, pseudorabies virus. Although it has not been immediately apparent why viruses that replicate their genome entirely in the cytoplasm, typically RNA viruses, would target the nucleolus, numerous reports are now available describing interactions of proteins from cytoplasmic viruses with nucleoli/nucleolar components. It has been noted that many of the RNA virus-expressed proteins reported to target nucleoli are capsid proteins (also known as core/nucleoprotein/nucleocapsid), which bind to the viral RNA to form nucleocapsid structures (Matthews et al., 2011); these proteins often can also bind cellular RNA and many are small enough to enter the nucleus by diffusion through the NPC. It has thus been suggested that nucleolar localization could relate to a non-specific binding to the RNA dense, membrane-less nucleolus, with questionable functional significance. However, it would seem likely that a rapidly evolving RNA virus would quickly take advantage of this fortuitous localization to a central cellular regulator in order to usurp one or more of its functions, and several clear examples of important nucleolar functions of capsid proteins have been described. Importantly, a significant number of cytoplasmic RNA viruses have now been reported to express proteins that undergo specific trafficking between the nucleus and cytoplasm using virus-encoded NLSs and nuclear export sequences to interact with the cellular nuclear trafficking machinery, and thereby target the nucleolus, suggestive of the evolution of highly regulated mechanisms to interact with this compartment distinct from cytoplasmic sites of replication (Fazakerley et al., 2002; Mori et al., 2005; Pei et al., 2008). Furthermore, while many capsid proteins of positive stranded RNA (+ssRNA) viruses target nucleoli, a number of other proteins of +ss and −ssRNA viruses also target nucleoli, including P3 protein of the rabies virus (RV; Rhabdoviridae family) and the non-structural nsP2 protein of Semliki Forest virus (SFV; Togaviridae family) (Fazakerley et al., 2002; Oksayan et al., 2015). Thus, nucleolar targeting by cytoplasmic viruses appears to be a complex and selective process. Viral control of nucleolar function in cellular stress responses, the cell cycle and apoptosis Among the most significant recently elucidated nucleolar functions are in stress sensing, regulation of the cell cycle and apoptosis (Boulon et al., 2010; James et al., 2014; Tsai and Pederson, 2014). A primary mediator of these interconnected stress response processes is the tumour suppressor protein, p53, which when activated can trigger cell cycle arrest, senescence or apoptosis. Under normal conditions, p53 levels are low due to the E3 ubiquitin ligase ability of mouse double minute 2 homolog (MDM2 or HDM2) that marks p53 for proteasomal destruction (Kruse and Gu, 2009). Early studies showed that disruption of nucleoli (e.g. through ultraviolet irradiation to induce DNA damage) stabilizes p53 to induce cell cycle arrest by activation of the cell cycle inhibitor/apoptotic inhibitor, p21 (Rubbi and Milner, 2003). The primary link between the nucleolus and p53 activation is thought to be via nucleolar factors that inhibit MDM2 function, including cyclin-dependent kinase inhibitor 2A (CDKN2A or ARF). CDKN2A localizes to the nucleolus through its interaction with the core/resident protein, B23, and is released from the nucleolus under stress conditions, resulting in binding to nuclear MDM2 and inhibition of its p53 ubiquitin ligase activity (Kruse and Gu, 2009; Lee and Gu, 2010). Cell cycle, apoptosis and cell stress responses are common targets of viruses as their manipulation can induce a cellular environment more conducive to viral production and/or propagation and enable inhibition/evasion of cellular antiviral responses (Whelan, 2013; Bagga and Bouchard, 2014). The +ssRNA flavivirus family member, West Nile virus (WNV), directly antagonizes the p53/MDM2 pathway via its nucleolar-localizing capsid protein. WNV induces apoptosis in several cell lines, as well as in mouse brain and skeletal muscles (Parquet et al., 2001; Yang et al., 2002; Chu and Ng, 2003). Capsid was shown to bind to MDM2 and mediate its sequestration to the nucleolus, thereby preventing MDM2-mediated p53 ubiquitination to stabilize p53 and induce p53-mediated apoptosis (Yang et al., 2008). Consistent with this, WNV was less pathogenic in p53-null mouse embryonic fibroblasts or p53-knockdown SH-SYS5 cells. The core protein of another flavivirus, hepatitis C virus (HCV), is capable of targeting nucleoli (Falcon et al., 2003), and in transiently transfected cells, core can bind p53 and increase its DNA-binding activity, thereby increasing the expression of p21 (Otsuka et al., 2000). Furthermore, transiently expressed HCV core protein can induce protein kinase R (PKR)-dependent apoptosis, with a truncated core protein shown to enhance PKR translocation into nucleoli (Realdon et al., 2004), although whether this occurs during a viral infection is not yet clear. However, this may suggest that core activation of p53 may be mediated through PKR, as p53 is a component of the PKR apoptotic pathway (Yeung and Lau, 1998). Antagonism of the MDM2/p53 pathway has also been reported for the −ssRNA nuclear-replicating virus influenza virus A (IAV, family Orthomyxoviridae) nucleoprotein (NP), which encapsidates the viral genome and is known to enter the nucleoli (Davey et al., 1985; Emmott et al., 2010a). NP impaired MDM2-mediated p53 ubiquitination by associating with p53 to prevent interaction with MDM2 (Wang et al., 2012). IVA non-structural protein 1 (NS1) also enters the nucleolus (Melen et al., 2007; 2012; Emmott et al., 2010a) and associates with p53 (Terrier et al., 2013) but, in contrast to NP, inhibits p53-mediated transcriptional activity and apoptosis (Wang et al., 2010b). Thus, it appears that IVA might mediate a complex-regulatory interplay with the nucleolus/p53 to regulate apoptosis, possibly to inhibit antiviral apoptosis during early stages of infection, while inducing proapoptotic effects late in infection to facilitate viral spread. Many viruses manipulate/induce arrest of the cell cycle, probably to allow access to cellular factors/conditions associated with specific stages of the cell cycle (Bagga and Bouchard, 2014). The +ssRNA alphavirus M1 (M1; Togaviridae family) induces S-phase arrest and subsequent apoptosis in malignant glioma cells (Hu et al., 2009), which was suggested to relate to down-regulation of p21 protein. Intriguingly, p21 was also mislocalized to the nucleolus in M1-infected cells, where it bound to B23. The precise role of this nucleolar localization remains unclear but it is tempting to speculate that the nucleoli may be used as inhibitory platforms to provide an additional level of efficient down-regulation of p21 function, whereby nucleolar targeting of p21 affects the sequestration out of the nucleoplasm where its anti-apoptotic functions are mediated. Alternatively, the nucleolus may facilitate virus-induced p21 ubiquitination to affect p21 degradation, as several ubiquitin ligases localize to the nucleolus (Mekhail et al., 2005). How M1 virus affects p21 mislocalization is not yet clear, although the capsid and nsP2 proteins of another alphavirus, SFV, have been reported to localize to the nucleolus (Michel et al., 1990; Rikkonen et al., 1992), suggesting that nucleolar localization of viral proteins may be important. Viral modulation of host cell transcription and translation Many viruses modify host transcription pathways to facilitate viral replication/propagation by inhibiting antiviral responses and allowing access to higher levels of cellular resources (Lyles, 2000). In some cases, this is achieved by global shutdown of cellular transcription through mechanisms involving nucleolar targeting. The +ssRNA virus poliovirus (PV; Picornaviridae family) shuts off host–cell transcription early in infection (Weidman et al., 2003). This is achieved at several levels through inhibition of RNA polymerases (RNA Pol) I, II and III. Inhibition of RNA Pol I, which synthesizes rRNA in the nucleolus, is achieved by the PV protease, 3Cpro, through modification and inactivation of factors essential for rRNA transcription, upstream binding factor (UBF) and selectivity factor 1 (SL1) possibly involving direct protein cleavage (Banerjee et al., 2005). Targeting of host transcription appears to be a general phenomenon among picornaviruses (Chase and Semler, 2012), and several picornavirus proteins are known to localize to the nucleolus (Table 1). Proteins 2A, as well as 3BVPg, 3Cpro and 3Dpol (all of which are formed by autoproteolysis of a precursor protein 3BCD) of the picornavirus encephalomyocarditis virus localize to the nucleolus at early times post-infection (Aminev et al., 2003b). Nucleolar targeting of 2A and the precursor protein 3BCD (which may be mediated by interaction of 3BCD with B23) have been linked to inhibition of RNA Pol II-mediated mRNA transcription, but not rRNA synthesis; nevertheless, rRNA was reduced during infection, suggestive of additional mechanisms of inhibition. 2A nucleolar targeting has also been linked to inhibition of cap-dependent mRNA translation, representing an additional level of control over cellular gene expression (Aminev et al., 2003b). The precursor proteins 3CD and 3CD′ of the protease 3Cpro of the picornavirus human rhinovirus 16 (RV16) also localize to the nucleoli of the infected cells, which has been linked to the cleavage of Octamer-Binding Transcription Factor 1 (OCT-1) that is implicated in the shutdown of cellular transcription (Amineva et al., 2004). Although elucidation of the precise molecular mechanisms of the shutdown of cellular transcription and translation by picornaviruses awaits further research, the nucleolus is clearly a primary interface in this process. In contrast to the examples mentioned earlier, a number of viruses have been shown to activate transcription by mechanisms involving interaction with the nucleolus (Fig. 1 and Table 1). HCV core protein activates RNA Pol I (Kao et al., 2004) by enhancing the binding of UBF and RNA Pol I to the rRNA promoter. The core appears to become integrated into the RNA Pol I multiprotein complex via association with SL1 through direct interaction with the SL1 component, TATA-box binding protein (TBP). TBP is involved in transcription of all three RNA Pols, regulated through TBP-association factors, and it is believed that through interaction with TBP, the core is also able to activate RNA Pol II and III. This has potential implications for the role of core in cell growth and proliferation (Moriya et al., 1998), and possibly in liver cancer disease caused by HCV infection (Kao et al., 2004). The role of nucleoli in this process is indicated not only because rRNA transcription occurs in the nucleolus, but also because core protein can localize to the nucleolus potentially through its interaction with the major nucleolar protein B23 (Mai et al., 2006). Notably, the interaction of HCV core with B23 is also implicated in relieving the transcriptional repression on B23 expression that is mediated by YY1, a transcription factor that regulates many promoters (Mai et al., 2006). This occurs through the formation of a complex of core, B23, YY1 and p300 (a chromatin-modifying enzyme), which binds to the YY1-responsive element of B23. B23 has roles in ribosome biogenesis and transport, among other functions, and its overexpression is often associated with cellular proliferation and transformation (Okuwaki, 2008). Thus, HCV core may have multiple distinct roles in effecting proliferation of HCV-infected cells and liver cancer development. Direct roles of nucleolar components in the viral life cycle Distinct from roles of viral protein targeting of nucleoli in modifying host cell functions, several cytoplasmic viruses directly recruit/exploit specific nucleolar components to facilitate/medi