Cellular Microbiology (2015) 17(8), 1108–1120

doi:10.1111/cmi.12465 First published online 26 June 2015

Microreview The nucleolar interface of RNA viruses Stephen M. Rawlinson and Gregory W. Moseley* Viral Pathogenesis Laboratory, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Australia 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 positivestranded 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

Received 16 April, 2015; revised 27 May, 2015; accepted 1 June, 2015. *For correspondence. E-mail gregory.moseley@ unimelb.edu.au; Tel. (+61) 83442288; Fax (+61) 93481421.

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.

© 2015 John Wiley & Sons Ltd

cellular microbiology

Nucleolar targeting of RNA 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). 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 © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1108–1120

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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. Although it has not been immediately apparent why viruses that replicate their genome entirely in the cytoplasm, typically RNA viruses, would target the nucleolus,

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S. M. Rawlinson and G. W. Moseley –ssRNA

+ssRNA

Cyto Cy topl plas asm m Entry

JEV: Nucleolin

Nucl Nu cleu eus s

HPIV-3: Nucleolin

Entry

Nucl Nu cleo eolu lus s

PV: Nucleolin

Host transcription shutdown PV 3Cpro : UBF, SL1

First transcription

Cell cycle arrest

Translation

Translation

M1 virus : p21 NDV M : B23 RABV P3 : nucleolin Hendra/Nipah M : several proteins? Apoptosis HCV core : PKR, p53

Replication

JEV core: B23

? ? Replication

Cell growth/ proliferation HCV core : SL1, TBP

Assembly/ Release

Assembly/ Release DV: Nucleolin

FC DFC GC

WNV:DDX56

Fig. 1. 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.

© 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1108–1120

Replication site

Cytoplasm

Cytoplasm Cytoplasm

Picornaviridae

Togaviridae Coronaviridae

© 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1108–1120

Cytoplasm Nucleus Nucleus

Rhabdoviridae

AAV2 HIV-1

Nucleus

Nucleus/ cytoplasm

Parvoviridae Retroviruses Retroviridae Tat, Rev

AAP, VP3

E6, E7

hRIP

B23, CDKN2A, UBF1 n.d.

Nucleolin Nucleolin

hTREX n.d.

Several – strain specific (e.g. ADAR1)

Trafficking of intronless viral mRNA from the nucleus occurs via the nucleolus

Nucleolus required for viral mRNA nuclear export Mutation of the NoLS affects viral gene expression, DNA synthesis and viral production Nucleolin required to maintain the architecture of replication compartments Nucleolin binding to EBNA-1 important for EBNA-1 role in EBV episome binding, maintenance and transcription E6/E7 up-regulates B23, important for proliferation and inhibition of differentiation; E7 stimulates UBF-1-mediated rDNA gene transcription Viral capsid assembly occurs in the nucleolus

Infection induced significant changes to nucleolar proteome

M localizes to nucleolus early during infection; B23 redistributed to nucleoplasm late in infection; B23 is important for replication and cytopathic effect Nucleolin acts as cellular receptor for entry Nucleolin expression required for virus production Site of RNA transcription and replication Inhibits p53 activity and apoptosis; nucleolar targeting differs between viral subtypes

M protein of a number of paramyxoviruses localizes to the nucleolus; proteomic data indicate binding to several nucleolar proteins

Cell surface-expressed nucleolin required for efficient entry into lung cells

Nucleolin stimulates viral IRES-mediated translation Shutdown of RNA Pol I transcription Shutdown of RNA Pol II transcription, and cap-dependent translation Shutdown of cellular transcription Facilitates virus entry Induces S-phase arrest and apoptosis Nuclear/nucleolar localization linked to replication/pathogenesis Cell cycle-dependent nucleolar localization; affects nucleolar morphology

Michienzi et al. (2000); Sanchez-Velar et al. (2004)

Sonntag et al. (2010)

McCloskey et al. (2010); Dichamp et al. (2014)

Strang et al. (2010; 2012); Bender et al. (2014) Chen et al. (2014)

Boyne and Whitehouse (2006) Li et al. (2011)

Emmott et al. (2010a)

Tayyari et al. (2011) Oksayan et al. (2015) Pyper et al. (1998) Melen et al. (2007; 2012); Wang et al. (2010b; 2012)

Peeples et al. (1992); Duan et al. (2014)

Pentecost et al. (2015)

Bose et al. (2004)

Waggoner and Sarnow (1998); Izumi et al. (2001) Weidman et al. (2003) Aminev et al. (2003a,b) Amineva et al. (2004) Su et al. (2015) Hu et al. (2009) Yoo et al. (2003); Lee and Gu (2010) Dove et al. (2006); Cawood et al. (2007)

Kao et al. (2004) Otsuka et al. (2000); Realdon et al. (2004) Yu et al. (2005) Hirano et al. (2003); Shimakami et al. (2006); Kusakawa et al. (2007)

Tsuda et al. (2006) Mai et al. (2006)

Yang et al. (2008) Xu et al. (2011); Xu and Hobman (2012) Mori et al. (2005)

References

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.

HPV

UL44, UL84 EBNA-1

HCMV EBV

Nucleus

ORF57 UL54

Papillomaviridae

B23

M Nucleolin Nucleolin n.d. Nucleolin, Fibrillarin

Several unconfirmed

M

G P3 N, M NS1 PB2, HA, NP, M1, NS1, NS2

Nucleolin

F

Nucleolin UBF, SL1 B23 OCT-1 Nucleolin p21 Fibrillarin n.d.

HCV IRES NS5B 3’NCR 3Cpro 2A, 3BCD 3CDpro VP1 n.d. N N

Regulates transcriptional pathways; promotes cell growth and proliferation Core protein induces apoptosis Role in IRES-mediated translation initiation Interaction of NS5B and nucleolin critical for replication

SL1, TBP PKR, p53 Nucleolin Nucleolin

HVS PRV

Nucleus

RSV RV BDV IAV

HPIV-3 Hendra, Nipah, others NDV

EMCV RV16 EV71 M1 PPRSV IBV

PV

B23 important for viral replication Activates transcription of B23

B23 B23, YY1

HCV

Core

Capsid

JEV

Functional data

Capsid activates p53-mediated apoptosis Role in virus assembly Nuclear/nucleolar targeting linked to pathogenesis in mice

Host protein involved/targeted

MDM2 DDX56 n.d.

Capsid

Viral factor

WNV

Virus

Herpesviridae

DNA viruses

Orthomyxoviridae

Cytoplasm

Paramyxoviridae

RNA, negative-stranded

Cytoplasm

Flaviviridae

RNA, positive-stranded

Family

Table 1. Selected virus–nucleolar interactions with related functional significance/insights.

Nucleolar targeting of RNA viruses 1111

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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 virusencoded 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 p53mediated 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 complexregulatory interplay with the nucleolus/p53 to regulate © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1108–1120

Nucleolar targeting of RNA viruses 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., © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1108–1120

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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.

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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/mediate essential steps of the viral life cycle; examples of this have been reported at almost all stages of the infectious cycle (see Fig. 1 and Table 1). Among these, the core proteome of the flavivirus, Japanese encephalitis virus (JEV), mislocalizes B23 from nucleoli to the cytoplasm, with overexpression of a truncated ‘dominant negative’ form of B23 able to impair JEV replication (Tsuda et al., 2006). The nucleolar helicase, DDX56, has been shown to interact with the capsid protein of WNV and to become relocalized from the nucleolus to the cytoplasm in infected cells (Xu et al., 2011); this appears to be related to viral particle assembly, for which helicase activity of DDX56 was essential (Xu and Hobman, 2012). However, the best characterized nucleolar protein implicated in facilitation of viral life cycles is nucleolin.

Direct roles of nucleolin in the life cycle of RNA viruses Nucleolin is one of the best characterized and most abundant nucleolar proteins and localizes principally to the nucleolus, although it can also be detected in the nucleoplasm, cytoplasm and cell surface (Mongelard and Bouvet, 2007). Due to its strong nucleolar localization, early studies of nucleolin focused on roles in ribosome biogenesis; however, it is now clear that nucleolin is highly multifunctional, due in part to its ability to bind both RNA and DNA, and is involved in processes including transcription, ribosome assembly, mRNA stability, nucleocytoplasmic transport, DNA replication, telomere maintenance and chromatin remodelling (Mongelard and Bouvet, 2007; Abdelmohsen and Gorospe, 2012). This multifunctionality is likely to account for its apparently diverse roles in the viral life cycle. This includes a number of clear examples among DNA viruses, including roles in replication and transcription (see summaries in Table 1), while it is clear that it is also important at many stages of RNA virus life cycles (Fig. 1). Cell entry. Cell surface-expressed nucleolin is involved in cell entry by a number of RNA viruses. It was reported to have co-receptor activity for diverse cytoplasmic viruses including the paramyxovirus human parainfluenza virus type 3 (HPIV-3) (Bose et al., 2004); the picornavirus, enterovirus 71 (EV71) (Su et al., 2015); Crimean-Congo haemorrhagic fever virus (Bunyaviridae family) (Xiao et al., 2011); and the flavivirus, JEV (Thongtan et al., 2012). The paramyxovirus, respiratory syncytial virus was reported to use nucleolin as a cellular receptor, which is bound by the

viral envelope glycoprotein (G) (Tayyari et al., 2011). As diverse viruses have been reported to utilize nucleolin for entry, it has been proposed to represent a promising target for therapeutic intervention. Supporting this idea, infection by the retrovirus human immunodeficiency virus (HIV), which uses nucleolin for cell attachment (Nisole et al., 1999), was inhibited by treatment of cells with a non-toxic pseudopeptide that prevents HIV–nucleolin interaction at the plasma membrane (Nisole et al., 2002). The effectiveness of this approach for inhibition of infection by other viruses, however, is currently unclear. Viral translation/replication. Nucleolin has been implicated in the replication of several cytoplasmically replicating viruses, which is generally considered to relate to its RNA-binding activity. In PV, nucleolin binds to the 3′ noncoding region (NCR) of the RNA genome and is required early in infection when it is redistributed to the cytoplasm in infected cells, indicative of specific viral mechanisms for sequestration/recruitment (Waggoner and Sarnow, 1998). Nucleolin also stimulates the PV internal ribosome entry site (IRES); the use of IRESs is a common viral strategy to mediate cap-independent translation initiation, which for PV allows translation of viral proteins even when host cell translation is shut-off by the virus (Izumi et al., 2001). Similarly, nucleolin enhances the activity of the HCV IRES, with which it associates (Yu et al., 2005). Furthermore, nucleolin interacts with the HCV polymerase, NS5B (Hirano et al., 2003), and is redistributed to the perinuclear regions; although the precise mechanistic outcomes are unclear, the interaction was shown to be indispensible for HCV replication (Shimakami et al., 2006; Kusakawa et al., 2007). Viral assembly/egress. Roles of nucleolin in virion assembly and egress have been reported for the flavivirus, dengue virus (DV). DV capsid protein interacts with and colocalizes with nucleolin in the nucleoli of transfected cells, and knockdown of nucleolin or treatment with a nucleolin-binding aptamer reduced viral titers (Balinsky et al., 2013). As viral protein and RNA levels were unaffected, this implies specific role(s) in virion assembly/ release. The precise function of nucleolin in DV assembly is not known, although it has been speculated that it may act as a chaperone that assists capsid in forming stable nucleocapsids, as particle stability was affected following treatment with the nucleolin aptamer (Balinsky et al., 2013). Nucleolar targeting by negative-sense cytoplasmic RNA viruses Until recently, reports of cytoplasmic virus targeting of the nucleolus has been largely restricted to +ssRNA viruses, © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1108–1120

Nucleolar targeting of RNA viruses with examples of −ssRNA viruses mostly limited to the nuclear-replicating viruses influenza A virus and Borna disease virus (Bornaviridae family) (BDV) (Pyper et al., 1998). However, a number of recent studies have indicated that cytoplasmic −ssRNA viruses can target and, potentially, subvert nucleolar functions/proteins (Fig. 1 and Table 1). The P3 protein, a truncated isoform of the phosphoprotein (P) of RV can access the nucleus via a highly regulated NLS in the N-terminal region (Oksayan et al., 2015) and subsequently localizes to the nucleolus via a distinct sequence in the C-terminal domain, suggestive of a distinct NoLS. Full-length P protein, P3 and the C-terminal domain alone could interact with nucleolin, and nucleolin knockdown inhibited viral protein expression and virus production (Oksayan et al., 2015) indicative of roles either in viral modulation of cell biology or direct functions in the life cycle. The matrix (M) protein of the paramyxovirus Newcastle disease virus (NDV) localizes to the nucleolus through an interaction with B23, and knockdown of B23 results in reduced cytopathic effect and inhibition of replication (Duan et al., 2014). It is now becoming clear that M proteins of a number of paramyxoviruses can localize to nucleoli in infected cells, including the highly lethal viruses, Nipah and Hendra as well as Sendai and Mumps viruses (Pentecost et al., 2015). Furthermore, proteomic analysis has identified interaction of paramyxovirus M proteins with several host nucleolar proteins (Sun et al., 2014; Pentecost et al., 2015). Although many of these interactions require further validation and functional characterization, these data clearly suggest that the nucleolus/ nucleolar proteins have important roles in infection by diverse cytoplasmic −ssRNA viruses. Mutant viruses deficient in the nucleolar interface: roles in pathogenesis A genuine appreciation of the roles of nucleoli/nucleolar proteins in viral infection and disease requires the generation of viruses containing mutations specifically affecting the nucleolar interface. Only a few such studies have been described, most likely because the identification of mutations able to impact nucleolar interactions without impacting other functions is challenging, particularly as many nucleolar viral proteins including capsid, M and P proteins are multifunctional with critical roles in processes such as viral replication/structure, assembly and innate immune evasion. In addition, while reverse genetic systems are now available for many important animal viruses, they can often be technically challenging to work with (Condit, 2013), with the lack of suitable host model systems to analyse disease often restricting analysis. Nevertheless, two key studies have provided indications that nucleolar targeting is important in cytoplasmic virus infection and pathogenesis. © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1108–1120

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Porcine reproductive and respiratory syndrome virus (PPRSV) (family Coronaviridae) causes severe respiratory disease in pigs, and its nucleocapsid (N) protein localizes strongly to the nucleus, in particular the nucleolus, requiring an Arg/Lys-rich region, suggesting that this region represents a NLS/NoLS sequence (Rowland et al., 2003). Viruses generated by reverse genetic approaches in which nuclear/nucleolar localization of nucleocapsid was impaired by mutation of this sequence displayed 100-fold reduction in viral titers in culture and showed significantly reduced viremia in the natural host (pigs) with significantly higher neutralizing antibody titers compared with wild type, indicating that nuclear/nucleolar localization may be important to evasion of host immune responses (Lee et al., 2006). Importantly, all reversion mutants observed included mutations in the NLS/NoLS sequence that restored N nuclear and nucleolar targeting, clearly linking these processes to pathogenesis. The core protein of JEV localizes strongly to the nucleolus dependent on residues Gly-42 and Pro-43 (Mori et al., 2005). Mutation of these residues to Ala blocked nuclear/ nucleolar core targeting and resulted in impaired replication and protein synthesis in infected mammalian cells, but, intriguingly, had no effect in mosquito cells. Neurovirulence in mice infected intracerebrally or intraperitoneally remained similar to wild type but neuroinvasiveness was substantially affected (Mori et al., 2005). Thus nucleolar targeting of the core appears to play an important host-specific role in infection, significant to pathogenesis and encephalitis. While these studies are suggestive, it should be noted that in both cases the mutations affected nucleolar and nuclear localization; as many NoLSs closely associate/ overlap with NLSs, this presents an additional challenge to direct analysis of the roles of the nucleolar interface in infection. Nevertheless, there are instances where the NLS and NoLS have been found to be physically/ functionally separate, enabling analysis of the distinct roles of nuclear and nucleolar targeting. In the case of the DNA virus, pseudorabies virus (Herpesviridae family), the NLS and NoLS sequences of UL54 protein were found to be independent and such that mutagenic analysis enabled the identification of a specific role for nucleolar interaction, whereby NoLS mutation reduced viral production (Li et al., 2011). Similar studies have not yet been performed for cytoplasmic RNA viruses and await further detailed molecular analysis of the specific sequences involved. Proteomics approaches to elucidate the cytoplasmic virus–nucleolar interface The majority of research into the virus–nucleolar interaction has used classical methods to investigate specific

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interactions of viral proteins with nucleoli or nucleolar components. However, several recent studies have begun to apply nucleolar proteomics approaches to perform global, non-biased investigation of nucleoli during viral infection, including the nuclear-replicating viruses human adenovirus (Lam et al., 2010) and IAV (Emmott et al., 2010a), and the cytoplasmic avian infectious bronchitis virus (IBV, family Coronaviridae) (Emmott et al., 2010b,c). Given the profound impact that viral infection can have on cellular biology, including the activation of signalling networks, regulation of transcription, translation and cell cycle, similar extensive changes to those observed in response to stimuli such as DNA damage and inhibition of rRNA production might be expected (Andersen et al., 2005). Intriguingly, however, relatively limited changes have been observed, with only small fractions of the nucleolar proteome showing significant changes in abundance. For example, only 7% of nucleolar proteins showed a twofold change in adenovirus-infected cells, whereas 30% of proteins were significantly altered by inhibition of rRNA synthesis by actinomycin D. Notably, many of the changes observed in virus-infected cells differ between viruses, and even between different strains of the same virus (Emmott et al., 2010a). In cells infected by human H1N1 strain (PR8), which is regarded as a highly laboratory-adapted strain, only four viral proteins were identified in nucleolar fractions (HA, NP, M1 and NS1) and only three host proteins showed a >2-fold change in nucleolar abundance. In contrast, in cells infected by the human H3N2 strain (Udorn), which retains characteristics of low passage clinical isolates (e.g. filamentous morphology), six viral proteins were detected in nucleolar fractions (PB2, HA, NP, M1, NS1 and NS2) and 21 nucleolar proteins showed a >2-fold change in abundance. Nucleolar abundance of two host proteins were significantly affected by both strains, although the abundance of one of these, Lupus La, was increased in cells infected by PR8 and decreased in cells infected with Udorn. Thus, these early studies indicate that viral changes to the nucleolus are not due to general effects on cells by viral infection and are instead consistent with the idea that viruses selectively target specific components/pathways in the nucleolus. Furthermore, the data for the IVA strains suggest that the quantitative and qualitative nature of the interaction with nucleoli and effects on the nucleolar proteome might correlate with pathogenicity. As more studies become available, it will be of great interest to determine how diverse the effects of different viruses on nucleoli are and how these effects might correlate with specific families or genera, or strains of differing pathogenicity. Increased knowledge of how viruses affect the molecular composition of nucleoli, the nucleolar interactome of viral proteins and the corresponding effects on cellular functions should provide key insights into the molecular mechanisms of

disease and may also provide greater understanding of the molecular biology of nucleoli. Perspectives We are now moving beyond the phenomenological observations of early studies of virus–nucleolar interactions, with recent research beginning to demonstrate critical roles in infection by a range of different viruses. Although these studies are providing tantalizing clues as to the importance of this emerging virus–host interface, understanding of the precise significance of the interactions is still very much in its infancy. The application of quantitative proteomics has highlighted the value of systems biology approaches to elucidate the biology of nucleoli, with analyses of viral infection providing key indications that there exists a highly specific relationship between viruses and the nucleolar proteome. Genuine appreciation of the functional importance of the interface will require the combination of systems approaches, with classical virology and molecular biology techniques. In particular, the identification of mutations able to specifically modify viral protein–nucleolar interactions is of great importance to evaluating the roles of the interface in cellular processes of infection, as well as in diseases in animal models. Such studies should validate the critical importance of nucleolar interaction in infection, as well as demonstrating the potential of these interactions as targets for the development of novel antivirals and attenuated vaccines. Initial studies have supported this idea, including through the development of potential therapeutics for HIV, which inhibit an essential nucleolar step in trafficking of unspliced HIV RNA from the nucleus (Michienzi et al., 2000). A more comprehensive analysis of viral interactions with the nucleolus should provide new targets for interventions, including through the identification of virus-specific interfaces and, potentially, commonly targeted nucleolar factors/pathways, which could provide opportunities for broad-spectrum antiviral approaches. References Abdelmohsen, K., and Gorospe, M. (2012) RNA-binding protein nucleolin in disease. RNA Biol 9: 799–808. Ahmad, Y., Boisvert, F.M., Gregor, P., Cobley, A., and Lamond, A.I. (2009) NOPdb: nucleolar proteome database – 2008 update. Nucleic Acids Res 37: D181–D184. Aminev, A.G., Amineva, S.P., and Palmenberg, A.C. (2003a) Encephalomyocarditis viral protein 2A localizes to nucleoli and inhibits cap-dependent mRNA translation. Virus Res 95: 45–57. Aminev, A.G., Amineva, S.P., and Palmenberg, A.C. (2003b) Encephalomyocarditis virus (EMCV) proteins 2A and 3BCD localize to nuclei and inhibit cellular mRNA transcription but not rRNA transcription. Virus Res 95: 59–73. © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1108–1120

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