Borna disease virus nucleoprotein requires both nuclear localization and export activities for viral nucleocytoplasmic shuttling

Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV) belongs to the order Mononegavirales and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.     

Interactions and nuclear import of the N and P proteins of sonchus yellow net virus, a plant nucleorhabdovirus

We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization.     

Two nuclear localization signals required for transport from the cytosol to the nucleus of externally added FGF-1 translocated into cells

Externally added FGF-1 is transported into the nucleus of cells. It was earlier shown that FGF-1 contains an N-terminal nuclear localization signal (NLS) implicated in the stimulation of DNA synthesis. We here provide evidence that FGF-1 contains a second putative NLS (NLS2), which is located near the C-terminus. It is a bipartite NLS consisting of two clusters of lysines separated by a spacer of 10 amino acids. A fusion protein of GFP and the bipartite NLS was more efficiently transported into the nucleus than GFP alone, indicating that it can act as an NLS in the living cell. FGF-1 mutated in the N-terminal NLS (NLS1) or in the first cluster of the bipartite NLS2 bound to heparin and FGF receptors and activated downstream signaling similarly to the wild-type growth factor. Mutations in the second cluster of NLS2 resulted in impaired interaction with heparin and reduced stability. When radiolabeled FGF-1 with mutated NLS1 or the first lysine cluster of NLS2 was added to NIH/3T3 cells, it was translocated into the cytosol, but not transported efficiently to the nucleus. Phosphorylation of FGF-1 occurs normally in the nucleus, and while wild-type FGF-1 was phosphorylated after addition to cells, the NLS mutants were not. It therefore appears that both NLS1 and NLS2 are important for efficient transport of FGF-1 to the nucleus. Stimulation of DNA synthesis by FGF-1 with mutations in both NLSs was reduced considerably indicating that efficient transport to the nucleus may be involved in the stimulation of DNA synthesis.     

Heat shock cognate protein 70 controls Borna disease virus replication via interaction with the viral non-structural protein X

Borna disease virus (BDV) is a non-segmented, negative-sense RNA virus and has the property of persistently infecting the cell nucleus. BDV encodes a 10-kDa non-structural protein, X, which is a negative regulator of viral polymerase activity but is essential for virus propagation. Recently, we have demonstrated that interaction of X with the viral polymerase cofactor, phosphoprotein (P), facilitates translocation of P from the nucleus to the cytoplasm. However, the mechanism by which the intracellular localization of X is controlled remains unclear. In this report, we demonstrate that BDV X interacts with the 71 kDa molecular chaperon protein, Hsc70. Immunoprecipitation assays revealed that Hsc70 associates with the same region of X as P and, interestingly, that expression of P interferes competitively with the interaction between X and Hsc70. A heat shock experiment revealed that BDV X translocates into the nucleus, dependent upon the nuclear accumulation of Hsc70. Furthermore, we show that knockdown of Hsc70 by short interfering RNA decreases the nuclear localization of both X and P and markedly reduces the expression of viral genomic RNA in persistently infected cells. These data indicate that Hsc70 may be involved in viral replication by regulating the intracellular distribution of X.     

Two major histocompatibility complex class I-restricted epitopes of the Borna disease virus p10 protein identified by cytotoxic T lymphocytes induced by DNA-based immunization

Borna disease virus (BDV) infection of Lewis rats is the most studied animal model of Borna disease, an often fatal encephalomyelitis. In this experimental model, BDV-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a prominent role in the immunopathogenesis of infection by the noncytolytic, persistent BDV. Of the six open reading frames of BDV, CTLs to BDV X (p10) and the L-polymerase have never been studied. In this study, we used plasmid immunization to investigate the CTL response to BDV X and N. Plasmid-based immunization was a potent CTL inducer in Lewis rats. Anti-X CTLs were primed by a single injection of the p10 cDNA. Two codominant p10 epitopes, M(1)SSDLRLTLL(10) and T(8)LLELVRRL(16), associated with the RT1.A(l) major histocompatibility complex class I molecules of the Lewis rats, were identified. In addition, immunization with a BDV p40-expressing plasmid confirmed the previously reported RT1.A(l)-restricted A(230)SYAQMTTY(238) peptide as the CTL target for BDV N. In contrast to the CTL responses, plasmid vaccination was a poor inducer of an antibody response to p10. Three injections of a recombinant eukaryotic expression plasmid of BDV p10 were needed to generate a weak anti-p10 immunoglobulin M response. However, the antibody response could be optimized by a protein boost after priming with cDNA.     

Brap2 functions as a cytoplasmic retention protein for p21 during monocyte differentiation

The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation.     

Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.     

14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1)

p27(Kip1) (p27), a CDK inhibitor, migrates into the nucleus, where it controls cyclin-CDK complex activity for proper cell cycle progression. We report here that the classical bipartite-type basic amino-acid cluster and the two downstream amino acids of the C-terminal region of p27 function as a nuclear localization signal (NLS) for its full nuclear import activity. Importin alpha3 and alpha5, but not alpha1, transported p27 into the nucleus in conjunction with importin beta, as evidenced by an in vitro transport assay. It is known that Akt phosphorylates Thr 157 of p27 and this reduces the nuclear import activity of p27. Using a pull-down experiment, 14-3-3 was identified as the Thr157-phosphorylated p27NLS-binding protein. Although importin alpha5 bound to Thr157-phosphorylated p27NLS, 14-3-3 competed with importin alpha5 for binding to it. Thus, 14-3-3 sequestered phosphorylated p27NLS from importin alpha binding, resulting in cytoplasmic localization of NLS-phosphorylated p27. These findings indicate that 14-3-3 suppresses importin alpha/beta-dependent nuclear localization of Thr157-phosphorylated p27, suggesting implications for cell cycle disorder in Akt-activated cancer cells.     

The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A.nidulans and human

The heterotrimeric CCAAT-binding complex is evolutionarily conserved in eukaryotic organisms, including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the subunits HapB, HapC and HapE. All three subunits are necessary for DNA binding. HapB contains two putative nuclear localisation signal sequences (NLSs) designated NLS1 and NLS2. Previously, it was shown that only NLS2 was required for nuclear localisation of HapB. Furthermore, HapC and HapE are transported to the nucleus only in complex with HapB via a piggy back mechanism. Here, by using various GFP constructs and by establishing a novel marker gene for transformation of A.nidulans, i.e. the pabaA gene encoding p-aminobenzoic acid synthase, it was shown that the HapB homologous proteins of both Saccharomyces cerevisiae (Hap2p) and human (NF-YA) use an NLS homologous to HapB NLS1 for nuclear localisation in S.cerevisiae. Interestingly, for A.nidulans HapB, NLS1 was sufficient for nuclear localisation in S.cerevisiae. In A.nidulans, HapB NLS1 was also functional when present in a different protein context. However, in A.nidulans, both S.cerevisiae Hap2p and human NF-YA entered the nucleus only when HapB NLS2 was present in the respective proteins. In that case, both proteins Hap2p and NF-YA complemented, at least in part, the hap phenotype of A.nidulans with respect to lack of growth on acetamide. Similarly, A.nidulans HapB and human NF-YA complemented a hap2 mutant of S.cerevisiae. In summary, HapB, Hap2p and NF-YA are interchangeable. Because the A.nidulans hapB mutant was complemented, at least in part, by both the human NF-YA and S.cerevisiae Hap2p this finding suggests that the piggy-back mechanism of nuclear transport found for A.nidulans is conserved in yeast and human.     

A methionine-rich domain mediates CRM1-dependent nuclear export activity of Borna disease virus phosphoprotein

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that replicates and transcribes in the nucleus of infected cells. Recently, we have demonstrated that BDV phosphoprotein (P) can modulate its subcellular localization through binding to the protein X, which is encoded in the overlapping open reading frame (T. Kobayashi et al., J. Virol. 77:8099-8107, 2003). This observation suggested a unique strategy of intracellular trafficking of a viral protein that is essential for the formation of a functional BDV ribonucleoprotein (RNP). However, neither the mechanism nor the consequences of the cytoplasmic retention or nuclear export of BDV X-P complex have been elucidated. In this study, we show that BDV P contains a bona fide nuclear export signal (NES) and can actively shuttle between the nucleus and cytoplasm. A transient transfection analysis of cDNA clones that mimic the BDV bicistronic X/P mRNA revealed that the methionine-rich (MetR) domain of P is responsible for the X-dependent cytoplasmic localization of the protein complex. Mutational and functional analysis revealed that the methionine residues within the MetR domain are critical for the activity of the NES of P. Furthermore, leptomycin B or small interfering RNA for inhibition of CRM1 strongly suggested that a CRM1-dependent pathway mediates nuclear export of P. Fluorescence loss in photobleaching analysis confirmed the nucleocytoplasmic shuttling of P. Moreover, we revealed that the nuclear export of P is not involved in the inhibition of the polymerase activity by X in the BDV minireplicon system. Our results may provide a unique strategy for the nucleocytoplasmic transport of viral RNP, which could be critical for the formation of not only infectious virions in the cytoplasm but also a persistent viral state in the nucleus.     

Signals that dictate nuclear localization of human papillomavirus type 16 oncoprotein E6 in living cells

Human papillomavirus (HPV) type 16 E6 (16E6) is an oncogenic, multifunctional nuclear protein that induces p53 degradation and perturbs normal cell cycle control, leading to immortalization and transformation of infected keratinocytes and epithelial cells. Although it is unclear how 16E6 disrupts the epigenetic profile of host genes, its presence in the nucleus is a key feature. The present report describes intrinsic properties of 16E6 that influence its nuclear import in living cells. When the coding region of full-length 16E6 was inserted in frame into the C terminus of green fluorescent protein (GFP), it effectively prevented the 16E6 pre-mRNA from being spliced and led to the expression of a GFP-E6 fusion which localized predominantly to the nucleus. Further studies identified three novel nuclear localization signals (NLSs) in 16E6 that drive the protein to accumulate in the nucleus. We found that all three NLS sequences are rich in positively charged basic residues and that point mutations in these key residues could abolish the retention of 16E6 in the nucleus as well as the p53 degradation and cell immortalization activities of the protein. When inserted into corresponding regions of low-risk HPV type 6 E6, the three NLS sequences described for 16E6 functioned actively in converting the normally cytoplasmic HPV type 6 E6 into a nuclear protein. The separate NLS sequences, however, appear to play different roles in nuclear import and retention of HPV E6. The discovery of three unique NLS sequences in 16E6 provides new insights into the nuclear association of 16E6 which may reveal other novel activities of this important oncogenic protein.     

A role for Hsc70 in regulating nucleocytoplasmic transport of a temperature-sensitive p53 (p53Val-135)

Mouse temperature-sensitive p53(Val-135) accumulates in the nucleus and acts as a "wild-type" at 32 degrees C while it is sequestered in the cytoplasm at 37 degrees C. The cytoplasmic p53(Val-135) relocalized into the nucleus upon inhibition of the nuclear export at 37 degrees C, whereas a mutation in a major bipartite nuclear localization signal (NLS) caused constitutive cytoplasmic localization, indicating that it shuttled between the cytoplasm and the nucleus by its own nuclear export signal and NLS rather than tethered to cytoplasmic structures. Although the full-length p53(Val-135) did not bind the import receptor at 37 degrees C, a C-terminally truncated p53(Val-135) lacking residues 326-390 did bind it. Molecular chaperones such as Hsc70 were associated with p53(Val-135) at 37 degrees C but not at 32 degrees C. When the nuclear export was blocked by leptomycin B, only a fraction lacking Hsc70 was specifically accumulated in the nucleus. Immunodepletion of Hsc70 from the reticulocyte lysate caused p53(Val-135) to bind the import receptor. This binding was blocked by supplying the cell extract containing Hsc70 but not by the addition of recombinant Hsc70 alone. We suggest that the association with the Hsc70-containing complex prevents the NLS from the access of the import receptor through the C-terminal region of p53(Val-135) at 37 degrees C, whereas its dissociation at 32 degrees C allows rapid nuclear import.