key: cord-0703410-u6x5zd4i
authors: Taliansky, M.E.; Brown, J.W.S.; Rajamäki, M.L.; Valkonen, J.P.T.; Kalinina, N.O.
title: Involvement of the Plant Nucleolus in Virus and Viroid Infections: Parallels with Animal Pathosystems
date: 2010-10-15
journal: Adv Virus Res
DOI: 10.1016/b978-0-12-385034-8.00005-3
sha: 75a3617fb5f0c407284fe76d2b6ece6f763bbba3
doc_id: 703410
cord_uid: u6x5zd4i

The nucleolus is a dynamic subnuclear body with roles in ribosome subunit biogenesis, mediation of cell-stress responses, and regulation of cell growth. An increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. This chapter will highlight studies showing how plant viruses recruit nucleolar functions to facilitate virus translation and replication, virus movement and assembly of virus-specific ribonucleoprotein (RNP) particles, and to counteract plant host defense responses. Plant viruses also provide a valuable tool to gain new insights into novel nucleolar functions and processes. Investigating the interactions between plant viruses and the nucleolus will facilitate the design of novel strategies to control plant virus infections.

The nucleolus is a dynamic subnuclear body with roles in ribosome subunit biogenesis, mediation of cell-stress responses, and regulation of cell growth. An increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. This chapter will highlight studies showing how plant viruses recruit nucleolar functions to facilitate virus translation and replication, virus movement and assembly of virus-specific ribonucleoprotein (RNP) particles, and to counteract plant host defense responses. Plant viruses also provide a valuable tool to gain new insights into novel nucleolar functions and processes. Investigating the interactions between plant viruses and the nucleolus will facilitate the design of novel strategies to control plant virus infections. 

The interactions between a virus and its host cell play a central role in the viral infection cycle. The analysis of virus-host interactions is critical for understanding the mechanisms of viral infections and for the development of novel antiviral strategies. Viruses are obligate intracellular pathogens with small genomes and, therefore, are reliant on subverting of the cellular The Nucleolus and Plant Viruses functions and machineries to facilitate their own replication. The cell nucleus is one of the key features of eukaryotic cells. It is a highly dynamic, membrane-bound organelle that hosts major cellular events, including DNA replication, messenger RNA synthesis and processing, and ribosome subunit biogenesis (Trinkle-Mulcahy and . Given the pivotal role of the nucleus in cell host function, it is not surprising that viruses interact with this organelle and its compartments, and that such interactions play a crucial role in the virus infection cycle. Indeed, certain viruses including plant DNA containing begomoviruses (Rojas et al., 2001; Sharma and Ikegami, 2009) , RNA reverse-transcribing caulimoviruses (CaMVs) (Haas et al., 2005) , and negative-strand RNA rhabdoviruses belonging to the genus Nucleorhabdovirus (Tsai et al., 2005) replicate in the nucleus, and therefore it makes sense that these viruses modify nuclear structures to usurp some of the nuclear functions and provide an appropriate environment for their own replication. In contrast, single-stranded positive-sense RNA [(þ)ssRNA] viruses replicate in the cytoplasm. Therefore, the rational for RNA viruses targeting the nucleus and its compartments is not so evident. However, an increasing number of reports clearly show that cytoplasmic RNA viruses including plant viruses can also target nuclear compartments and, nucleoli, in particular (reviewed by Hiscox 2002 Hiscox , 2007 Greco, 2009 ). Thus, investigating why and how plant RNA viruses interact with the nucleolus to meet their own needs will contribute to the better understanding of molecular biology of plant viruses and facilitate the design of novel strategies for virus control. These studies may also teach us about the fundamental principles of plant nucleolar structure and functions. The nucleolus is a prominent subnuclear compartment formed around the clusters of genes (rDNA) coding for ribosomal RNA (rRNA), and is the site of rDNA transcription, rRNA processing, and ribosome assembly (Olson, 2004; Rubbi and Milner, 2003; Sirri et al., 2008) . Indeed, for many years the exclusive function of the nucleolus was thought to be rRNA synthesis and ribosome biogenesis. However, several insights from the past decade have dramatically changed this traditional view and implicated the nucleolus in many other aspects of cell function such as cellcycle regulation, gene silencing, telomerase activity, senescence, stress responses, and biogenesis of multiple ribonucleoprotein (RNP) particles (Boisvert et al., 2007; Olson and Dundr, 2005; Sirri et al., 2008) .

This chapter briefly reviews the structure and composition of the nucleolus and, subsequently, data implicating the nucleolus in the infection cycles of animal and plant viruses and also viroids. Most of the current knowledge on nucleolar localization and functions of viral proteins has been gained in studies on animal viruses and has also been reviewed comprehensively (Greco, 2009; Hiscox, 2002 Hiscox, , 2007 . Therefore, the main emphasis of this review will be on what is known about the different aspects of interactions between plant virus proteins and the nucleolus, of which the functional significance in control of virus movement and interference with host antiviral defense has started to appear only recently. We also discuss recent findings on the potential role of Cajal bodies (CBs), another type of small subnuclear bodies structurally and functionally associated with the nucleolus.

The nucleus is a highly structured organelle responsible for chromosome organization, replication and division, for gene expression and regulation, and the integration of a vast array of activities required for cellular function. The nucleus contains chromatin-rich regions, made up of condensed heterochromatin, more dispersed euchromatin, and interchromatin regions, and chromatin organization is responsible for regulating gene expression, DNA replication, and chromosome segregation (Schneider and Grosscheld, 2007; Trinkle-Mulcahy and Lamond, 2008) . In addition, it contains many other structures or bodies of different numbers and sizes which vary between cell types, at different stages in the cell cycle and under different conditions. The most prominent of these structures is the nucleolus but many other bodies [e.g., CBs, splicing speckles, paraspeckles, PML (pro-myeloid leukemia) bodies, etc.] have now been identified and are being characterized on the basis of their protein and RNA components and functions (Boisvert et al., 2007; Cioce and Lamond, 2005; Lamond and Spector, 2003; Matera et al., 2009; Rippe, 2007) .

The nucleolus is where rRNA genes are transcribed and processed, and rRNAs are assembled, along with ribosomal proteins, into the small and large subunits of the ribosome (Boisvert et al., 2007; Fatica and Tollervey, 2002; Granneman and Baserga, 2004) . The nucleolus contains three different regions on the basis of their appearance in the transmission electron microscope: fibrillar centers (FC), the dense fibrillar component (DFC), and the granular component (GC). In the mammalian nucleolus, the FCs are small, lightly staining structures surrounded by the more densely stained DFC. The DFC in turn is surrounded a particulate region-the GC. The plant nucleolus tends to be more regular in its structure (usually near to spherical) than animal nucleoli. The organization of the nucleolar regions is different in plant nucleoli in that the DFC is not as densely stained as in animal nucleoli and occupies much more of the nucleolar volume (up to 70%). Transcription of precursor rRNAs (pre-rRNAs) occurs at multiple sites (200-400) within the DFC regions and pre-rRNAs undergo processing in the DFC and GC. Indeed, the localization of pre-rRNAs and different small nucleolar RNAs (snoRNAs) and proteins has allowed early and late pre-rRNA cleavage events to be correlated to the DFC and GC, respectively, suggesting a vectorial model for the production and maturation of rRNAs. Plant nucleoli also often contain a prominent central region called the nucleolar cavity (Brown and Shaw, 1998; Shaw and Brown, 2004) . The function of the nucleolar cavity is currently unknown, and it remains to be seen whether the differences in organization of plant and animal nucleoli influence the type and range of other functions which nucleoli carry out and interactions with different viruses.

The major function of the nucleolus is the transcription and processing of precursor rRNAs and ribosomal subunit assembly. The dynamic assembly pathway involves a series of intermediate pre-ribosomal complexes of the 40S and 60S ribosomal subunits and in addition to ribosomal proteins, requires up to 200 accessory proteins (Grannemann and Baserga, 2004) . Processing of the pre-rRNAs involves both cleavage of the precursor transcript and nucleotide modifications, the majority of which are 2 0 -O-ribose methylation and pseudouridylation. Some cleavage reactions and the modifications require snoRNPs. SnoRNPs consist of a snoRNA and associated proteins (Kiss, 2002) . By the nature of the major function in rRNA production and ribosomal subunit assembly, there is a huge flux of proteins and RNA complexes into and out of the nucleolus. The highly dynamic movement of proteins and complexes has been illustrated in animal cells by quantitative nucleolar proteomic analyses Lam et al., 2007) . In particular, ribosomal proteins are highly expressed and rapidly accumulate in the nucleolus where they are incorporated into ribosomal subunits or rapidly degraded (Lam et al., 2007) .

Three of the most abundant and well-studied nucleolar proteins are fibrillarin, nucleolin, and B23. Fibrillarin, one of the major proteins of the nucleolus, is a core component of box C/D snoRNPs and is required for rRNA processing (Venema and Tollervey, 1999) . Fibrillarin has methyltransferase activity directing 2 0 -O-ribose methylation of rRNA (Barneche et al., 2000; Cioce and Lamond, 2005; Matera and Shpargel, 2006) . Fibrillarin is highly conserved in sequence, structure, and function in eukaryotes. The N-terminal region of fibrillarin comprises a glycine-and arginine-rich (GAR) domain (Barneche et al., 2000) . The GAR domain is methylated at arginine residues (Liu and Dreyfuss, 1995) and is responsible for interactions with various proteins including the survival motor neuron (SMN) gene product ( Jones et al., 2001) and the nuclear DEAD (Asp-Glu-Ala-Asp) box protein p68 (Nicol et al., 2000) . Fibrillarin contains a centrally located RNA-binding domain which together with the C-terminal helix domain and the intervening spacer constitutes a methyltransferase-like domain that contains an S-adenosyl methionine binding motif and is responsible for fibrillarin methyltransferase activity (Wang et al., 2000a) . The C-terminal helix domain appears to target fibrillarin to CBs (Snaar et al., 2000) . Although it is well established that fibrillarin plays a role in ribosome biogenesis within the nucleolus, its role in CBs is not well understood but it is presumably responsible for the 2 0 -O-ribose methylation of small nuclear RNAs (snRNAs).

Nucleolin is an abundant, ubiquitously expressed protein, which is highly phosphorylated, methylated, and also can be ADP-ribosylated (Ginisty et al., 1999) . Nucleolin is found in various cell compartments, and it is especially abundant in the nucleolus. Nucleolin has three well-defined domains. The N-terminal domain with alternating acidic and basic stretches is involved in rDNA transcription by interacting with rDNA repeats and histone H1 and in nuclear localization. The central portion is the RNAbinding domain, whereas the C-terminal part contains a GAR domain involved in interaction with the ribosomal proteins (Tuteja and Tuteja, 1998) . Nucleolin is involved in ribosome biogenesis (Mongelard and Bouvet, 2007) , affects transcription, processing and modification of rRNA and nuclear-cytosolic transport of ribosomal proteins and ribosomal subunits by shuttling between the nucleus and the cytoplasm (Tuteja and Tuteja, 1998) .

B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly, binding to other nucleolar proteins, nucleocytoplasmic shuttling (Li et al., 1996) , and possibly regulating transcription of rDNA by mediating structural changes in chromatin (Okuwaki et al., 2001) . Two isoforms of B23 have been identified: the major form (B23.1) is predominately located in the nucleolus and the minor form (B23.2) resides in the cytoplasm (reviewed by Hiscox, 2002) .

Small proteins of less than 40-60 kDa can enter the nucleus through nuclear pore complexes by passive diffusion (Hiscox, 2007; Nigg, 1997) . RNA-binding proteins that diffuse into the nucleus may therefore nonspecifically target the nucleolus as it contains a large amount of rRNA. The nuclear import of larger proteins is mediated by nuclear localization signals (NLSs), composed of one (monopartite) or two (bipartite) stretches of basic amino acid (arginine and/or lysine) residues of a given size (Hiscox, 2007; Macara, 2001) . Nucleocytoplasmic shuttling proteins also contain specific nuclear export signals (NESs), usually leucine-rich amino acid motifs (Hiscox, 2007; Macara, 2001) .

How proteins may be further delivered to the nucleolus is poorly understood. The nucleolus does not have apparent membrane or other barriers, and entry into it does not require energy, unlike entry to the nucleus. It seems conceivable that viral proteins localize to the nucleolus, firstly, as a result of targeting the nucleus via classical NLS followed by direct or indirect interactions between the viral molecules (via various nucleolar localization signals, NoLSs) and components that make up the nucleolus (Hiscox, 2002 (Hiscox, , 2007 . The structure of NoLSs is not well defined and depends on different factors including whether the protein associates with another nucleolar-bound protein or alternatively traffics to the nucleolus on its own or associates with RNA transcripts that are being transcribed in the nucleolus. At least in some cases, NoLS are rich in arginine and lysine residues and can overlap with NLSs (Hiscox, 2002 (Hiscox, , 2007 .

The second most studied nuclear body is CBs which are frequently associated with the nucleolus and found in animal and plant nuclei. CBs are involved in snRNP and snoRNP maturation and transport, with snRNPs and snoRNPs accumulating in CBs before appearing in speckles or the nucleolus, respectively (Cioce and Lamond, 2005) . As mentioned above, spliceosomal snRNAs are also modified and contain 2 0 -O-ribose methylations and pseudouridines. Modification of nucleotides in snRNAs is guided by small CB-specific RNAs (scaRNAs) (Darzacq et al., 2002; Jady et al., 2003) . A major component of CBs is the protein, coilin, which is required for their formation. In plants, mutants are available which knock out CBs or alter their size and number, two of which are due to mutations in coilin (Collier et al., 2006) . The number of CBs per nucleus can vary in different cell types and is under developmental control (Cioce and Lamond, 2005) .

Besides rRNA transcription and processing and the production of ribosomal subunits, the nucleolus is also involved in many other aspects of RNA processing and RNP assembly as well as cellular functions (Boisvert et al., 2007; Olson, 2004; Pedersen, 1998; Raška et al., 2006; Rubbi and Milner, 2003) . For example, the nucleolus has a role in the maturation, assembly, and export of RNP particles. The signal recognition particle (SRP) has a nucleolar phase in its assembly with particular protein constituents of the SRP being localized to the nucleolus. Similarly, telomerase RNP, required for chromosome replication, may be assembled in the nucleolus, and the nucleolus may also have a role in sequestering telomerase RNP to avoid inappropriate nucleation of telomere structures. Spliceosomal snRNPs may also undergo part of their assembly in the nucleolus, and processing of pre-tRNAs and U6snRNA occurs in the nucleolus. In addition, the nucleolus is a site of sequestration of particular proteins to regulate, for example, the cell cycle or cell death, and acts as a sensor of cellular stress (Boisvert et al., 2007; Olson, 2004; Pedersen, 1998; Raška et al., 2006; Rubbi and Milner, 2003) .

Characterization of the protein composition of the nucleolus under different conditions has provided support for or suggested new functions for the nucleolus. Initial proteomic analyses of human cells identified a few hundred proteins which included not only well-known nucleolar proteins such as ribosomal proteins, fibrillarin, nucleolin, B23, etc. but also, for example, splicing and translation factors (Andersen et al., 2002) . Advances in proteomics has now allowed the identification of around 4500 proteins in the human nucleolar proteome (Ahmad et al., 2008) and the dynamic behavior of nucleolar components and complexes can now be determined Lam et al., 2007) . In plants, a partial proteomic analysis of the nucleolus identified many expected ribosomal and nucleolar proteins but also found splicing and translation factors (Pendle et al., 2005) . In particular, exon junction complex proteins (associated with mRNAs following splicing) were identified in the nucleolar proteome and in the nucleolus by fluorescence microscopy (Pendle et al., 2005) . One of these proteins, eIF4A-III, a core protein of the exon junction complex, was shown to redistribute from the nucleoplasm to the nucleolus and finally to splicing speckles under stress conditions of hypoxia . Similar dynamic distribution, involving the nucleolus, of proteins that interact with mRNAs has been demonstrated and the nucleolus and CBs have been shown to be involved in U1snRNP production in plants (Lorković and Barta, 2008; Tillemans et al., 2006) .

In plants, novel functions for the plant nucleolus, CBs, and another largely nucleolar-associated body, the D-body, have been described. Firstly, the production of heterochromatic siRNAs, which are involved in transcriptional silencing, is thought to occur in a region of the nucleolus or in D bodies due to the localization of protein components of the machinery and of siRNAs (Pontes and Pikaard, 2008) . Secondly, maturation of microRNAs (miRNAs) may occur in D-bodies as precursor miR-NAs as well as Dicer-like ribonuclease 1 (DCL1) have been located to these structures (Pontes and Pikaard, 2008) . Similarly, in mammalian cells, some precursor and mature miRNAs have recently been found in the nucleus with some being enriched in the nucleolus (Politz et al., 2009; Scott et al., 2009) . Of particular interest was the enrichment of some precursors in the GC suggesting that miRNA processing could occur in the nucleolus, or these miRNAs may be involved in ribosome synthesis or other nucleolar functions (Politz et al., 2009) . The link between the nucleolus and miRNA production is illustrated by the evolutionary relationship between some snoRNAs and miRNA precursors. For example, a human snoRNA was shown to be processed by Dicer to generate small RNAs which were associated with argonaute proteins (AGOs) and caused reduced expression of gene targets (Ender et al., 2008) . In addition, numerous snoRNA-derived small RNAs from different organisms (including Arabidopsis) were associated with components of RNA silencing pathways (Taft et al., 2009) and many miRNA precursors have retained snoRNA features (Scott et al., 2009 ). Finally, a third novel function for the plant nucleolus is in mRNA biogenesis, surveillance, or nonsensemediated decay (NMD). Biochemical fractionation of nucleoplasm and nucleoli and subsequent sequencing of isolated cDNAs have shown that the plant nucleolus not only contains mRNAs but that aberrant mRNAs are enriched in the nucleolus ). The aberrant mRNAs show splicing defects, the majority of which would introduce premature termination codons and therefore be expected to be substrates for NMD. Using upf mutants (affected UPF proteins, key components of the NMD mechanism), the correlation between enrichment of aberrant mRNAs in the nucleolus and turnover by NMD was corroborated and was further supported by the localization of the NMD proteins, UPF2 and UPF3, to the nucleolus .

Before the observation that the plant nucleolus contained mRNAs and aberrant mRNAs, some spliced mRNAs (e.g., c-myc) were localized to the nucleolus while their unspliced versions were found in the nucleoplasm in mammalian cells (Pedersen, 1998; Olson, 2004) . The nucleolus has also been associated with mRNA export on the basis of accumulation of polyAþ RNA upon disruption of export factors, nucleolar structure, or stress conditions in yeast and animal cells, although this could reflect increased polyadenylation prior to degradation (Ideue et al., 2004; Pederson, 1998; Schneiter et al., 1995) . More recently, the nucleolus has been proposed to be involved in the formation of mRNPs which are localized to specific regions of the cytoplasm for translation. In yeast, ASH1 mRNA enters the nucleolus bound to specific RNA-binding proteins at which time translation repressor proteins are loaded onto the mRNA. The mRNP is then exported to the cytoplasm, transported to its final destination, and then translation is activated (Du et al., 2008; Jellbauer and Jansen, 2008) . A similar trafficking pathway may also operate in mammals for mRNPs associated with the nucleolar protein, Staufen, which is involved in transport of mRNAs in neurons .

Thus, the nucleolus has numerous functions related to RNA biogenesis and different RNA processing and RNP maturation pathways. It contains many RNA-interacting proteins involved in these processes, including highly abundant RNA-binding proteins (such as fibrillarin and nucleolin) involved in rRNA and ribosomal subunit production. It is therefore a dynamic hub of RNA processing activity, RNA:protein interaction and complex formation. These characteristics, in addition to the potential involvement of the nucleolus in mRNA biogenesis and, particularly, the transport of mRNAs and mRNPs to and from the nucleolus to other parts of the cell, make the nucleolus a prime target for exploitation by viruses. It is therefore not surprising that viruses have taken advantage of the nucleolus and nucleolar components for production and distribution of viral RNAs and RNPs.

Several excellent reviews have documented various aspects of the involvement of the nucleolus in the infection cycle of animal and human viruses (Greco, 2009; Hiscox, 2002 Hiscox, , 2007 Matthews and Olson, 2006; Stark and Taliansky, 2009 ). This section therefore briefly summarizes the main findings in this research area to illuminate the nucleolar functions involved in virus infections that are conserved between plant and animal kingdoms.

Certain animal viruses such as DNA containing adenoviruses that replicate in the nucleus interact with and disrupt the nucleolus. As a result, synthesis of rRNA is disrupted in adenovirus-infected cells. Adenovirus infection also causes the redistribution of nucleolin and B23 (Matthews, 2001) . Interestingly, B23 has been shown to stimulate adenovirus replication. RNA virus replication may also be facilitated by nucleolar proteins. Indeed, the HDAg protein encoded by hepatitis delta virus (a (À)ssRNA virus which is a subviral satellite of the DNA virus, hepatitis B virus) also binds to nucleolin and B23. It has been proposed that HDAg interacts with both these proteins in a complex that promotes viral RNA replication, presumably as a result of the helicase activity of nucleolin (Huang et al., 2001) . Nucleolar proteins may also be involved in virus assembly and egress. For example, accumulation and assembly of structural (Cap) proteins of adeno-associated virus take place in nucleoli (Bevington et al., 2007) . Further export of the assembled Cap proteins from the nucleolus is mediated by another type of virus-encoded packaging (Rep) proteins, which should form a complex with Cap proteins. Remarkably, formation of this complex is mediated by B23. After export of complexes containing Rep, B23, and Cap proteins from the nucleolus to the nucleoplasm, viral DNA encapsidation occurs (Bevington et al., 2007) .

Another animal virus, the (À)ssRNA containing Borna disease virus, uses the nucleolus as a site of genome replication (Pyper et al., 1998 ). An RNA-binding protein encoded by this virus has the appropriate trafficking signals for import and export to and from the nucleus (Cros and Palese, 2003) 

Many proteins encoded by animal viruses that replicate mainly or exclusively in the cytoplasm have also been shown to localize to the nucleolus, cause the relocalization of nucleolar proteins and disruption of nucleolar architecture and function. These include the (þ)ssRNA virus proteins such as nucleocapsid proteins encoded by coronavirus and arterivirus, the dengue virus core protein, the alphavirus capsid protein and nonstructural nsP2 protein, the (À)ssRNA virus proteins, including the influenza virus nucleoprotein (NP) and nonstructural protein 1 (NS1), Newcastle disease virus matrix protein, and retrovirus proteins such as human immunodeficiency virus (HIV) Rev and the transactivator Tat proteins (reviewed by Greco, 2009; Hiscox, 2002 Hiscox, , 2007 . Although functional relevance of why these proteins localize to the nucleus or nucleolus and how this relates to their functions in virus replication in many cases is largely unknown, several reports reveal significant progress in this area as exemplified below.

Infection of cells with poliovirus (picornavirus) results in the inactivation of the nucleolar protein, RNA polymerase I upstream binding (transcription) factor, which inhibits transcription of rRNAs in the host cell (Banerjee et al., 2005) . Poliovirus is also able to interact with nucleolin causing its selective redistribution from the nucleolus to the cytoplasm (Waggoner and Sarnow, 1998) . After relocalization, nucleolin binds to the internal ribosome entry sites (IRESs) at the 5 0 untranslated region of poliovirus genomic RNA, and this interaction stimulates IRES-dependent translation (Hellen and Sarnow, 2001) . This also occurs in hepatitis C virus (Izumi et al., 2001) and represents an alternative translation initiation strategy as compared to the classical eukaryotic Cap-dependent translation (Hellen and Sarnow, 2001) . Nucleolin is also able to interact with the 3 0 -untranslated region of poliovirus RNA, which controls synthesis of negative strand RNA (Waggoner and Sarnow, 1998) . Interaction of picornaviruses with the nucleolus could also down-regulate host gene expression. For example, the human rhinovirus 3C protease precursors that are localized in the nucleolus at early stages of the infection inhibit cellular RNA by cleavage of vital transcription factors (Amineva et al., 2004) .

The nucleocapsid proteins encoded by porcine arterivirus and avian coronavirus (infectious bronchitis virus, IBV) interact with nucleolin and fibrillarin (Chen et al., 2002; Yoo et al., 2003) , and as a result may disrupt the normal functions of these proteins. For instance, by altering the distribution of fibrillarin, viruses might be reducing polI transcription, that is, the synthesis of rRNA, as blocking fibrillarin with antibody prevented its translocation to nucleoli and resulted in the reduction or inhibition of polI transcription (Fomproix et al., 1998) . The IBV infection leads to disruption of the nucleolus (Dove et al., 2006a) and arrest of the cell cycle and cytokinesis (Chen et al., 2002; Dove et al., 2006b; Wurm et al., 2001) . Therefore, disruption of nucleolar architecture and function might be common in cells infected with viruses interacting with the nucleolus. The loss of essential nucleolar function in its turn may play an important role during virus infection toward an active production of the virus.

One of the most studied viruses in terms of viral interactions with the nucleolus is HIV, a retrovirus. HIV RNAs are reverse transcribed in the cytoplasm of infected cells and trafficked to the nucleus. After transcription in the nucleus, progeny RNA molecules are transported back to the cytoplasm. One of the functions of the Rev protein is to export unspliced or partially spliced viral mRNA from the nucleus (reviewed by Greco, 2009; Hiscox, 2007) . Before nuclear export, HIV RNA passes through the nucleolus. Rev binds to a cis-acting RNA element (Rev-response element), which is found in all unspliced and incompletely spliced viral mRNAs, and this promotes the translocation of these RNAs from the nucleus (Cantó-Nogués et al., 2001; Michienzi et al., 2000) . The nucleolus plays a central role in this process, and the nucleolar trafficking of Rev and viral RNA is critical for the outcome of infection.

Thus, many animal viruses, whether they replicate or not in the nucleus, have evolved a nucleolar phase for part of their infection cycle to prevent unwanted interference from the cell. Alternatively, they use nucleolar functions for their own benefit. Recruitment of nucleolar proteins is especially beneficial for viruses, and in particular for RNA containing viruses, as these proteins possess many crucial functions in cellular RNA biosynthesis, processing, translation, and trafficking. Indeed, during virus infections of mammalian cells, various viral components traffic to and from the nucleolus where they interact with different host factors. Certain nucleolar proteins are redistributed into other cell compartments or are modified, and some cellular proteins are relocalized in the nucleolus of infected cells (reviewed by Greco, 2009; Hiscox, 2002 Hiscox, , 2007 . Well-documented studies have established that several of these nucleolar modifications play a role in some steps of the viral infection cycle such as viral attachment and entry, intracellular trafficking, transcription, translation, replication, virus assembly, and regress (reviewed by Greco, 2009 ). The virally induced nucleolar modifications could also affect fundamental cellular pathways including the initiation of transcription from the DNA promoter of the rRNA genes, cell-cycle regulation, and apoptosis (reviewed by Greco, 2009) .

Although some steps (replication, translation) of the infection cycles of plant and animal/human viruses are essentially similar, there is a fundamental difference in some other mechanisms employed to enter host cells and spread from cell to cell between viruses infecting animal cells and viruses infecting plants. This is because animal cells are separated by barriers far less formidable than the thick, rigid, and impermeable cell walls consisting of cellulose and pectin that separate plant cells from one another. Other differences relate to defense strategies employed by humans/animals and plants against viral infections. For example, in mammals the interferon (IFN) pathway plays a key role in the innate antiviral immune response, whereas plants do not display such an activity. Instead, plants primarily rely on other natural defense strategies such as RNA silencing. Interestingly, functional links between plant virus infection cycle and the nucleolus have been described for both common and plant-specific virus infection steps.

Certain plant virus proteins localize to the nucleolus with examples from single-stranded DNA viruses, para-retroviruses and negative-strand and positive-strand RNA viruses (Table I) . The most common technique for studying the nucleolar targeting of plant virus proteins is based on the confocal microscopy localization of the proteins which have been tagged with a fluorescent fusion protein (such as green fluorescent protein, GFP). Such proteins are usually larger than the size-exclusion limit ($ 40-60 kDa) and hence prevented from nonspecific protein diffusion into the nucleus. Moreover, in many cases the specific nucleolar localization of plant virus proteins has been supported by identification of NoLSs (Table II) . Although no overall conserved nucleolar trafficking motif has been identified in these NoLSs, they presumably resemble host NoLSs.

Thus plant viral nucleolar trafficking might use a form of molecular mimicry as has earlier been proposed for animal viruses (Table II) . However, in the case of GRV ORF3, in addition to the arginine-rich domain (NLS), a leucine residue at position 149 (L149) residing inside the leucine-rich domain (NES) is also essential for nucleolar targeting of ORF3 protein (Table II; Kim et al., 2007a) . The fact that viral proteins contain NoLSs is a strong indication that viruses have evolved specific nucleolar functions. Viral proteins might also traffic to the nucleolus through association with host proteins. One example of such an association may be the interaction of various plant virus proteins with the major nucleolar protein, fibrillarin. The first description of this interaction was the demonstration that the umbravirus GRV ORF3 long-distance movement protein (MP) binds to fibrillarin in vivo and in vitro (Kim et al., 2007a,b) . For example, the leucine-rich domain (and L149, in particular) of ORF3 is involved in direct interaction with fibrillarin (Kim et al., 2007b) . Mutations in the leucine-rich domain prevent fibrillarin from binding to ORF3 and nucleolar trafficking (Kim et al., 2007b) . By implication, this may relate fibrillarin binding to nucleolar trafficking.

Other plant virus MPs which interact with fibrillarin are the pomovirus PMTV triple gene block protein 1 (TGB1) and hordeivirus PSLV TGB1 (N. O. Kalinina and D. Rakitina, unpublished results). As their name suggests, MPs are involved in virus spread in infected plants, and the potential role of fibrillarin in this process will be discussed in Section IV.B. The multifunctional PVA (potato virus A)-encoded viral genomelinked protein (VPg) is also able to interact with fibrillarin (Rajamäki and Bonfiglioli et al. (1996) , Qi and Ding (2003) Valkonen, 2009). However, the role of this interaction is likely to be different from that in virus movement as this process is not compromised by fibrillarin depletion (Rajamäki and Valkonen, 2009 ; Section IV.C). Another example of a viral protein that interacts with fibrillarin in the nucleolus is a silencing suppressor of CMV (cucumber mosaic virus), the 2b protein (González et al., 2010 ; Section IV.C). Collectively, these data indicate that interaction with fibrillarin is a general property of various plant virus proteins that is not restricted to one or two virus taxonomic groups. However, such interactions may have quite diverse molecular implications for different viruses being required at various phases of virus infection cycle. This may also suggest novel, unexpected natural functions for fibrillarin that are hijacked or affected by plant viruses at different stages of infection for needs of the viruses.

Viroids are small, circular, self-replicating, and non-coding RNA molecules that cause plant diseases (reviewed by Tabler and Tsagris, 2004) . Viroids do not replicate in the cytoplasm like conventional plant RNA viruses but replicate in either the nucleus or the chloroplast. Nuclear viroids such as PSTVd and CCCVd have a nucleolar phase in their replication cycle (Table I ; Qi and Ding, 2003; Schumacher et al., 1983) . 

Virus acronyms and other abbreviations are as in Table I . a NLSs are also required for the nucleolar localization. b Nuclear and nucleolar localization is controlled independently by the same NLS regions. c L149 (italic) residing inside the ORF3 NES is also essential for nucleolar localization of the ORF3 protein.

In early experiments using in situ hybridization, PSTVd RNA [including both (þ) and (À) RNA strands] was localized in nucleoli of nuclei isolated from infected plants (Harders et al., 1989) , suggesting that the nucleolus is the replication site of the viroid. However, later using improved sample preparation and in situ hybridization protocols, Qi and Ding (2003) have found that the (À) strand of PSTVd localizes in the nucleoplasm but not in the nucleolus. By contrast, the (þ) strand of PSTVd localizes in the nucleolus as well as in the nucleoplasm, with various distinct spatial patterns. These experiments are suggestive of successive stages of nuclear involvement in viroid replication: (1) synthesis of the (À) and (þ) strands of PSTVd occurs in the nucleoplasm; (2) the (À) strand RNA is anchored in the nucleoplasm; (3) the (þ) strand RNA is transported selectively into the nucleolus; and (4) some (þ) strand RNA traffics from the nucleolus back into the nucleoplasm and further into the cytoplasm for spreading into neighboring cells. The significance of (þ) strand PSTVd RNA trafficking into the nucleolus remains to be determined. However, nucleolar machineries for processing of rRNAs, snoRNAs, and tRNAs make the nucleolus an attractive candidate site for PSTVd processing. The specific intranuclear localization of (À) and (þ) strands of PSTVd may have some implication for pathogenicity. Assuming that the (À) and (þ) strands each can interact with specific cellular factors to disrupt normal cell functions to cause symptoms, the differential subnuclear localization of the (þ) and (À) strands of PSTVd may suggest different cellular targets for these RNAs.

(þ) strand of PSTVd RNA has been shown to interact with a bromodomain-containing protein (a member of a family of transcriptional regulators associated with chromatin remodeling), termed viroid RNA binding protein 1 (VIRP1) (Martínez de Alba et al., 2003; Tabler and Tsagris, 2004) . VIRP1 contains an NLS and therefore might transfer the viroid RNA to the nucleus and bring it into contact with transcription units associated with chromatin (Martínez de Alba et al., 2003; Tabler and Tsagris, 2004) . However, mechanisms for selective nucleolar trafficking of (þ) strand PSTVd are still unknown.

The nucleolus and its factors may be used not only by viruses and viroids for their own needs in replication, but also by plant host defense systems. For example, one of the major nucleolar proteins, nucleolin, binds to the 3 0 non-coding region of the tomato bushy stunt virus, tombusvirus, RNA ( Jiang et al., 2010) . This leads to significant inhibition of tombusviral RNA replication and may thus represent one of the innate immunity systems of plant hosts.

Plant viruses enter cells either through sites of mechanical injury to plant tissues or during the feeding by a specific vector organism (insect, nematode, or soil microbes belonging to protocists). To induce disease, after replication in the initially infected cells, plant viruses must spread to the rest of the plant. The systemic spread of plant viruses proceeds in two stages: (i) cell-to-cell movement through plasmodesmata (PD) and (ii) long-distance movement through vascular tissues. First, the virus (in the form of virions or nucleic acid protein complexes) moves intracellularly from the sites of replication to PD, which are unique intercellular membranous channels that span cell walls linking the cytoplasm of contiguous cells. The virus then transverses the PD to spread intercellularly. It is generally accepted that viral cell-to-cell movement involves virus-encoded MPs as well as host-encoded components (reviewed by Lucas, 2006) .

Virus systemic movement between organs (long-distance movement) occurs through the phloem, the specialized vascular system used by plants for the transport of assimilates and macromolecules (reviewed by Lucas, 2006; Oparka, 2004) . Viruses enter, move through, and exit from the vascular system, which is usually surrounded by bundle sheath cells and contains various cell types including vascular parenchyma cells, companion cells, and enucleate sieve elements. Thus, transport of a virus into and within vascular tissue implies movement from mesophyll cells to bundle sheath cells, from bundle sheath cells to vascular parenchyma and/or companion cells, and into sieve elements. Virus exit from vascular tissue presumably involves the same steps in reverse order. Coat protein (CP) is essential for efficient long-distance transport of plant viruses, with only few exceptions (reviewed by Lucas, 2006 ).

One of the most well-studied plant viruses in terms of viral interactions with the nucleolus is GRV, an umbravirus [(þ)ssRNA virus]. Umbraviruses differ from most other viruses in that they do not encode a CP such that conventional virus particles are not formed in infected plants. Umbraviral genomes encode at least three proteins. In GRV, two ORFs at the 5 0 -end of the RNA are expressed by a frameshift mechanism as a single protein that appears to be an RNA replicase . The other ORFs (ORF3 and ORF4) overlap each other. ORF4 encodes the MP that mediates the cell-to-cell movement of viral RNA via PD (Ryabov et al., 1998) . ORF3 protein is the long-distance movement factor that facilitates trafficking of viral RNA through the phloem (Ryabov et al., 1999) . Umbraviral ORF3 proteins (26) (27) (28) (29) show no significant similarity with any other recorded or predicted proteins . The GRV ORF3 protein interacts with viral RNA in vivo to form filamentous RNP particles, which have elements of regular helical structure, but not the uniformity typical of virus particles . The RNPs accumulate in cytoplasmic inclusions which have been detected in all cell types and were abundant in phloem cells . They serve to protect viral RNA and move it through the phloem to cause systemic infection. Remarkably, in addition to its presence in the cytoplasm, the ORF3 protein was also found in nuclei and predominantly in nucleoli (Ryabov et al., 1998 (Ryabov et al., , 2004 .

Studies of the biology of GRV infection have provided molecular insights into how and why viruses may target the nucleolus (Canetta et al., 2008; Kim et al., 2007a,b) . It has been demonstrated that the GRV ORF3 protein traffics to the nucleolus via a mechanism involving the reorganization of CBs into multiple Cajal body-like structures (CBL) and their fusion with the nucleolus. Nucleolar localization and further trafficking of ORF3 protein from the nucleolus back to the cytoplasm is essential for the umbravirus infection. The integral connection between nucleolar targeting of the ORF3 protein and its biological function in virus long-distance spread is demonstrated by mutagenesis of the arginine-and leucine-rich domains that block nucleolar localization or nuclear export of the ORF3 protein, and which prevent the formation of cytoplasmic viral RNPs and their long-distance movement (Kim et al., 2007a) .

Although the mechanisms by which the ORF3 protein targets CBs and produces CBLs are unknown, it could be suggested that targeting of CBs by the ORF3 protein may utilize elements of existing CB-trafficking pathways. For example, part of the maturation pathway of snRNPs in mammalian cells occurs in the cytoplasm and involves a complex containing the SMN protein (SMN complex) which together with the snRNP are reimported into the nucleus and targeted to CBs (Matera and Shpargel, 2006; Navascues et al., 2004; Sleeman and Lamond, 1999) . Particular snRNP proteins contain modified symmetric di-methyl arginines (sDMAs) which enhance the formation of snRNPs and interaction with SMN (Paushkin et al., 2002) . Preliminary experiments have identified sDMAs in the ORF3 protein (M. Taliansky, unpublished results) , suggesting that targeting of the ORF3 protein to CBs could involve interactions with the SMN protein. Although SMN protein has yet to be identified in plants, the existence of an orthologue has been suggested (Collier et al., 2006) .

The formation of CBLs may involve either fragmentation of CBs into multiple bodies by the ORF3 protein or the redistribution of CB components into new structures containing the ORF3 protein. Interestingly, the multiple CBL phenotype, described here, is similar to that of the poly Cajal bodies (pcb) mutant of Arabidopsis (Collier et al., 2006) . As the protein normally encoded by a gene controlling the pcb phenotype, appears to regulate CB formation, ORF3 protein may interfere with the function of this or other proteins to affect the integrity and number of CBs in nuclei. The second possibility is that the ORF3 protein causes the redistribution of CB components, such as coilin, U2B 00 and fibrillarin, to form CBLs with the ORF3 protein.

ORF3 protein trafficking to the nucleolus uses a novel pathway of nucleolar import by causing the fusion of CBLs with the nucleolus. The physical and functional association of the nucleolus and CBs is welldocumented and is controlled by complex molecular interactions among CB and nucleolar proteins such as coilin, SMN, fibrillarin, and Nopp140 (Cioce and Lamond, 2005; Ogg and Lamond, 2002) . Expression of mutant versions of some of these proteins has profound effects on CB structure and function causing disruption or dispersal and compositional changes ( Jones et al., 2001; Pellizzoni et al., 2001; Tucker et al., 2001) . Moreover, phosphorylation of coilin is an important factor determining physical interactions and trafficking of CBs (Cioce and Lamond, 2005; Ogg and Lamond, 2002) . For example, CBs form within the nucleolus of HeLa cells upon treatment with okadaic acid (an inhibitor of protein phosphatase) and with transient expression of coilin mutated at a single serine residue (Lyon et al., 1997; Sleeman et al., 1998) . CBs have also been observed within nucleoli in human breast carcinomas (Ochs et al., 1994) and in liver cells of hibernating dormice (Malatesta et al., 1994) . Therefore, the ORF3 protein may interfere with normal protein-protein interactions or posttranslational modifications causing the reorganization and fusion of CBs with the nucleolus.

The last stage of the nuclear voyage of the ORF3 protein is its nuclear export leading to the redistribution of some fibrillarin from the nucleolus to cytoplasm (fibrillarin normally does not accumulate in cytoplasm) (Kim et al., 2007a) . This redistribution is mediated by the direct interaction between the ORF3 protein and fibrillarin (Kim et al., 2007b) . Taking into account the long-distance movement function of GRV ORF3, it could be expected that fibrillarin is directly involved in this particular viral function. Further support of a role for fibrillarin in umbravirus systemic infection has been provided by the fibrillarin knock-down experiments (Kim et al., 2007b) . Silencing of the fibrillarin gene has indeed prevented formation of RNP particles and long-distance movement of GRV but does not affect viral replication or cell-to-cell movement. Thus, it has been concluded that fibrillarin is needed for formation of cytoplasmic RNP particles that are capable of long-distance movement and causing systemic viral infection such that the redistribution of fibrillarin with ORF3 protein is a prerequisite to form RNPs (Kim et al., 2007a,b) . Further experiments have shown that fibrillarin, in combination with ORF3 protein and viral RNA in vitro, produced filamentous RNP structures with structural properties similar to in vivo RNPs (as discussed in detail in Section IV.E). Fibrillarin, an RNA-binding protein, may bind the viral RNA or act as a chaperone to permit or catalyze the regular assembly of proteins around viral RNA. Although fibrillarin and ORF3 protein are sufficient to form functional viral RNP particles in vitro, additional proteins may also be associated with the in vivo particles. When formed in phloem companion cells, the viral RNPs are able to enter sieve elements and move through the plant to cause systemic infection. Hence formation of the fibrillarin-ORF3 protein complexes appears to be the key prerequisite for formation of GRV RNP particles and their long-distance movement through the phloem.

Poleroviruses are (þ) ssRNA viruses that are mainly restricted to cells in the vascular system. Besides a major CP PLRV encodes a minor CP, an extended version of the major CP produced by occasional translational ''readthrough'' of the CP gene (CP-RT) (Bahner et al., 1990) . Both CP and CP-RT contain the same NoLS, and are targeted to the nucleolus when they are individually expressed in plant tissues (Haupt et al., 2005) . However, CP-RT but not PLRV CP loses its nucleolar localization in the presence of replicating PLRV. It has been suggested that the CP-RT protein does not accumulate in the nucleolus in the presence of PLRV infection because PLRV RNA or PLRV-encoded or -induced factors retain this protein outside the nucleolus (Haupt et al., 2005) . Like GRV, PLRV has been unable to cause systemic infection in the fibrillarin-silenced plants, although accumulation of PLRV in the inoculated leaves was not affected (Kim et al., 2007b) suggesting that fibrillarin is also involved in PLRV long-distance movement.

A role of nucleolar functions in systemic infection has also been suggested for plant viruses that require TGB for movement. The genomes of some viruses, such as potexviruses, hordeiviruses, and pomoviruses, contain so-called triple gene block, TGB that encodes three proteins required for cell-to-cell and long-distance movement (reviewed by Morozov and Solovyev, 2003) .

The TGB1 protein encoded by the TGB-containing virus, PMTV (pomovirus) localizes to the nucleus and nucleolus. Deletion of 84 Nterminal amino acids abrogates its nucleolar localization. Northern blots of RNA from inoculated and upper non-inoculated leaves of plants infected with clones carrying the TGB1 N-terminal deletion mutant reveal that long distance movement of viral RNAs has also been abolished, but this mutant is still competent for cell-to-cell movement (L. Torrance, personal communication). Moreover, the PMTV TGB1 protein has been shown to interact with fibrillarin in vitro. Interestingly, TGB1 protein encoded by the hordeivirus, PSLV can also interact with fibrillarin both in vitro and in vivo (N. O. Kalinina, unpublished results) . Thus it appears that some TGB-encoding viruses such as PMTV and PSLV may represent another example of plant viruses that require association with the nucleolus to control long distance movement of their genomes.

Potyviruses form the largest group of plant-infecting RNA viruses (Rajamäki et al., 2004) . They have a polyadenylated (þ) ssRNA genome of ca. 9500-10,000 nucleotides that is encapsidated into a 680-900 nm long, filamentous particle. The genome is translated into a large polyprotein of about 3000-3350 amino acids, which is subsequently processed into up to ten mature proteins by three virus-encoded proteinases (Rajamäki et al., 2004) . Additionally, a 25-kDa protein produced from the P3 cistron by frameshifting was recently identified (Chung et al., 2008) . Potyviruses replicate in membranous structures in the cytoplasm (Cotton et al., 2009; Schaad et al., 1997a) . However, two potyviral replication-associated proteins, the nuclear inclusion protein a (NIa) and nuclear inclusion protein b (NIb), accumulate in the nucleus of virus-infected cells (Baunoch et al., 1991; Dougherty and Hiebert, 1980; Hajimorad et al., 1996; Knuhtsen et al., 1974; Restrepo et al., 1990) . In addition, NIa localizes in the nucleolus and CBs (Baunoch et al., 1991; Rajamäki and Valkonen, 2009; Restrepo et al., 1990) . The NIb of TEV (tobacco etch virus) has also been detected in the nucleolus (Baunoch et al., 1991; Restrepo et al., 1990) . Potyviruses may also induce nuclear inclusions, which consist of NIa and NIb (Baunoch et al., 1991; Dougherty and Hiebert, 1980; Edwardson and Christie, 1983; Knuhtsen et al., 1974; Martin et al., 1992) . The significance of these nuclear inclusions is unknown but they may simply represent inactivated protein complexes, because they seem to form only at late stages of virus infection (Baunoch et al., 1991) .

NIa is multifunctional and consists of two domains. The N-proximal half of NIa is the VPg that is covalently linked to the 5 0 -end of viral genomic RNA (Oruetxebarria et al., 2001; Siaw et al., 1985) . The C-proximal part (NIa-Pro) is the main viral proteinase (Dougherty et al., 1989) . The two domains are separated by a suboptimal proteolytic cleavage site, the slow processing of which is essential for efficient viral replication Schaad et al., 1996) . The majority of full-length NIa is found in the nucleus of virus-infected cells . However, NIa can also exist as a polyprotein with the 6K2 protein located upstream of NIa in the viral polyprotein. The 6K2 protein impedes nuclear localization of the 6K2-NIa polyprotein (Restrepo-Hartwig and Carrington, 1992) and directs NIa to cytoplasmic membrane vesicles, the sites of viral replication Cotton et al., 2009; Restrepo-Hartwig and Carrington, 1994; Schaad et al., 1997a) . Many lines of evidence suggest that NIa is part of the viral replication complex involving several viral and host proteins Fellers et al., 1998; Li et al., 1997; Murphy et al., 1996; Puustinen and Mäkinen, 2004; Schaad et al., 1996) . The VPg domain has NTP binding activity, is uridylylated by NIb, and may act as a primer for synthesis of viral RNA (Anindya et al., 2005; Murphy et al., 1996; Puustinen and Mäkinen, 2004; Schaad et al., 1996) . In addition, VPg is involved in viral cell-to-cell and long-distance movement (Nicolas et al., 1997; Valkonen, 1999, 2002; Schaad et al., 1997b) . The NLSs and NoLS of potyviral NIa map to the N-proximal part of VPg (Carrington et al., 1991; Rajamäki and Valkonen, 2009; Schaad et al., 1996) . The signal controlling nuclear localization of NIa is bipartite (Carrington et al., 1991; Rajamäki and Valkonen, 2009; Schaad et al., 1996) . The regions of NIa controlling nucleolar targeting of NIa have been studied in PVA and found to map to the same regions as the NLSs. However, mutations in both NLS regions are required to abolish nuclear targeting of PVA NIa, whereas mutations in either NLS prevent nucleolar localization of NIa (Rajamäki and Valkonen, 2009) . The most N-terminal NLS controls also localization of PVA NIa to CBs (Rajamäki and Valkonen, 2009) . Mutations that affect nuclear localization of NIa are detrimental for genome amplification of TEV and PVA (Rajamäki and Valkonen, 2009; Schaad et al., 1996) , suggesting that nuclear/nucleolar localization of NIa has an important role in virus infection.

Potyviral NIa (or VPg) has been shown to interact with several host proteins (Dunoyer et al., 2004; Huang et al., 2010; Léonard et al., 2004; Rajamäki and Valkonen, 2009; Schaad et al., 2000; Thivierge et al., 2008; Wittmann et al., 1997) . The structure of VPg is intrinsically disordered (Grzela et al., 2008; Rantalainen et al., 2008) , which may provide flexibility for many types of interactions. One of the interaction partners of VPg and/or NIa is the eukaryotic translation initiation factor eIF4E or its isoform (Robaglia and Caranta, 2006; Schaad et al., 2000; Wittmann et al., 1997) and the interaction appears important for virus infectivity (Léonard et al., 2000; Robaglia and Caranta, 2006) . Remarkably, interaction of TuMV NIa with eIF(iso)4E and the poly(A) binding protein 2 (PABP2) has been detected in the nucleus and nucleolus using bimolecular fluorescence complementation (BiFC) and colocalization experiments . By contrast, interaction with the 6K2-NIa polyprotein targets eIF(iso)4E and PABP2 to cytoplasmic membrane vesicles . The data suggest that these host proteins are needed in different viral processes. In healthy plants, most of PABP is cytoplasmic but some of the protein is redistributed to the nucleolus probably by the NIa . NIa appears to mediate also eIF (iso)4E localization to the nucleolus and could interact simultaneously with eIF(iso)4E and PABP2 as shown by in vitro binding assays and BiFC and colocalization experiments . Hence, all three proteins may be part of the same complex. Although the role of interaction between NIa and eIF(iso)4E and/or PABP2 in the nucleus is currently unclear, some possibilities can be suggested. The nuclear pool of eIF4E takes part in mRNA export from the nucleus but may also be involved in nuclear translation and NMD of RNA (Strudwick and Borden, 2002) . PABP regulates the initiation of protein synthesis, nuclear export of mature mRNAs, and mRNA stability and decay (Mangus et al., 2003) . Interaction with NIa might disrupt some of these functions. As mentioned above, some animal viruses are known to modulate and inhibit activities of translation initiation factors in order to favor viral replication and translation (reviewed by Thompson and Sarnow, 2000) .

Recent data connect nuclear/nucleolar targeting of plant viral proteins to interference with host antiviral defense. For example, the potyviral VPg protein has been observed to accumulate in the companion cells of upper leaves of a wild potato species ahead of virus infection, which suggested that VPg might interfere with host defense and hence facilitate infection of the cells that receive the virus via systemic transport (Rajamäki and Valkonen, 2003) . Indeed, this hypothesis gained support in the experiments in which overexpression of VPg in leaf tissues temporarily interfered with cosuppression of gene silencing (i.e., RNA silencing), whereas NLS mutants of VPg, which exhibited reduced nuclear and nucleolar localization, were not able to suppress RNA silencing (Rajamäki and Valkonen, 2009) .

RNA silencing is a sophisticated, sequence-specific RNA degradation mechanism operating in the cytoplasm (Ruiz-Ferrer and Voinnet, 2009) . A key feature of the RNA silencing pathway is the generation of dsRNA that corresponds in sequence to the target (virus) RNA. This dsRNA is cleaved into siRNAs by DCLs and these mediate the target specificity for RNA degradation (for reviews, see Carrington, 2000; Ruiz-Ferrer and Voinnet, 2009; Vance and Vaucheret, 2001; Voinnet, 2001) . RNA silencing is a natural anti-viral defense system of plants but is also involved in gene regulation in a wide range of developmental and pathogen defense processes (Ruiz-Ferrer and Voinnet, 2009) . To combat host defense RNA silencing, most plant viruses encode silencing suppressor proteins. The potyviral helper-component proteinase (HC-Pro) was the first detected viral suppressor of RNA silencing (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau and Carrington 1998) and the potyviral P1 protein may enhance its activity (Rajamäki et al., 2005) . HC-Pro acts in the cytoplasm and, therefore, the association of VPg with RNA silencing suppression when localized in the nucleolus is intriguing because many host proteins involved in RNA silencing as well as the processing centers for small RNAs are located in the nucleus, nucleolus, and CBs as discussed in Section II (Pontes and Pikaard, 2008) .

In contrast, nuclear/nucleolar localization of the P19 protein of TBSV, tombusvirus, mediated by plant ALY proteins (mRNA-processing and -export factors) may be a defense mechanism of the plant to down-regulate the silencing suppressor activity of P19 (Canto et al., 2006) .

Previously, suppression of RNA silencing has been found to be connected to nuclear localization of another viral protein: the 2b protein, silencing suppressor of CMV. CMV 2b localizes in the nucleus and the nucleolus where it interacts with Argonaute 1 (AGO1), the core component of the RNA-induced silencing complex (González et al., 2010; Lucy et al., 2000; Zhang et al., 2006) . More recently, it has also been shown that CMV 2b interacts with AGO4 (González et al., 2010) . However, these interactions are not sufficient for suppression of RNA silencing, and hence their biological relevance remains so far unclear (González et al., 2010) .

The VPg of PVA interacts with fibrillarin in the nucleolus and CBs as detected by BiFC experiments (Rajamäki and Valkonen, 2009 ). Depletion of fibrillarin reduces PVA accumulation in Nicotiana benthamiana, suggesting a role for fibrillarin in virus infection (Rajamäki and Valkonen, 2009 ). As mentioned above, in GRV, fibrillarin is recruited for viral long-distance transport (Canetta et al., 2008; Kim et al., 2007a,b) , but in potyviruses the role is likely to be different as long-distance transport of PVA is not compromised by depletion of fibrillarin (Rajamäki and Valkonen, 2009) . Taking into account the silencing suppression activity of VPg, it could be suggested that VPg-fibrillarin might contribute to such an activity. Alternatively, the fibrillarin-VPg interaction may disrupt certain nucleolar functions (e.g., host transcription or pre-mRNA processing), which might explain the observed shutdown of host gene expression during potyvirus infection (Wang and Maule, 1995) .

In mammals, the IFN pathway plays a key role in the innate antiviral immune response whereas involvement of RNA silencing is still controversial (reviewed by Hale et al., 2008) . However, emerging evidence suggests some parallels in how plant and animal viruses could use the nucleolus to counteract host defense. For example, the NS1 protein of influenza A virus is an important factor in counteracting IFN-based cellular antiviral mechanisms (Hale et al., 2008) . In addition, NS1 is also able to suppress RNA silencing in plant, insect, and mammalian cells (Bucher et al., 2004; de Vries et al., 2009; Delgadillo et al., 2004; Haasnoot et al., 2007; Li et al., 2004) . Remarkably, NS1 localizes to the nucleolus and interacts with nucleolin (Murayama et al., 2007) .

NIb is a viral RNA-dependent RNA polymerase of potyviruses and hence involved in the replication complex (Hong and Hunt, 1996; Schaad et al., 1997a) . While viral replication takes place in the cytoplasm on cellular

The Nucleolus and Plant Viruses membranes, NIb is also targeted to the nucleus and the nucleolus (Baunoch et al., 1991; Restrepo et al., 1990) . Nuclear targeting of NIb appears to be highly sensitive to alterations in protein conformation and is eliminated by deletions, dipeptide insertions and amino acid substitutions introduced also into parts of NIb other than the NLSs (Li and Carrington, 1993) . Nuclear and nucleolar localization of NIb may be important for the viral infection cycle, because mutations in the NLSs abolish infectivity of TEV (Li and Carrington, 1995; Li et al., 1997) . Three host proteins, the PABP2, the heat shock cognate 70 protein (Hsc70-3) , and the eukaryotic translation elongation factor 1A, eIF1A, interact with NIb Thivierge et al., 2008; Wang et al., 2000b) . However, none of these protein interactions was found in the nucleolus, shedding no light on the role of the nucleolar localization of NIb. Besides nuclear inclusion proteins, the P3 protein of TEV is targeted to the nucleus and nucleolus of virus-infected tobacco cells, as detected by immunogold labeling (Langenberg and Zhang, 1997) . P3 is a nonstructural protein with no well-characterized function. However, it is involved in virus multiplication (Kekarainen et al., 2002) and virus-host interactions (Chu et al., 1997; Eggenberger et al., 2008; Jenner et al., 2003; Johansen et al., 2001) . A role for nuclear/nucleolar localization of P3 is currently unknown.

The multifunctional nucleocytoplasmic shuttling P6 protein encoded by plant para-retrovirus CaMV has also been found to localize to the nucleolus (Haas et al., 2005) . Although a NoLS has not been identified for this protein, its nucleolar import might be facilitated by its interaction with ribosomal proteins of the 60S ribosomal subunit (Haas et al., 2008) . The presence of P6 in the nucleolus, where assembly of ribosomal subunits occurs, raises the possibility that P6 might interact directly with ribosomes before their export to render them competent for translation of the CaMV polycistronic mRNA. Indeed, P6 interacts with the ribosomal proteins L18 (Leh et al., 2000) , L24 (Park et al., 2001) , and L13 (Bureau et al., 2004) , which hence may be the potential targets because they participate in the formation of the 60S subunit in the nucleolus (Andersen et al., 2002) . Another functional activity of CaMV P6 is suppression of RNA silencing. This protein interferes with RNA-directed RNA polymerase 6 (RDR6)dependent RNA silencing via inhibition of the dsRNA-binding protein DRB4, a protein normally enhancing DCL4-mediated dicing. However, unlike nuclear targeting, the nucleolar localization of P6 is completely dispensable for its silencing suppression function (Haas et al., 2008) .

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant DNA viruses such as the begomoviruses. CPs of monopartite begomoviruses (such as TYLCV and ToLCJAV) are nucleocytoplasmic shuttling proteins thought to be involved in this process (Rojas et al., 2001; Sharma and Ikegami, 2009 ). Interestingly, these proteins also contain NoLSs targeting them to the nucleolus (Sharma and Ikegami, 2009 ). However, the biological significance of nucleolar localization of begomoviral CP is unclear.

NLSs have been identified in three of the seven proteins encoded by plant nucleorhabdovirus, MFSV. Remarkably, two of them, the nucleocapsid (N) protein and phosphoprotein (P), localize to the nucleolus but only when they are coexpressed in plant tissues (as fusions with GFP) using the Agrobacterium system; when expressed individually these proteins do not target nucleoli (Tsai et al., 2005) . This clearly indicates the interdependent character of nucleolar targeting for the N and P proteins. However, the molecular mechanisms responsible for this effect and its biological relevance remain to be explored.

Another plant virus protein with nucleolar localization is the CP of SPMV (satellite panicum mosaic virus) (Qi et al., 2008) . Some of the functional and biochemical properties of the SPMV CP including the nucleolar association of SPMV CP, its RNA binding activity (Desvoyes and Scholthof, 2000) , and the involvement of the CP in systemic movement (Omarov et al., 2005) are similar to those of GRV ORF3. By analogy therefore, the authors have suggested that nucleolar localization of SPMV CP may be essential for its role in the systemic movement of SPMV as in the case of GRV ORF3. Collectively, all of these findings support an active role of the nucleolus and fibrillarin in various aspects of the virus infection cycle and interactions with host cells promoting systemic infections with plant viruses belonging to various taxonomic groups.

The nucleolus contains a complex machinery for rRNA modification and rRNP assembly and may provide an environment which allows other forms of functional RNPs, such as SRP, telomerase, splicing snRNPs, and viral RNPs to exploit the nucleolus or nucleolar components in their biogenesis pathways (reviewed by Boisvert et al., 2007) . For example, fibrillarin is one of the four core protein components of a box C/D snoRNP complex (reviewed by Tran et al., 2004) . Being a methyltransferase, fibrillarin (as a component of box C/D snoRNPs) functions in the 2 0 -in vitro-methylation and processing of pre-rRNA. In addition, human fibrillarin forms a subcomplex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins a3 and b1 that is independent of its association with snoRNPs, suggesting that fibrillarin may also coordinate protein methylation during the processes of ribosome biogenesis . Furthermore fibrillarin interacts with some other cellular proteins such as SMN protein ( Jones et al., 2001) and the nuclear DEAD box protein p68, an RNA-dependent ATPase and RNA helicase (Nicol et al., 2000) . However, the physiological role of these interactions is unclear and may be based on novel natural functions of fibrillarin that remain to be established. The most studied viral RNP complex containing fibrillarin is the GRV ORF3 complex with elements of regular helical structure that is capable of long-distance movement via the phloem. Assembly of GRV RNP particles occurs in the cytoplasm and requires fibrillarin relocalized from the nucleolus (Kim et al., 2007a,b; ; also see above). RNP particles similar in architecture and infectivity to the viral RNPs formed in vivo, have been reconstituted in vitro from fibrillarin, the ORF3 protein and viral RNA (Kim et al., 2007a,b) . Taking the study further, the in vitro experiments have shown that the ORF3-fibrillarin interaction occurs between the leucine-rich region (L149 in particular) of the ORF3 protein and the GAR domain of fibrillarin (Kim et al., 2007b) known to be responsible for interaction with other proteins such as SMN ( Jones et al., 2001) . This interaction leads to formation of single-layered ring-like complexes of ORF3 with fibrillarin as was shown by atomic force microscopy (Canetta et al., 2008) . The diameter of these ORF3 protein-fibrillarin rings is 18-22 nm which correlates with that of the filamentous RNP particles (Canetta et al., 2008) . It thus appears that the ORF3 protein fibrillarin rings interact with viral RNA, encapsidating it and reorganizing it into helical structures, and thereby play a key role in the assembly of umbraviral RNP complexes. These results demonstrate that, in addition to traditional functions in rRNA processing and modification, fibrillarin possesses completely novel functions in mediating assembly of umbraviral RNPs. These functions are presumably based on the ability of the ORF3 protein to interact and form ring-like complexes with fibrillarin such that the virus alters and exploits the properties of fibrillarin for successful virus propagation.

Other viral proteins, such as the nucleocapsid protein encoded by porcine arterivirus also interact with fibrillarin in an RNA-dependent manner . However, the structure and architecture of these complexes and how they impact the viral infection cycle remain unknown.

The nucleolus is a highly conserved feature of eukaryotic cells that has a key role as the site of ribosome subunit production. However, recent multiple lines of investigation have confirmed and characterized additional roles for nucleoli in other important cellular processes including cell-cycle control, stress responses, and coordination of the biogenesis of a number of other functional RNPs. The nucleus has also been shown to play a crucial role in the infection cycle of various viruses, and the nucleolar localization of viral proteins has recently been described as a pan-virus phenomenon (Hiscox, 2002 (Hiscox, , 2007 . In this regard, plant viruses are not different from other eukaryotic viruses. The past few years have brought remarkable progress in our understanding of why and how some plant viruses (in particular, umbraviruses and potyviruses) target the nucleolus and the functional role of the interaction between viral and nucleolar proteins in the plant virus infection cycle. For many other plant virus proteins, nucleolar localization and interaction with nucleolar components have also been demonstrated, and functional implications of these findings is a challenge for future research. We anticipate that more information will emerge about the mechanisms involved in regulating nucleolar function and structure in response to plant virus infections and hijacking nucleolar functions for the virus infection cycle. There are now several examples in which the plant viruses also target other subnuclear bodies, such as CBs. In particular, for umbraviruses the role of CBs in nucleolar trafficking of the ORF3 protein has been established. The potential role of sub-nuclear structures in other plant virus infections will be addressed in the future.

The study of viral interactions with the nucleolus also provides unique and valuable tools to gain new insights into novel nucleolar functions and processes. For example, as previously discussed, the major nucleolar protein fibrillarin, is involved in formation and long-distance movement of umbraviral RNP particles. These functions as well as other potential yet unrecognized natural functions of nucleolar proteins will be the focus of much future research. On a practical level, both the plant cell and viral biology of the nucleolus can, and hopefully will be exploited for the design of novel strategies to control plant virus infections.

NOPdb: Nucleolar proteome database -2008 update

Rhinovirus 3C protease precursors 3CD and 3CD' localize to the nuclei of infected cells

The Nucleolus and Plant Viruses

A viral suppressor of gene silencing in plants

Directed proteomic analysis of the human nucleolus

Nucleolar proteome dynamics

Tyrosine 66 of Pepper vein banding virus genome-linked protein is uridylylated by RNA-dependent RNA polymerase

Expression of the genome of potato leafroll virus: readthrough of the coat protein termination codon

Modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of RNA polymerase I transcription

Fibrillarin genes encode both a conserved nucleolar protein and a novel small nucleolar RNA involved in ribosomal RNA methylation in Arabidopsis thaliana

A temporal study of the expression of the capsid, cytoplasmic inclusion and nuclear inclusion proteins of tobacco etch potyvirus in infected cells

The poly(A) binding protein is internalized in virus-induced vesicles or redistributed to the nucleolus during Turnip mosaic virus infection

Visualization of the interaction between the precursors of VPg, the viral protein linked to the genome of Turnip mosaic virus, and the translation eukaryotic initiation factor iso 4E in planta

Adeno-associated virus interactions with B23/Nucleophosmin: identification of sub-nucleolar regions

The multifunctional nucleolus

Tissue and intra-cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy

Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana

Small nucleolar RNAs and pre-rRNA processing in plants

The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants

P6 protein of Cauliflower mosaic virus, a translation reinitiator, interacts with ribosomal protein L13 from Arabidopsis thaliana

A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro

Translocation of Tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity

Ultrastructural localization of the RNA of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes

RNA silencing. Moving targets

Bipartite signal sequence mediates nuclear translocation of the plant potyviral NIa protein

Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Two separate regions in the genome of the tobacco etch virus contain determinants of the wilting response of Tabasco pepper

An overlapping essential gene in the Potyviridae

Cajal bodies: a long history of discovery

A distant coilin homologue is required for the formation of Cajal bodies in Arabidopsis

Turnip mosaic virus RNA replication complex vesicles are mobile, align with microfilaments and are each derived from a single viral genome

Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses

Cajal body-specific small nuclear RNAs: a novel class of 2 0 -O-methylation and pseudouridylation guide RNAs

Differential RNA silencing suppression activity of NS1 proteins from different influenza A virus strains

Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants

RNA: protein interactions associated with satellites of panicum mosaic virus

Translation of potyvirus RNA in a rabbit reticulocyte lysate: Identification of nuclear inclusion proteins as products of tobacco etch virus RNA translation and cylindrical inclusion protein as a product of the potyvirus genome

Characterization of catalytic residues of the tobacco etch virus 49-kDa proteinase

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

Nuclear transit of the RNA-binding protein She2 is required for translational control of localized ASH1 mRNA

Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

A cysteine-rich plant protein potentiates Potyvirus movement through an interaction with the virus genomelinked protein VPg

Cytoplasmic cylindrical and nucleolar inclusions induced by potato virus-A

Gain of virulence on Rsv1-genotype soybean by an avirulent Soybean mosaic virus requires concurrent mutations in both P3 and HC-Pro

A human snoRNA with microRNA-like functions

Making ribosomes

In vitro interactions between a potyvirus-encoded, genome-linked protein and RNA-dependent RNA polymerase

Effect of anti-fibrillarin antibodies on building of functional nucleoli at the end of mitosis

Structure and functions of nucleolin

Cucumber mosaic virus 2b protein subcellular targets and interactions: Their significance to its RNA silencing suppressor activity

Ribosome biogenesis: of knobs and RNA processing

Involvement of the nucleolus in replication of human viruses

Virulence factor of potato virus Y, genome-attached terminal protein VPg, is a highly disordered protein

The open reading frame VI product of cauliflower mosaic virus is a nucleocytoplasmic protein: Its N terminus mediates its nuclear export and formation of electron-dense viroplasms

Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4

RNA interference against viruses: Strike and counterstrike

NIa and NIb of peanut stripe potyvirus are present in the nucleus of infected cells, but do not form inclusions

The multifunctional NS1 protein of influenza A viruses

Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy

Nucleolar localization of potato leafroll virus capsid proteins

Internal ribosome entry sites in eukaryotic mRNA molecules

Brief review: The nucleolus -a gateway to viral infection?

RNA viruses: Hijacking the dynamic nucleolus

RNA polymerase activity catalyzed by a potyvirusencoded RNAdependent RNA polymerase

The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication

A host RNA helicase-like protein, AtRH8, interacts with the potyviral genome-linked protein, VPg, associates with the virus accumulation complex, and is essential for infection

The nucleolus is involved in mRNA export from the nucleus in fission yeast

Nucleolin stimulates viral internal ribosome entry site-mediated translation

Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm

A putative function of the nucleolus in the assembly or maturation of specialised messenger ribonucleoprotein complexes

The dual role of the potyvirus P3 protein of Turnip mosaic virus as a symptoms and avirulence determinant in Brassicas

Nucleolin/Nsr1p binds to the 3 0 noncoding region of the tombusvirus RNA and inhibits replication

Recessive resistance in Pisum sativum and potyvirus pathotype resolved in a gene-for-cistron correspondence between host and virus

Direct interaction of the spinal muscular atrophy disease protein SMN with the small nucleolar RNAassociated protein fibrillarin

A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing

Functional genomics on potato virus A: Virus genome-wide map of sites essential for virus propagation

Cajal bodies and the nucleolus are required for a plant virus systemic infection

Interaction of a plant virus-encoded protein with the major nucleolar protein fibrillarin is required for systemic virus infection

Aberrant mRNA transcripts and the nonsensemediated decay proteins UPF2 and UPF3 are enriched in the Arabidopsis nucleolus

Small nucleolar RNAs: An abundant group of non-coding RNAs with diverse cellular functions

Partial purification and some properties of tobacco etch virus induced intranuclear inclusions

Dynamic behaviour of the eIF4A-III putative core protein of the exon junction complex: Fast relocation to nucleolus and speckles under hypoxia

Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins

Nuclear speckles: A model for nuclear organelles

Immunocytology shows the presence of tobacco etch virus P3 protein in nuclear inclusions

The Cauliflower mosaic virus translational transactivator interacts with the 60S ribosomal subunit protein L18 of Arabidopsis thaliana

Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity

Interaction of VPg-Pro of Turnip mosaic virus with the translation initiation factor 4E and the poly(A)-binding protein in planta

Nuclear transport of tobacco etch potyviral RNAdependent RNA polymerase is highly sensitive to sequence alterations

Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells

C23 interacts with B23, a putative nucleolar-localization-signal-binding protein

Functions of the tobacco etch virus RNA polymerase (NIb): Subcellular transport and protein-protein interaction with VPg/proteinase (NIa)

Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing

In vivo and in vitro arginine methylation of RNA-binding proteins

Role of Cajal bodies and nucleolus in the maturation of the U1 snRNP in Arabidopsis

Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes

Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus

Inhibition of protein dephosphorylation results in the accumulation of splicing snRNPs and coiled bodies within the nucleolus

Transport into and out of the nucleus. Microbiol

Ultrastructural location of non-structural protein 3A of cucumber mosaic virus in infected tissue using monoclonal antibodies to a cloned chimeric fusion protein

Cytochemical and immunocytochemical characterization of nuclear bodies during hibernation

Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression

Intracellular localization of three non-structural plum pox potyvirus proteins by immunogold labelling

A bromodomaincontaining protein from tomato specifically binds potato spindle tuber viroid RNA in vitro and in vivo

Pumpimg RNA: Nuclear bodybuilding along the RNP pipeline

Nuclear bodies: random aggregates of sticky proteins or crucibles of macromolecular assembly?

Adenovirus protein V induces redistribution of nucleolin and B23 from nucleolus to cytoplasm

What's new in the nucleolus?

Rybozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA

Nucleolin: a multiFACeTed protein

Triple gene block: Modular design of a multifunctional machine for plant virus movement

Influenza A virus non-structural protein 1 (NS1) interacts with cellular multifunctional protein nucleolin during infection

Replacement of the tyrosine residue that links a potyviral VPg to the viral RNA is lethal

Targeting SMN to Cajal bodies and nuclear gems during neuritogenesis

The nuclear DEAD box RNA helicase p68 interacts with the nucleolar protein fibrillarin and colocalizes specifically in nascent nucleoli during telophase

Variations in the VPg protein allow a potyvirus to overcome va gene resistance in tobacco

Nucleocytoplasmic transport: signals, mechanisms and regulation

Coiled bodies in the nucleolus of breast cancer cells

Cajal bodies and coilin-moving towards function

Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins

Nontraditional roles of the nucleolus

The moving parts of the nucleolus

The capsid protein of satellite panicum mosaic virus contributes to systemic invasion and interacts with its helper virus

Getting the message across: How do plant cells exchange macromolecular complexes?

Identification of the genome-linked protein in virions of potato virus A, with comparison to other members in genus Potyvirus

A plant viral 'reinitiation' factor interacts with the host translational machinery

The SMN complex, an assemblysome of ribonucleoproteins

The plurifunctional nucleolus

The survival of motor neurons (SMN) protein interacts with the snoRNP proteins fibrillarin and GAR1

Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions

MicroRNAs with a nucleolar location

siRNA and miRNA processing: New functions for Cajal bodies

Uridylylation of the potyvirus VPg by viral replicase NIb correlates with the nucleotide binding capacity of VPg

The nucleolus is the site of Borna disease virus RNA transcription and replication

Differential subnuclear localization of RNA strands of opposite polarity derived from an autonomously replicating viroid

The complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection

The 6K2 protein and the VPg of potato virus A are determinants of systemic infection in Nicandra physaloides

Viral genome-linked protein (VPg) controls accumulation and phloem-loading of a potyvirus in inoculated potato leaves

Localization of a potyvirus and the viral genome-linked protein in wild potato leaves at an early stage of systemic infection

Control of nuclear and nucleolar localization of nuclear inclusion protein a of picorna-like Potato virus A in Nicotiana species

Infection with potyviruses

A novel insertion site inside the potyvirus P1 cistron allows expression of heterologous proteins and suggests some P1 functions

Potato virus A genome-linked protein VPg is an intrinsically disordered molten globule-like protein with a hydrophobic core

Structure and function of the nucleolus in the spotlight

Nuclear transport of plant potyviral proteins

Regulation of nuclear transport of a plant potyvirus protein by autoproteolysis

The tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication

Dynamic organisation of the cell nucleus

Translation initiation factors: a weak link in plant RNA virus infection

Functional analysis of proteins involved in movement of the monopartite begomovirus, tomato yellow leaf curl virus

Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences

Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

Roles of plant small RNAs in biotic stress responses

Intracellular location of two groundnut rosette umbravirus proteins delivered by PVX and TMV vectors

A plant virus-encoded protein facilitates long-distance movement of heterologous viral RNA

Identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance RNA movement protein

Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing and genome amplification

Formation of plant RNA virus replication complexes on membranes: role of an endoplasmid reticulum-targeted viral protein

VPg of tobacco etch potyvirus is a host genotype-specific determinant for long-distance movement

Strain-specific interaction of the tobacco etch virus NIa protein with the translation initiation factor eIF4E in the yeast twohybrid system

Dynamics and interplay of nuclear architecture, genome organisation, and gene expression

mRNA transport in yeast: Time to reinvestigate the functions of the nucleolus

Subcellular localization of viroids in highly purified nuclei from tomato leaf tissue

Human miRNA precursors with box H/ACA snoRNA features

Characterization of signals that dictate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of tomato leaf curl Java virus associated with DNAb satellite

Plant nuclear bodies

Identification of a protein covalently linked to the 5 0 -terminus of tobacco vein mottling virus RNA

Nucleolus: The fascinating nuclear body

Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway

Dynamic interactions between slicing snRNPs, coiled bodies and nucleoli revealed using snRNP protein fusions to the green fluorescent protein

Mutational analysis of fibrillarin and its mobility in living human cells

Old and new faces of the nucleolus. Workshop on the nucleolus and disease

The emerging roles of translation factor eIF4E in the nucleus

Viroids: petite RNA pathogens with distinguished talents

Small RNAs derived from snoRNAs

Molecular biology of umbraviruses: Phantom warriors

An umbraviral protein, involved in long-distance RNA movement, binds viral RNA and forms unique, protective ribonucleoprotein complexes

Eukaryotic elongation factor 1A interacts with Turnip mosaic virus RNA-dependent RNA polymerase and VPg-Pro in virus-induced vesicles

Regulation of host cell translation by viruses and effects on cell function

Insights into nuclear organisation in plants as revealed by the dynamic distribution of Arabidopsis SR splicing factors

Evolutionary origins of the RNA-guided nucleotide-modification complexes: from the primitive translation apparatus?

Toward a high-resolution dynamics view of nuclear dynamics

Nuclear functions in space and time: Gene expression in a dynamic

Complete genome sequence and in planta subcellular localization of maize fine streak virus proteins

Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product

Nucleolin: a multifunctional major nucleolar phosphoprotein

RNA silencing in plants -defense and counterdefense

Ribosome synthesis in Saccharomyces cerevisiae

RNA silencing as a plant immune system against viruses

Viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells

Inhibition of host gene expression associated with plant virus replication

Crystal structure of a fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 Å resolution

Interaction between zucchini yellow mosaic potyvirus RNA-dependent RNA polymerase and host poly-(A) binding protein

Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system

Localisation to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division

Human fibrillarin forms a sub-complex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins alpha 3 and beta 1 that is independent of its association with preribosomal ribonucleoprotein complexes

Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNAassociated protein fibrillarin

Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense