Higher detection of JC polyomavirus in colorectal cancerous tissue after pretreatment with topoisomerase I enzyme; colorectal tissue serves as a JCPyV persistence site.

BACKGROUND: The JC polyomavirus has been blamed to contribute in colorectal cancer (CRC), however, the topic is still controversial. Varying detection rate of JCPyV genome has been reported mainly due to technical reasons. Here, we provide summative data on the topic, with emphasize on technical issues. METHODS: Formalin-fixed paraffin-embedded tissue samples from 50 patients with CRC, consisting of tumoral and non-cancerous marginal tissue (totally 100 samples) were included in the study. After DNA extraction, specific JCPyV T-Ag sequences were targeted using Real-time PCR. To unwind the supercoiled JCPyV genome, pretreatment with topoisomerase I, was applied. Immunohistochemical (IHC) staining was performed using an anti-T-Ag monoclonal antibody. RESULTS: In the first attempts, no samples were found to be positive in Real-time PCR assays. However, JCPyV sequences were found in 60% of CRC tissues and 38% of non-cancerous colorectal mucosa after application of pre-treatment step with topoisomerase I enzyme (P = 0.028). T-Ag protein was found in the nuclear compartment of the stained cells in IHC assays. CONCLUSIONS: The presence of JCPyV in CRC tissues, as well as T-Ag localization in the nucleolus, where its oncogenic effect takes place, may provide supporting evidence for JCPyV involvement in CRC development. The study highlights the importance of using topoisomerase I to enhance JCPyV genome detection. Also, colorectal tissue is one of the permissive human tissue for JC resistance after preliminary infection.

Plant viral proteins and fibrillarin: the link to complete the infective cycle.

The interaction between viruses with the nucleolus is already a well-defined field of study in plant virology. This interaction is not restricted to those viruses that replicate in the nucleus, in fact, RNA viruses that replicate exclusively in the cytoplasm express proteins that localize in the nucleolus. Some positive single stranded RNA viruses from animals and plants have been reported to interact with the main nucleolar protein, Fibrillarin. Among nucleolar proteins, Fibrillarin is an essential protein that has been conserved in sequence and function throughout evolution. Fibrillarin is a methyltransferase protein with more than 100 methylation sites in the pre-ribosomal RNA, involved in multiple cellular processes, including initiation of transcription, oncogenesis, and apoptosis, among others. Recently, it was found that AtFib2 shows a ribonuclease activity. In plant viruses, Fibrillarin is involved in long-distance movement and cell-to-cell movement, being two highly different processes. The mechanism that Fibrillarin performs is still unknown. However, and despite belonging to very different viral families, the majority comply with the following. (1) They are positive single stranded RNA viruses; (2) encode different types of viral proteins that partially localize in the nucleolus; (3) interacts with Fibrillarin exporting it to the cytoplasm; (4) the viral protein-Fibrillarin interaction forms an RNP complex with the viral RNA and; (5) Fibrillarin depletion affects the infective cycle of the virus. Here we review the relationship of those plant viruses with Fibrillarin interaction, with special focus on the molecular processes of the virus to sequester Fibrillarin to complete its infective cycle.

HBV Core Protein Is in Flux between Cytoplasmic, Nuclear, and Nucleolar Compartments.

Hepatitis B virus (HBV) core protein (Cp) can be found in the nucleus and cytoplasm of infected hepatocytes; however, it preferentially segregates to a specific compartment correlating with disease status. Regulation of this intracellular partitioning of Cp remains obscure. In this paper, we report that cellular compartments are filled and vacated by Cp in a time- and concentration-dependent manner in both transfections and infections. At early times after transfection, Cp, in a dimeric state, preferentially localizes to the nucleolus. Later, the nucleolar compartment is emptied and Cp progresses to being predominantly nuclear, with a large fraction of the protein in an assembled state. Nuclear localization is followed by cell-wide distribution, and then Cp becomes exclusively cytoplasmic. The same trend in Cp movement is seen during an infection. Putative nucleolar retention signals have been identified and appear to be structure dependent. Export of Cp from the nucleus involves the CRM1 exportin. Time-dependent flux can be recapitulated by modifying Cp concentration, suggesting transitions are regulated by reaching a threshold concentration.IMPORTANCE HBV is an endemic virus. More than 250 million people suffer from chronic HBV infection and about 800,000 die from HBV-associated disease each year. HBV is a pararetrovirus; in an infected cell, viral DNA in the nucleus is the template for viral RNA that is packaged in nascent viral capsids in the cytoplasm. Inside those capsids, while resident in cytoplasm, the linear viral RNA is reverse transcribed to form the circular double-stranded DNA (dsDNA) of the mature virus. The HBV core (or capsid) protein plays a role in almost every step of the viral life cycle. Here, we show the core protein appears to follow a programmed, sequential localization from cytoplasmic translation then into the nucleolus, to the nucleus, and back to the cytoplasm. Localization is primarily a function of time, core protein concentration, and assembly. This has important implications for our understanding of the mechanisms of antivirals that target HBV capsid assembly.

Regions of bovine adenovirus-3 IVa2 involved in nuclear/nucleolar localization and interaction with pV.

Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24-48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1-25 and 373-448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4-18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1-25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1-25 and 120-140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.

Epitope mapping and cellular localization of swine acute diarrhea syndrome coronavirus nucleocapsid protein using a novel monoclonal antibody.

A swine acute diarrhea syndrome coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in Southern China in 2017. To develop an antigen that is specific, sensitive, and easy to prepare for serological diagnosis, antigenic sites in the SADS-CoV nucleocapsid (N) protein were screened. We generated and characterized an N-reactive monoclonal antibody (mAb) 3E9 from mice immunized with recombinant N protein. Through fine epitope mapping of mAb 3E9 using a panel of eukaryotic-expressed polypeptides with GFP-tags, we identified the motif 343DAPVFTPAP351 as the minimal unit of the linear B-cell epitope recognized by mAb 3E9. Protein sequence alignment indicated that 343DAPVFTPAP351 was highly conserved in different SADS-CoV strains and SADS-related coronaviruses from bat, with one substitution in this motif in HKU2-related bat coronavirus. Using mAb 3E9, we observed that N protein was expressed in the cytoplasm and was in the nucleolus during SADS-CoV replication. N protein was immunoprecipitated from SADS-CoV-infected Vero E6 cells. Taken together, our results indicated that 3E9 mAb could be a useful tool to investigate the structure and function of N protein during viral replication.

Non-structural protein 1 of H3N2 influenza A virus induces nucleolar stress via interaction with nucleolin.

The nucleolus is a stress sensor associated with cell cycle progression and a central hub for the replication of pathogenic RNA viruses. However, the role of nucleolus in influenza A virus infection has not been well studied. Here we show that the interaction between NS1 protein of influenza A/Shantou/602/06 (H3N2) and nucleolin, a ubiquitous protein of nucleolus repressed RNA Pol I-dependent transcription via establishing hyper-methylation in the UCE of rRNA gene promoter. NS1 expressed cells showed significant association of ribosomal proteins with MDM2, and p53 accumulation, suggesting induced nucleolar stress. Disruption of the interaction of NS1 with nucleolin or overexpression of nucleolin in NS1 expressed cells revived RNA Pol I-dependent transcription, indicating nucleolin could be one target for NS1 to repress rRNA synthesis of host cells. Our present study suggests that NS1 protein of H3N2 could induce nucleolar stress based on epigenetic alteration of rRNA gene promoter via interaction with nucleolin.

Molecular cloning and characterization of the genes encoding the proteins of Zika virus.

Zika virus (ZIKV) encodes a precursor protein (also called polyprotein) of about 3424 amino acids that is processed by proteases to generate 10 mature proteins and a small peptide. In the present study, we characterized the chemical features, suborganelle distribution and potential function of each protein using Flag-tagged protein expression system. Western blot analysis revealed the molecular weight of the proteins and the polymerization of E, NS1, and NS3 proteins. In addition, we performed multi-labeled fluorescent immunocytochemistry and subcellular fractionation to determine the subcellular localization of these proteins in host cells. We found that 1) the capsid protein colocalizes with 3 different cellular organelles: nucleoli, Golgi apparatus, and lipid droplet; NS2b and NS4a are associated with the Golgi apparatus; 2) the capsid and NS1proteins distribute in both cytoplasm and nucleus, NS5 is a nuclear protein; 3) NS3 protein colocalizes with tubulin and affects Lamin A; 4) Envelope, PrM, and NS2a proteins co-localize with the endoplasmic reticulum; 5) NS1 is associated with autophagosomes and NS4b is related to early endosome; 6) NS5 forms punctate structures in the nucleus that associate with splicing compartments shown by SC35, leading to reduction of SC35 protein level and trafficking of SC35 from the nucleus to the cytoplasm. These data suggest that ZIKV generates 10 functional viral proteins that exhibit distinctive subcellular distribution in host cells.

Cleavage of bovine adenovirus type 3 non-structural 100K protein by protease is required for nuclear localization in infected cells but is not essential for virus replication.

The L6 region of bovine adenovirus type 3 (BAdV-3) encodes a non-structural protein named 100K. Rabbit antiserum raised against BAdV-3 100K recognized a protein of 130 kDa at 12-24 h and proteins of 130, 100, 95 and 15 kDa at 36-48 h after BAdV-3 infection. The 100K species localized to the nucleus and the cytoplasm of BAdV-3-infected cells. In contrast, 100K localized predominantly to the cytoplasm of the transfected cells. However, BAdV-3 infection of cells transfected with 100K-enhanced yellow fluorescent protein-expressing plasmid detected fluorescent protein in the nucleus of the cells, suggesting that other viral proteins may be required for the nuclear localization of 100K. Interaction of BAdV-3 100K with BAdV-3 33K protein did not alter the cytoplasmic localization of 100K. However, co-expression of BAdV-3 100K and BAdV-3 protease localized 100K to the nucleolus of the transfected cells. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (aa 740-745 and 781-786) in transfected or BAdV-3-infected cells. The cleaved C terminus (107 aa) was localized to the nucleolus of the transfected cells. Further analysis suggested that the cleaved C terminus contains a bipartite nuclear localization signal and utilizes import receptor importin-α3 of the classical importin-α/ß transport pathway for nuclear transport. Successful isolation of recombinant BAdV-3 expressing mutant 100K (substitution of alanine for glycine in the potential protease cleavage site) suggested that cytoplasmic cleavage of BAdV-3 100K by adenoviral protease is not essential for virus replication.

Identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein.

UNLABELLED: Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2(144-184)). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and ß-galactosidase-AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2(144-184) has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly. IMPORTANCE: Adeno-associated virus (AAV) has become a popular and successful vector for in vivo gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of the assembly-activating protein (AAP), a nonstructural, nucleolar-localizing AAV protein essential for viral capsid assembly, has provided us a new opportunity to better understand the fundamental processes required for virion formation. Here, we identify clusters of basic amino acids in the carboxy terminus of AAP from AAV serotype 2 (AAV2) that act as nuclear and nucleolar localization signals. We also demonstrate their importance in maintaining AAP expression levels and efficient production of viral capsids. Insights into the functions of AAP can elucidate the requirements and process for AAV capsid assembly, which may lead to improved vector production for use in gene therapy. This study also contributes to the growing body of work on nuclear and nucleolar localization signals.

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement.

Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2-YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2-YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2-YFP fluorescence were affected in fibrillarin-silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.

Tobacco etch virus protein P1 traffics to the nucleolus and associates with the host 60S ribosomal subunits during infection.

UNLABELLED: The genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells. IMPORTANCE: In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly targeting the nucleolus. Our experiments also showed that P1 protein binds host ribosomes during infection. Based on these findings and other in vitro experiments we propose that P1 protein stimulates translation of viral proteins during infection.

Transcriptome profiling confirmed correlations between symptoms and transcriptional changes in RDV infected rice and revealed nucleolus as a possible target of RDV manipulation.

BACKGROUND: Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which limits rice production in many areas of south East Asia. Transcriptional changes of rice in response to RDV infection have been characterized by Shimizu et al. and Satoh et al.. Both studies found induction of defense related genes and correlations between transcriptional changes and symptom development in RDV-infected rice. However, the same rice cultivar, namely Nipponbare belonging to the Japonic subspecies of rice was used in both studies. METHODS: Gene expression changes of the indica subspecies of rice, namely Oryza sativa L. ssp. indica cv Yixiang2292 that show moderate resistance to RDV, in response to RDV infection were characterized using an Affymetrix Rice Genome Array. Differentially expressed genes (DEGs) were classified according to their Gene Ontology (GO) annotation. The effects of transient expression of Pns11 in Nicotiana benthaminana on the expression of nucleolar genes were studied using real-time PCR (RT-PCR). RESULTS: 856 genes involved in defense or other physiological processes were identified to be DEGs, most of which showed up-regulation. Ribosome- and nucleolus related genes were significantly enriched in the DEGs. Representative genes related to nucleolar function exhibited altered expression in N. benthaminana plants transiently expressing Pns11 of RDV. CONCLUSIONS: Induction of defense related genes is common for rice infected with RDV. There is a co-relation between symptom severity and transcriptional alteration in RDV infected rice. Besides ribosome, RDV may also target nucleolus to manipulate the translation machinery of rice. Given the tight links between nucleolus and ribosome, it is intriguing to speculate that RDV may enhance expression of ribosomal genes by targeting nucleolus through Pns11.

The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process.

De-repression of RaRF-mediated RAR repression by adenovirus E1A in the nucleolus.

Transcriptional activity of the retinoic acid receptor (RAR) is regulated by diverse binding partners, including classical corepressors and coactivators, in response to its ligand retinoic acid (RA). Recently, we identified a novel corepressor of RAR called the retinoic acid resistance factor (RaRF) (manuscript submitted). Here, we report how adenovirus E1A stimulates RAR activity by associating with RaRF. Based on immunoprecipitation (IP) assays, E1A interacts with RaRF through the conserved region 2 (CR2), which is also responsible for pRb binding. The first coiled-coil domain of RaRF was sufficient for this interaction. An in vitro glutathione-S-transferase (GST) pull-down assay was used to confirm the direct interaction between E1A and RaRF. Further fluorescence microscopy indicated that E1A and RaRF were located in the nucleoplasm and nucleolus, respectively. However, RaRF overexpression promoted nucleolar translocation of E1A from the nucleoplasm. Both the RA-dependent interaction of RAR with RaRF and RAR translocation to the nucleolus were disrupted by E1A. RaRF-mediated RAR repression was impaired by wild-type E1A, but not by the RaRF binding-defective E1A mutant. Taken together, our data suggest that E1A is sequestered to the nucleolus by RaRF through a specific interaction, thereby leaving RAR in the nucleoplasm for transcriptional activation.

The HBx protein of hepatitis B virus confers resistance against nucleolar stress and anti-cancer drug-induced p53 expression.

The nucleolus is a stress sensor associated with cell cycle progression and a viral target. However, the role of the nucleolus during hepatitis B virus infection has not been studied. Here we show that under nucleolar stress, the HBx oncoprotein down-regulates p53 and p21(waf1) levels by disrupting the interaction between ribosomal protein L11 and MDM2. Further, HBx inhibited Act D-mediated down-regulation of proliferative factors such as c-Myc and cyclin E and revived RNA pol I-dependent transcription under these conditions. Importantly, HBx also countered the action of anticancer drug Paclitaxel suggesting its possible role in drug resistance. Thus, HBx not only can facilitate cell proliferation under stress conditions but can confer resistance against anticancer drugs.

NC-mediated nucleolar localization of retroviral gag proteins.

The assembly and release of retrovirus particles from the cell membrane is directed by the Gag polyprotein. The Gag protein of Rous sarcoma virus (RSV) traffics through the nucleus prior to plasma membrane localization. We previously reported that nuclear localization of RSV Gag is linked to efficient packaging of viral genomic RNA, however the intranuclear activities of RSV Gag are not well understood. To gain insight into the properties of the RSV Gag protein within the nucleus, we examined the subnuclear localization and dynamic trafficking of RSV Gag. Restriction of RSV Gag to the nucleus by mutating its nuclear export signal (NES) in the p10 domain or interfering with CRM1-mediated nuclear export of Gag by leptomycin B (LMB) treatment led to the accumulation of Gag in nucleoli and discrete nucleoplasmic foci. Retention of RSV Gag in nucleoli was reduced with cis-expression of the 5' untranslated RU5 region of the viral RNA genome, suggesting the psi (Ψ) packaging signal may alter the subnuclear localization of Gag. Fluorescence recovery after photobleaching (FRAP) demonstrated that the nucleolar fraction of Gag was highly mobile, indicating that there was rapid exchange with Gag proteins in the nucleoplasm. RSV Gag is targeted to nucleoli by a nucleolar localization signal (NoLS) in the NC domain, and similarly, the human immunodeficiency virus type 1 (HIV-1) NC protein also contains an NoLS consisting of basic residues. Interestingly, co-expression of HIV-1 NC or Rev with HIV-1 Gag resulted in accumulation of Gag in nucleoli. Moreover, a subpopulation of HIV-1 Gag was detected in the nucleoli of HeLa cells stably expressing the entire HIV-1 genome in a Rev-dependent fashion. These findings suggest that the RSV and HIV-1 Gag proteins undergo nucleolar trafficking in the setting of viral infection.

Disassociation of the SV40 genome from capsid proteins prior to nuclear entry.

BACKGROUND: Previously, we demonstrated that input SV40 particles undergo a partial disassembly in the endoplasmic reticulum, which exposes internal capsid proteins VP2 and VP3 to immunostaining. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection, as well as to detection by an ethynyl-2-deoxyuridine (EdU)-based chemical reaction. The cytoplasmic partially disassembled SV40 particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. FINDINGS: In the current study, we asked where in the cell the SV40 genome might disassociate from capsid components. We observed partially disassembled input SV40 particles around the nucleus and, beginning at 12 hours post-infection, 5-Bromo-2-deoxyuridine (BrdU)-labeled parental SV40 DNA in the nucleus, as detected using anti-BrdU antibodies. However, among the more than 1500 cells examined, we never detected input VP2/VP3 in the nucleus. Upon translocation of the BrdU-labeled SV40 genomes into nuclei, they were transcribed and, thus, are representative of productive infection. CONCLUSIONS: Our findings imply that the SV40 genome disassociates from the capsid proteins before or at the point of entry into the nucleus, and then enters the nucleus devoid of VP2/3.

The signal peptide of mouse mammary tumor virus-env: a phosphoprotein tumor modulator.

Mouse mammary tumor virus (MMTV) is associated primarily with mammary carcinomas and lymphomas. The signal peptide of the MMTV envelope precursor is uniquely targeted to nucleoli of cells that harbor the virus, where it can function as a nuclear export factor for intron-containing transcripts. Antibodies to this signal peptide, which we refer to as p14, were previously shown to label nucleoli in a subset of human breast cancers. To look for additional cellular functions of p14, different mutants were ectopically expressed in the MCF-7 human breast cancer cell line. This approach identified motifs responsible for its nucleolar targeting, nucleocytoplasmic shuttling, target protein (B23, nucleophosmin) binding, and phosphorylation at serine 18 and 65 both in situ and in vitro. To test the role of these phosphorylation sites, we carried out in vivo tumorigenesis studies in severe combined immunodeficient mice. The findings show that the p14-Ser65Ala mutation is associated with impaired tumorigenicity, whereas the p14-Ser18Ala mutation is associated with enhanced tumorigenicity. Microarray analysis suggests that phosphorylation at serine 18 or at serine 65 is associated with transcriptional regulation of the L5 nucleolar ribosomal protein (a p14 target) and the Erb-B signal transduction pathway. Taken together, these results show that the phosphorylation status of p14 determines whether it functions as a pro-oncogenic or antioncogenic modulator.

The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin.

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A (116)KSKRKKKNKK(125) and B (175)KKATKKESKKQTK(187) reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein-protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.

A viral assembly factor promotes AAV2 capsid formation in the nucleolus.

The volume available in icosahedral virus capsids limits the size of viral genomes. To overcome this limitation, viruses have evolved strategies to increase their coding capacity by using more than one ORF while keeping the genome length constant. The assembly of virus capsids requires the coordinated interaction of a large number of subunits to generate a highly ordered structure in which the viral genome can be enclosed. To understand this process, it is essential to know which viral and nonviral components are involved in the assembly reaction. Here, we show that the adeno-associated virus (AAV) encodes a protein required for capsid formation by means of a nested, alternative ORF of the cap gene. Translation is initiated at a nonconventional translation start site, resulting in the expression of a protein with a calculated molecular weight of 23 kDa. This protein, designated assembly-activating protein (AAP), is localized in the host cell nucleolus, where AAV capsid morphogenesis occurs. AAP targets newly synthesized capsid proteins to this organelle and in addition fulfils a function in the assembly reaction itself. Sequence analysis suggests that also all other species of the genus Dependovirus encode a homologous protein in their cap gene. The arrangement of different ORFs that encode capsid proteins and an assembly factor within the same mRNA facilitates a timely coordinated expression of the components involved in the assembly process.