Effects of a highly basic region of human immunodeficiency virus Tat protein on nucleolar localization.

Human immunodeficiency virus type 1 encodes a positive trans-activator protein, Tat, which is located predominantly in the cell nucleolus. To study the role of the basic region of Tat in nucleolar localization, we constructed fusion genes encoding serially deleted segments of Tat joined to the amino-terminal end of the Escherichia coli beta-galactosidase molecule. We show that the basic region of Tat was sufficient for nuclear localization but not for nucleolar localization. Addition of three amino acids (59, 60, and 61) of the Tat sequence at the C-terminal end of the basic region was necessary for the chimeric beta-galactosidase to localize in the nucleus as well as in the nucleolus. We demonstrate that a short amino acid sequence (G-48 RKKRRQRRRA HQ N-61), when fused to the amino terminus of beta-galactosidase, can act as a nucleolar localization signal.     

Inhibition of human immunodeficiency virus type 1 and type 2 Tat function by transdominant Tat protein localized to both the nucleus and cytoplasm.

We introduced various mutations into the activation and RNA binding domains of human immunodeficiency virus type 1 (HIV-1) Tat in order to develop a novel and potent transdominant Tat protein and to characterize its mechanism of action. The different mutant Tat proteins were characterized for their abilities to activate the HIV LTR and inhibit the function of wild-type Tat in trans. A Tat protein containing a deletion of the basic domain (Tat(delta)49-57) localized exclusively to the cytoplasm of transfected human cells was nonfunctional and inhibited both HIV-1 and HIV-2 Tat function in a transdominant manner. Tat proteins containing mutations in the cysteine-rich and core domains were nonfunctional but failed to inhibit Tat function in trans. When Tat nuclear or nucleolar localization signals were fused to the carboxy terminus of Tat(delta)49-57, the chimeric proteins localized to the nucleus or nucleolus, respectively, and remained capable of acting in a transdominant manner. Introduction of secondary mutations in the cysteine-rich and core domains of the various transdominant Tat proteins completely eliminated their abilities to act in a transdominant fashion. Our data best support a mechanism in which these transdominant Tat proteins squelch a cellular factor or factors that interact with the Tat activation domain and are required for Tat to function.     

Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication

Japanese encephalitis virus (JEV) core protein is detected not only in the cytoplasm but also in the nucleoli of infected cells. We previously showed that a mutant JEV lacking the nucleolar localization of the core protein impaired viral replication in mammalian cells. In this study, we identified a nucleolar phosphoprotein B23 as a protein binding with the core protein of JEV but not with that of dengue virus. The region binding with JEV core protein was mapped to amino acid residues 38 to 77 of B23. Upon JEV infection, some fraction of B23 was translocated from the nucleoli to the cytoplasm, and cytoplasmic B23 was colocalized with the core protein of wild-type JEV but not with that of the mutant JEV. Furthermore, overexpression of dominant negatives of B23 reduced JEV replication. These results suggest that B23 plays an important role in the intracellular localization of the core protein and replication of JEV.     

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.     

The RNA binding activity of a ribosome biogenesis factor,nucleophosmin/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA.     

Immunofluorescence studies on proteins B23 and C23 in nucleoli of human lymphocytes

Nucleoli of normal and leukemic lymphocytes were studied by cytochemical and immunofluorescence methods to provide more information on the nucleolar presence and distribution of proteins B23 and C23. Annular nucleoli of human lymphocytes represent a very convenient subject for such studies, since they consist of one centrally located large fibrillar center surrounded by RNP components. In such nucleoli, protein C23 was present mainly in the central nucleolar region and protein B23 was found mostly in the periphery. The nucleolar area immunostained for protein B23 was usually larger than that stained for protein C23. The distribution of protein C23 appeared to be similar to that of intensely stained nucleolar argyrophilic components. No substantial differences were found between the distribution of proteins B23 and C23 in nucleoli of normal and leukemic lymphocytes. In lymphocytes of patients treated with chemotherapy, the immunofluorescence was diminished for protein B23 and particularly so for protein C23.     

Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site.

The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins induce cytoplasmic expression of incompletely spliced viral mRNAs by binding to these mRNAs in the nucleus. Each protein binds a specific cis-acting element in its target RNAs. Both proteins also associated with nucleoli, but the significance of this association is uncertain because mutations that inactivate nucleolar localization signals in Rev or Rex also prevent RNA binding. Here we demonstrate that Rev and Rex can function when tethered to a heterologous RNA binding site by a bacteriophage protein. Under these conditions, cytoplasmic accumulation of unspliced RNA occurs without the viral response elements, mutations in the RNA binding domain of Rev do not inhibit function, and nucleolar localization can be shown to be unnecessary for the biological response.     

Essential role of the B23/NPM core domain in regulating ARF binding and B23 stability

How cells coordinate inhibition of growth and division during genotoxic events is fundamental to our understanding of the origin of cancer. Despite increasing interest and extensive study, the mechanisms that link regulation of DNA synthesis and ribosomal biogenesis remain elusive. Recently, the tumor suppressor p14(ARF) (ARF) has been shown to interact functionally with the nucleolar protein B23/NPM (B23) and inhibit rRNA biogenesis. However, the molecular basis of the ARF-B23 interaction is hitherto unclear. Here we show that a highly conserved motif in the B23 oligomerization domain is essential for mediating ARF binding in vivo. Mutagenesis of conserved B23 core residues (L102A, G105A, G107A) prevented B23 from interacting with ARF. Modeling of the B23 core indicated that substitutions in the GSGP loop motif could trigger conformational changes in B23 thereby obstructing ARF binding. Interestingly, the GSGP loop mutants were unstable, defective for oligomerization, and delocalized from the nucleolus to the nucleoplasm. B23 core mutants displayed increased ubiquitination and proteasomal degradation. We conclude that the functional integrity of the B23 core motif is required for stability, efficient nucleolar localization as well as ARF binding.     

Intrinsically disordered regions of nucleophosmin/B23 regulate its RNA binding activity through their inter- and intra-molecular association

Nucleophosmin (NPM1/B23) is a nucleolar protein implicated in growth-associated functions, in which the RNA binding activity of B23 plays essential roles in ribosome biogenesis. The C-terminal globular domain (CTD) of B23 has been believed to be the RNA binding domain because the splicing variant B23.2 lacking the CTD binds considerably less efficiently to RNA. However, the recognition of target RNAs by B23 remains poorly understood. Herein, we report a novel mechanism by which B23 recognizes specific RNA targets. We observed that the nucleolar retention of B23.3 lacking the basic region of B23.1 was lower than that of B23.1 because of its low RNA binding activity. Circular dichroism measurements indicated that the basic region and adjacent acidic regions of B23 are intrinsically disordered regions (IDRs). Biochemical analyses revealed that the basic IDR alone strongly binds to RNA with low specificity. The excessive RNA binding activity of the basic IDR was restrained by intra-molecular interaction with the acidic IDR of B23. Chemical cross-linking experiments and fluorescent labeling of bipartite tetracysteine-tagged proteins suggested that the inter- and intra-molecular interactions between the two IDRs contribute to the regulation of the RNA binding activity of CTD to control the cellular localization and functions of B23.     

Charge versus sequence for nuclear/nucleolar localization of plant ribosomal proteins

Ribosomal subunit assembly in the nucleolus is dependent on efficient targeting of ribosomal proteins (RPs) from the cytoplasm into the nucleus and nucleolus. Nuclear/nucleolar localization of a protein is generally mediated by one or more specific stretches of basic amino acids—nuclear/nucleolar localization signals (NLSs/NoLSs). Arabidopsis thaliana RPL23aA has eight putative NLSs/NoLSs (pNLSs/NoLSs). Here we mutated all eight NLS/NoLSs individually and in groups and showed, via transient expression in tobacco cells that nucleolar localization of RPL23aA was disrupted by mutation of various combinations of five or more pNLSs/NoLSs. Mutation of all eight pNLSs/NoLSs, a 50 % reduction in total basic charge of RPL23aA, resulted in a complete disruption of nucleolar localization, however, the protein can still localize to the nucleus. As no individual or specific combination of NoLSs was absolutely required for nucleolar localization, we suggest that nucleolar localization/retention of RPL23aA is dependent on the overall basic charge. In addition to the optimal basic charge conferred by these NoLSs, nucleolar localization/retention of RPL23aA also required a C-terminal putative 26S rRNA binding site. In contrast, in the RPs RPS8A and RPL15A, mutation of just two and three N-terminal pNLSs, respectively, disrupted both nuclear and nucleolar localization of these two RPs, indicating differential signal requirements for nuclear and nucleolar localization of the three Arabidopsis RPs RPL23aA, RPL15A and RPS8A.     

Topogenesis of a nucleolar protein: determination of molecular segments directing nucleolar association.

To identify the element(s) in nucleolar proteins which determine nucleolus-specific topogenesis, we have used different kinds of cDNA constructs encoding various chimeric combinations of mutants of the constitutive nucleolar protein NO38 (B23): 1) with an amino terminally placed short "myc tag"; 2) with two different carboxyl terminally attached large alpha-helical coiled coil structures, the lamin A rod domain or the rod domain of vimentin; 3) with the sequence-related nucleoplasmic histone-binding protein nucleo-plasmin; and 4) with the soluble cytoplasmic protein pyruvate kinase. To avoid the problem of formation of complexes with endogenous wild-type (wt) molecules and "piggyback" localization, special care was taken to secure that the mutants and chimeras used did not oligomerize as is typical of protein NO38 (B23). Using microinjection and transfection of cultured cells, we found that the segment comprising the amino-terminal 123 amino acids (aa) alone was sufficient to effect nucleolar accumulation of the construct molecules, including the chimeras with the entire rod domains of lamin A and vimentin. However, when the amino-terminal 109 aa were deleted, the molecules still associated with the nucleolus. The results of further deletion experiments and of domain swaps with nucleoplasmin all point to the topogenic importance of two independent molecular regions located at both the amino- and carboxyl-terminal end. Our definition of dominant elements determining the nucleolar localization of protein NO38 (B23) as well as of diverse nonnucleolar proteins will help to identify its local binding partner(s) and functions, the construction of probes examining other proteins or sequence elements within the nucleolar microenvironment, and the generation of cells with an altered nuclear architecture.     

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

ABSTRACT: BACKGROUND: Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. RESULTS: Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. CONCLUSION: NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin.     

Shiga toxin B-subunit binds to the chaperone BiP and the nucleolar protein B23

BACKGROUND INFORMATION: In many cell lines, such as HeLa cells, STxB (Shiga toxin B-subunit) is transported from the plasma membrane to the ER (endoplasmic reticulum), via early/recycling endosomes and the Golgi apparatus, bypassing the late endocytic pathway. In human monocyte-derived macrophages and dendritic cells that are not sensitive to Shiga toxin-induced protein biosynthesis inhibition, STxB is not detectably targeted to the retrograde route and is degraded in late endosomes/lysosomes. RESULTS: We have identified B-subunit interacting proteins in HeLa cells and macrophages. In HeLa cells, the ER-localized chaperone BiP (binding protein) was co-immunoprecipitated with the B-subunit. This interaction was not observed in macrophages, consistent with our previous trafficking results. In both cell types, the B-subunit also interacted with the nucleolar protein B23. Consistently, the B-subunit could be detected on nucleoli, suggesting that it could serve to bring the holotoxin to the site of synthesis of its molecular target, rRNA. The nucleolar localization data are critically discussed. CONCLUSION: The interaction of STxB with BiP, involved in the retrotranslocation process to the cytosol and nucleolar B23, as described in this study, might be of relevance for explaining the efficiency of even low doses of Shiga toxin to inactivate cellular ribosomes, and for the use of STxB as a vector for targeting antigens to cytosolic proteasomes of the MHC I-restricted antigen presentation pathway.     

Functional mapping of the human immunodeficiency virus type 1 Rev RNA binding domain: new insights into the domain structure of Rev and Rex.

Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the direct interaction of the viral trans-activator protein Rev with its cis-acting RNA sequence (Rev-response element [RRE]). A stretch of 14 amino acid residues of the 116-amino-acid Rev protein is sufficient to impose nucleolar localization onto a heterologous protein. Our results demonstrated that these same amino acid residues confer Rev-specific RRE binding to the heterologous human T-cell leukemia virus type I Rex protein. In addition, our results indicated that amino acids distinct from the nuclear localization signal are important for Rex-specific RRE RNA binding.     

Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23

Protein B23 is a multifunctional nucleolar protein whose molecular chaperone activity is proposed to play role in ribosome assembly. Previous studies (Szebeni, A., and Olson, M. O. J. (1999) Protein Sci. 8, 905-912) showed that protein B23 has several characteristics typical of molecular chaperones, including anti-aggregation activity, promoting the renaturation of denatured proteins, and preferential binding to denatured substrates. However, until now there has been no proposed mechanism for release of a bound substrate. Protein B23 can be phosphorylated by protein kinase CK2 (CK2) in a segment required for chaperone activity. The presence of bound substrate enhanced the rate of CK2 phosphorylation of protein B23 by 2-3-fold, and this enhancement was dependent on a nonpolar region in its N-terminal end. Formation of a complex between B23 and chaperone test substrates (rhodanese or citrate synthase) was inhibited by CK2 phosphorylation. Furthermore, CK2 phosphorylation of a previously formed B23-substrate complex promoted its dissociation. The dissociation of complexes between B23 and the human immunodeficiency virus-Rev protein required both CK2 phosphorylation and competition with a Rev nuclear localization signal peptide, suggesting that Rev binds B23 at two separate sites. These studies suggest that unlike many molecular chaperones, which directly hydrolyze ATP, substrate release by protein B23 is dependent on its phosphorylation by CK2.     

Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation

Bright nucleolar immunofluorescence was observed in HeLa S3 cells by immunostaining with a monoclonal antibody to the nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1). After 48 h of incubation in a serum-free medium, the nucleolar fluorescence was diminished and a general nuclear immunofluorescence was observed. This change in localization of fluorescence indicated that protein B23 had migrated out of the nucleoli. No gross morphological change in nucleoli was observed by light microscopy and the immunolocalization of another nucleolar phosphoprotein, C23, was unaffected by serum deprivation. Relocation of protein B23 in nucleoli was observed after refeeding with serum-containing medium. This re-entry process was not observed after treatment with actinomycin D (50 ng/ml-5 micrograms/ml), but the process was unaffected by cycloheximide (0.2 mM). Quantitation of protein B23 in the nucleoli of the control (fed) or starved HeLa cells was done by ELISA immunoassay. A marked decrease in the amount of protein B23 occurred in the nucleoli of the starved cells (11.8 micrograms B23/mgDNA) as compared with the control nucleoli (20.8 micrograms B23/mgDNA). The amount of protein B23 in the nucleoplasm (excluding nucleoli) was 70% higher in the starved cells. Protein B23 was analysed by one- and two-dimensional PAGE. Three components of protein B23 with slightly different molecular weights and pIs (37 kD/5.1, 35 kD/5.1 and 35 kD/5.3) were observed in nucleoli. The lower molecular weight components were predominantly found in the nucleoplasm.