The nucleolus and herpesviral usurpation

The nucleolus is a distinct subnuclear compartment known as the site for ribosome biogenesis in eukaryotes. Consequently, the nucleolus is also proposed to function in cell-cycle control, stress sensing and senescence, as well as in viral infection. An increasing number of viral proteins have been found to localize to the nucleolus. In this article, we review the current understanding of the functions of the nucleolus, the molecular mechanism of cellular and viral protein targeting to the nucleolus and the functional roles of the nucleolus during viral infection with a specific focus on the herpesvirus family.


Overview
The nucleolus is a large, dynamic subnuclear structure present in eukaryotes and its primary function is rRNA synthesis and ribosome biogenesis.Nucleoli disassemble during cell division and reform at the end of mitosis around tandemly repeated clusters of rRNA genes, which are known as nucleolar organizing regions.rRNA is synthesized and processed in the nucleolus with the help of RNA polymerase I (RNA pol I) (Caburet et al., 2005;Dimario, 2004;Leung et al., 2004;Olson & Dundr, 2005).Electron microscopy analysis has shown that the nucleolus can be divided into at least three different regions: a fibrillar centre (FC), a dense fibrillar component (DFC) and a granular component (GC) (Boisvert et al., 2007;Hernandez-Verdun, 2006).The FC contains the transcription factor upstream binding factor (UBF) and RNA pol I.The rDNA is transcribed at the border between the FC and the DFC (Cmarko et al., 2008;Raska et al., 2006).The DFC is usually associated with and surrounds the FC, within which pre-rRNA and small nucleolar ribonucleoproteins (snoRNPs) are accumulated and the post-transcriptional processing and modification takes place.Maturation of pre-rRNA in the DFC leads to formation of 18S, 5.8S and 28S rRNA (Ba ´rtova et al., 2010).The DFC contains fibrillarin, an RNA methyl-transferase, and nucleolin, a protein that has multiple roles in nucleolar and cellular biology (Mongelard & Bouvet, 2007).The GC, surrounding both the FC and the DFC, is the region where the late RNA processing takes place, such as pre-ribosomal particle maturation and ribosomal subunit synthesis and transportation (Hernandez-Verdun, 2006;Olson & Dundr, 2005).
Well-documented studies have established that nucleoli participate in gene silencing, senescence, cell-cycle regulation, stress responses and innate immune responses.In addition, numerous viral components have been demonstrated to target the nucleolus, manipulating the cell cycle and triggering apoptosis and the innate immunity of host cell.The discovery of this phenomenon provides a possible link between viral components and viral evasion from host innate immune responses, providing an impetus for further investigation (Boulon et al., 2010;Hernandez-Verdun, 2011;Pederson, 2011).

The multifunctional nucleolus
The nucleolus contains a number of nucleolar proteins, leading to a high degree of functional complexity that is restricted to the biosynthesis of ribosomes (Andersen et al., 2005;Andersen et al., 2002;Scherl et al., 2002).Recently, proteomic studies have shown that the nucleolus contains over 4500 identified proteins (Ahmad et al., 2009;Andersen et al., 2005;Boisvert et al., 2010;Leung et al., 2006).These proteins were found to be involved in cellcycle regulation, stress-specific responses, DNA processing and DNA damage response and repair, regulation of tumour suppressor and oncogene activities, signal recognition particle assembly, modification of small RNAs, control of ageing, senescence and modulation of telomerase function (Boisvert et al., 2007;Grisendi et al., 2006;Kar et al., 2011;Olson & Dundr, 2005;Ruggero & Pandolfi, 2003), as well as ribosome subunit production (Moore et al., 2011).
Small ubiquitin-like modifier (SUMO) modification is reported to affect recruitment of transcription factors and chromatin-modifying enzymes, often leading to transcriptional repression (Gill, 2004).Recently, a quantitative proteomics screen, using stable isotope labelling of amino acids in cell culture (SILAC) and mass spectrometry, revealed that the nucleoli contained some snoRNP proteins, specifically Nop58, Nhp2 and DKC1 as the substrates of SUMO1 or SUMO2 (Westman & Lamond, 2011).Together with the nucleolar targeting of SUMOspecific proteases SENP3 and SENP5 (Gong & Yeh, 2006;Nishida et al., 2000) and the co-localization of SUMO-1 and UBF in the GFC of neuronal cells (Casafont et al., 2007), this suggests a potential role of SUMOylation in the regulation of rDNA transcription, ribosome assembly and, thus, cellular translation, growth and proliferation; however, the molecular mechanisms require further investigation (Sirri et al., 2008;Westman & Lamond, 2011).
Nucleolin, fibrillarin and B23 are three of the most abundant, well-understood and multifunctional proteins in the nucleolus.Nucleolin (first known as C23) represents ~10 % of the total nucleolar protein content and is a ubiquitous, multifunctional and mobile protein.Nucleolin shuttles between the nucleolus, nucleoplasm, cytoplasm and cell surface, plays roles in interaction with viruses at the cellular membrane and regulates gene expression, chromatin remodelling, DNA recombination and replication, RNA synthesis by RNA pol I and II, rRNA processing, mRNA stabilization, cytokinesis and apoptosis (Calle ´et al., 2008;Mongelard & Bouvet, 2007;Storck et al., 2007).Nucleolin is modified by phosphorylation, methylation and ADP-ribosylation, which may enable it to target various compartments to exert different functions (Lo et al., 2006).Fibrillarin, a small nuclear RNP component with a highly conserved sequence, structure and function in eukaryotes, is a potential RNAbinding protein (Nicol et al., 2000).Fibrillarin is involved in many post-transcriptional processes including pre-rRNA processing, pre-rRNA methylation and ribosome assembly (Tollervey et al., 1993).B23 (also called NPM, nucleophosmin, NO38 or numatrin) is a highly conserved protein, which plays several important roles in ribosomal biogenesis in the nucleolus.Previous publications have proven that B23 has multiple functions, including ribosome assembly, binding to other nucleolar proteins, nucleocytoplasmic shuttling and possibly regulating transcription of rDNA by mediating structural changes in chromatin (Hiscox, 2002).Recent studies have indicated that B23 is implicated in the p53 network by its interaction with the ARF tumour suppressor protein, a major upstream activator of p53 (Boulon et al., 2010;Lindstro ¨m & Zhang, 2006).The subcellular localization of B23 is also regulated by SUMOylation, which occurs in close proximity to its nucleolar localization signal (NoLS) motif (Liu et al., 2007) and can affect the role of B23 in 28S rRNA maturation (Emmott & Hiscox, 2009;Haindl et al., 2008).
Proteins that are present in the nucleolus also have functions involved in cell growth control, telomere maintenance and protein degradation, tumour suppression, and apoptosis.For example, cyclin phosphatase Cdc14 and nucleostemin, a nucleolar protein highly expressed in stem cells and cancer cells, are involved in cell-cycle progression and cell division.Nucleostemin regulates the cell cycle perhaps as a consequence of its interaction with a multitude of proteins, including p53, the murine double minute 2 protein (MDM2), telomeric repeat-binding factor 1 (TRF1), alternative reading frame (ARF), ribosomal L1-domain-containing protein 1 (RSL1D1, also known as cellular senescence-inhibited gene or CSIG), and B23 (Pederson & Tsai, 2009).N-acetyltransferase 10 (NAT10), which has been shown to play a role in maintaining or enhancing the stability of a-tubulin, also influences the cell cycle.The depletion of NAT10 induces defects in nucleolar assembly and cytokinesis and decreases acetylated a-tubulin, leading to G2/M cell-cycle arrest or delay of mitotic exit (Shen et al., 2009).PICT1 (also known as GLTSCR2) is a nucleolar protein that is essential for embryogenesis and ES cell survival; it is a potent regulator of the MDM2-P53 pathway and promotes tumour progression by retaining RPL11 in the nucleolus (Sasaki et al., 2011).This apoptosis repressor, which contains a caspase-recruitment domain (ARC, also known as NOL3), is also an anti-apoptotic protein originally found to be involved in apoptosis of cardiac cells (Carter et al., 2011).NOL-6 and some other nucleolar proteins were reported to be involved in innate immunity.Mutation or silencing of NOL-6 could increase the transcriptional levels of genes regulated by CEP-1, a p53 homologue in Caenorhabditis elegans.It has been reported that activation of innate immunity by the inhibition of nucleolar proteins requires p53/CEP-1 and its transcriptional target SYM-1 (Fuhrman et al., 2009).

Nucleolus targeting of cellular and viral proteins
The nuclear localization signal (NLS) is crucial for proteins to be transported across the nuclear membrane into the nucleus.The NLS motifs, which are well-characterized and moderately conserved, are usually abundant in basic amino acids or can be predicted by suitable algorithms (Cokol et al., 2000;la Cour et al., 2004).Dedicated transport systems interact with the NLS and carry the protein into the nucleus (Boulikas, 1993;Emmott & Hiscox, 2009).Unlike the nucleus, the nucleolus is a non-membranous subnuclear region where any soluble molecule can spread into and out of.Even so, many researchers have reported that nucleolar localized proteins contain some distinct short sequences, which have been identified as functional NoLSs (Emmott & Hiscox, 2009;Scott et al., 2010).For example, the amino acid sequence RSRKYTSWYVALKR of fibroblast growth factor 2 (Sheng et al., 2004), the amino acid sequence KKLKKRNK at (position 466-473) of MDM2 (Lohrum et al., 2000) and the RKKRKKK residues in nuclear factor-kB-inducing kinase (Birbach et al., 2004) have been identified as functional NoLSs.It has been reported that in human immunodeficiency virus-1 (HIV-1)-infected HeLa cells, the expression of the viral regulatory protein Rev, which governs viral gene expression, induces nucleoporins Nup98 and Nup214 and a significant fraction of the nuclear export factor chromosome region maintenance protein 1 (CRM1) to relocalize into the nucleolus (Michienzi et al., 2000;Zolotukhin & Felber, 1999).Some nucleolus proteomics experiments have indicated that some host proteins enter into and accumulate in the nucleolus.For example, nuclear pore complex component (NUP210), rPIK3-p87 (also known as PIK3R6) and ribosomal protein S15a were more than twofold enriched in adenovirus-infected cell nucleoli compared with uninfected cell nucleoli (Lam et al., 2010).
During infection, many viral proteins also target to the nucleolus.For example, the HIV-1 transactivator of transcription protein TAT contains a NoLS amino acid sequence of RKKRRQRRRAHQ, which targets the nucleolus (Siomi et al., 1990).Herpes simplex virus type-1 (HSV-1) infected cell protein 27 (ICP27) is suggested to be a nucleolar targeting protein and contains a relatively short sequence, mapping to residues 110-152, which functions as an NoLS and plays important roles in efficient viral RNA export and regulation of HSV-1 replication (Mears et al., 1995).Analysis carried out in our lab identified an argininerich domain (RPRRPRRRPRRR) as a functional NoLS in bovine herpesvirus-1 infected cell protein 27 (BICP27), which localizes predominantly in the nucleolus (Guo et al., 2009).Recently, a genuine NoLS of the pseudorabies virus early protein UL54, with the amino acid sequence RRRRGGRGGRAAR, was identified in our lab and its nucleolar localization was found to regulate viral gene expression and DNA synthesis.Recombinant viruses with mutations of the NoLS in UL54 displayed a defect in viral gene expression and DNA synthesis (Li et al., 2011).It was previously established that the US11 protein of HSV-1 contains unique XPR repeats, which are responsible for its retention within the nucleolus (Catez et al., 2002), and in our lab, Xing et al. (2010) identified that, for what is believed to be for the first time, amino acids 84-125, 126-152 and 89-146 of the US11 XPR repeats are able to target reporter proteins at the nucleolus.Post-translational modifications might also influence the activity of an NoLS.A notable example of this can be found in the NoLS of the HSV-1 US11 protein, in which a single proline-rich motif, mediating nucleolar localization of US11 protein, is regulated by phosphorylation (Catez et al., 2002).
HSV-1 ICP34.5, a viral neurovirulence-associated protein, mediates eukaryotic translation initiation factor 2 subunit a (eIF-2a) dephosphorylation and prevents the shut-off of protein synthesis mediated by dsRNA-dependent protein kinase PKR.The ICP34.5 protein distributes throughout the nucleus, the nucleolus and the cytoplasm in transfected or infected cells, functioning by blocking the cellular antiviral response, viral egress, glycoprotein processing, DNA replication and cell-cycle regulation.A NoLS of the ICP34.5 protein was identified and mapped to amino acids 1-16, consisting of a cluster of arginine residues (MARR-RRHRGPRRPRPP).The function of the nucleolar-targeting protein ICP34.5 needs further investigation (Cheng et al., 2002).
The herpesvirus saimiri ORF57 protein is a nucleolar targeting protein, which redistributes the host-cell human transcription/export (TREX) proteins leading to ORF57mediated viral mRNA nuclear export.Its NoLS consists of two distinct NLSs, which function in tandem to localize ORF57 to the nucleolus (Boyne & Whitehouse, 2006).A homologous ORF57 protein from Kaposi's sarcomaassociated herpesvirus (KSHV) has been described as presenting a similar characteristic.NLS1, in combination with either NLS2 or NLS3 are able to function as NoLSs, which target the ORF57 protein to the nucleolus.Disruption of the nucleolus by actinomycin D or 5,6dichloro-1-b-D-ribofuranosylbenzimidazole leads to a reduction of KSHV ORF57-mediated intronless mRNA export, revealing that the intact nucleolus is essential for KSHV ORF57 to efficiently export the intronless mRNA (Hiscox et al., 2010;Boyne & Whitehouse, 2009).
NoLSs are, thus, emerging as a predominant mechanism in the targeting of proteins to the nucleolus.Determination of NoLSs is one way to identify potential nucleolar localization proteins from the large number of host and viral proteins (Scott et al., 2011;Simpson et al., 2001).Although NoLSs commonly vary in context and in length and sometimes overlap with each other (Emmott & Hiscox, 2009), Scott et al. (2011) collated and statistically analysed 46 human nucleolar localization sequences and developed a web server, the Nucleolar Localization Sequence Detector (NoD), and a command line program that predicts the presence of NoLSs in eukaryotic and viral proteins (Scott et al., 2011) based on that database.Further investigation is required to clarify whether NoLS predictions using this method are practical.
Many factors can determine the nucleolar localization of a protein.For instance, soluble proteins smaller than 40-60 kDa can passively diffuse through the nuclear pore complex and into, or out of, the nuclear compartment.Some RNA binding-domain-containing proteins can be found in nucleoli where the rRNA is present.Many proteins, including host proteins and viral proteins, diffuse through the nuclear pore into the nucleoplasm and localize in the nucleolus, associating with nucleolar factors (Hiscox, 2002).Mutagenesis studies have demonstrated that the nucleocapsid (N) protein of infectious bronchitis virus presents a motif eight amino acids long, which functions as an NoLS and is necessary and sufficient for nucleolar retention of the N protein and colocalization with nucleolin and fibrillarin, i.e. the NoLS is required for interaction with cell factors (Reed et al., 2006;Sirri et al., 2008).

Viral proteins interact with the nucleolus
Many viral proteins interact with and/or alter the nucleolus.These are produced by RNA viruses, retroviruses and DNA viruses (Hiscox, 2002(Hiscox, , 2007;;Hiscox et al., 2010) and function as part of their infection strategy.It is believed that localization of the viral proteins to the nucleolus results from one of three different direct or indirect types of interactions with rDNA, nucleolar RNA (consisting mainly of rRNA) or nucleolar protein components (Carmo-Fonseca et al., 2000;Scott et al., 2011;Wang et al., 2010).Thus, the viruses take over host-cell functions and recruit nucleolar proteins to help with viral replication.The HIV-1 Rev protein also contains an NoLS and predominantly localizes to the nucleolus (Kubota et al., 1999); meanwhile, B23 stimulates nuclear import of the HIV-1 Rev protein and interacts with Rev (Fankhauser et al., 1991;Szebeni et al., 1997).It has been wellestablished that a complex assembled within the nucleolus, composed of Rev, two nucleoporin proteins and nuclear export factor CRM1, helps HIV traffic its mRNA from the nucleolus to the cytoplasm (Daelemans et al., 2004).
Previous experiments demonstrated that HSV-1 induced modification of the nucleoli and ribosomes.Soon after HSV-1 infection, the nucleoli localize close to the nuclear membrane and finally fragment into small pieces.In addition, the non-reversible phosphorylation of ribosomal protein S6 is stimulated by infection (Diaz et al., 2002).
The ICP0 protein, which plays a crucial role during HSV-1 lytic cycle and reactivation, accumulates within the nucleoli of infected or transfected cells, presenting a particular spotted phenotype.The RING finger domain of ICP0 is essential for its nucleolar localization.It is described that ICP0 and the heat-shock protein 70 co-localize with each other in the nucleoli of infected cells during heat-shock stress (Burch & Weller, 2004).Although the nucleolar distribution of ICP0 partially overlaps with mRPA43, an essential subunit of Pol I that localizes in FC, ICP0 does not enhance Pol I activity compared to its activity on Pol II promoters, which suggests that the function of ICP0 in the nucleolus is still to be unravelled (Morency et al., 2005).
In addition to enhancing viral replication, nucleolar proteins are redistributed to alter cellular pathways during infection.For example, the redistribution of nucleolin from the nucleolus requires HSV-1 protein ICP4 or a factor(s) whose expression involves ICP4 (Calle ´et al., 2008).Apart from ICP4, several other HSV-1 proteins have also been shown to delocalize nucleolin.VP22 targets the nucleolus and disperses nucleolin and chromatin (Lo ´pez et al., 2008).UL24 has been reported to induce redistribution of nucleolin and B23 from the nucleolus during HSV-1 infection, promoting nuclear egress of nucleocapsids, possibly through its effect on the nucleolus (Pearson et al., 2011;Lymberopoulos et al., 2011).It has been reported that UL12 protein forms a complex with nucleolin in HSV-1-infected cells (Balasubramanian et al., 2010;Sagou et al., 2010).The amount of nucleolin increased progressively during HSV-1 infection, and co-localization between nucleolin and ICP8 was found to occur in the viral replication compartments, suggesting that nucleolin may take part in the HSV-1 replication process (Fig. 1).Knocking down nucleolin by using small interfering RNA inhibited the proliferation of HSV-1, demonstrating that viral replication requires a high level of nucleolin expression and nucleolar protein plays a direct role in HSV biology (Calle ´et al., 2008), contributing to efficient nuclear egress of HSV-1 nucleocapsids in infected cells.
UL12 protein is also proven to interact with the viral single-stranded DNA-binding protein ICP8, forming a two-subunit recombinase.UL12 protein is able to promote strand exchange in vitro in conjunction with ICP8, contributing to viral genome replication through a homologous recombination-dependent DNA replication mechanism (Balasubramanian et al., 2010).
Recent studies demonstrated a major tegument protein of human cytomegalovirus (HCMV), ppUL83 (pp65), located in the nucleolus, leading to cell-cycle arrest in G1-G1/S by HCMV and the promotion of viral infection.Simultaneously, nucleolin has been found to interact with pp65, revealing that pp65 might be involved in regulatory/signalling pathways related to nucleolar functions, such as cell-cycle control (Arcangeletti et al., 2011).In addition, nucleolin is also involved in viral DNA synthesis in HCMV-infected cells.During infection, the viral DNA polymerase processivity factor UL44 could co-localize with nucleolin.Treating HCMV-infected cells with small interfering RNA targeting nucleolin mRNA could impair viral DNA synthesis and there is a correlation between the efficacy of knock-down and effect on viral replication (Strang et al., 2010).Furthermore, Kuo et al. (2011) found that the Rta protein of Epstein-Barr virus (EBV), a transcription factor that activates the EBV lytic genes, interacts with MCRS2, a nucleolar protein promoting the transcription of the rRNA gene.

Perspectives
The nucleolar proteome in humans, cows and plants has shed light on the evolution and new function of the nucleolus.Microarrays, combined with high-throughput techniques and advanced bioinformatics tools, could be applied to study new functions of nucleolar proteins.Simultaneously, studies of the nucleolar proteome in uninfected cells are relatively well advanced but the application of these approaches to examine the role of the nucleolus in infection is just in its infancy and could, in turn, prove highly informative for the understanding of the entire role of the nucleolus in the eukaryotic cell.
It has been well-recognized that, in addition to lytic infection, herpesvirus, especially HSV-1, can establish an infection with life-long latency and may periodically reactivate from latency under certain conditions.As shown in Fig. 1, the nucleolus is involved in HSV-1 replication during lytic infection.Whether the nucleolus participates in the establishment of latent herpesvirus infection and its reactivation from latency still need further investigation.Furthermore, it has been well established that the repressor complex REST/CoREST/LSD1/HDAC plays a critical role during the initiation of HSV-1 IE gene expression.It is worth investigating whether the nucleolus functions in this process during the establishment of latent infection.This might open new avenues of research for investigation of HSV-1 infection and its latency.
Fig.1.HSV-1 proteins target or associate with the nucleolus.HSV-1 IE proteins ICP0, ICP4 and ICP27 targeting the nucleolus maybe related to transactivating the expression of HSV-1 genes.ICP4, VP22 and UL24 delocalize nucleolin from the nucleolus to the HSV-1 replication compartment.UL12 forms complex with nucleolin and colocalizes with ICP8 for viral replication.Other HSV-1 proteins, for example US11, ICP34.5, UL3 and US8.5, target to the nucleolus for as yet unknown functions.