CRM1 mediates nuclear export of nonstructural protein 2 from parvovirus minute virus of mice.

The nonstructural protein 2 (NS2) from parvovirus minute virus of mice (MVMp) is a 25-kDa polypeptide which localizes preferentially to the cytoplasm and associates with cellular proteins in cytoplasm. These lines of evidence suggest that NS2 is positively exported from the nucleus to cytoplasm and functions in cytoplasm. We report here that nuclear export of NS2 is inhibited by leptomycin B (LMB), a drug that specifically blocks nuclear export signal (NES)-chromosomal region maintenance 1 (CRM1) interactions. CRM1 binds specifically to the 81- to 106-amino-acid (aa) region of NS2, and the region of NS2 actually functions as a NES. Interestingly, this region appears to be distinct from a typical NES sequence, which consists of leucine-rich sequences. These results indicate that NS2 protein is continuously exported from the nucleus by a CRM1-dependent mechanism and suggest that CRM1 also exports to distinct type of NESs.     

A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP-USP21 and, to a lesser extent, GFP-OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.     

Ratjadone and leptomycin B block CRM1-dependent nuclear export by identical mechanisms

Research on the export of proteins and nucleic acids from the nucleus to the cytoplasm has greatly gained from the discovery that the actinobacterial toxin leptomycin B (LMB) specifically inactivates the export receptor chromosomal region maintenance 1 (CRM1). Recently, it was shown that myxobacterial cytotoxins, named ratjadones (RATs), also bind to CRM1 and inhibit nuclear export. However, the reaction mechanism of RATs was not resolved. Here, we show that LMB and RAT A employ the same molecular mechanism to inactivate CRM1. Alkylation of residue Cys528 of CRM1 determines both LMB and RAT sensitivity and prevents nuclear export of CRM1 cargo proteins.     

博尔纳病病毒磷蛋白细胞模型的构建与鉴定

目的建立博尔纳病病毒(Borna disease virus,BDV)磷蛋白的细胞模型,并对所建模型进行鉴定。方法重新扩增和鉴定已有的BDV磷蛋白(GFP-P24)质粒,使用转染试剂将该质粒转入PC12细胞,并用荧光定量PCR和ELISA的方法对所构建的细胞模型进行鉴定。结果重新扩增的质粒PCR鉴定阳性,其浓度符合转染的需要,测序后未发现核苷酸的突变;转染PC12细胞的效率高,荧光定量PCR和ELISA检测均为阳性。结论成功构建了BDV磷蛋白的细胞模型,为研究BDV感染过程中磷蛋白所起的作用奠定了基础。     

A CRM1-dependent nuclear export pathway is involved in the regulation of IRF-5 subcellular localization

Interferon regulatory factors (IRFs) are involved in gene regulation in many biological processes including the antiviral, growth regulatory, and immune modulatory functions of the interferon system. Several studies have demonstrated that IRF-3, IRF-5, and IRF-7 specifically contribute to the innate antiviral response to virus infection. It has been reported that virus-specific phosphorylation leads to IRF-5 nuclear localization and up-regulation of interferon, cytokine, and chemokine gene expression. Two nuclear localization signals have been identified in IRF-5, both of which are sufficient for nuclear translocation and retention in virus-infected cells. In the present study, we demonstrate that a CRM1-dependent nuclear export pathway is involved in the regulation of IRF-5 subcellular localization. IRF-5 possesses a functional nuclear export signal (NES) that controls dynamic shuttling between the cytoplasm and the nucleus. The NES element is dominant in unstimulated cells and results in the predominant cytoplasmic localization of IRF-5. Mutation of two leucine residues in the NES motif to alanine, or three adjacent Ser/Thr residues to the phosphomimetic Asp, results in constitutively nuclear IRF-5 and suggests that phosphorylation of adjacent Ser/Thr residues may contribute to IRF-5 nuclear accumulation in virus-induced cells. IKK-related kinases TBK1 and IKKepsilon have been shown to phosphorylate and activate IRF-3 and IRF-7, leading to the production of type 1 interferons and the development of a cellular antiviral state. We examined the phosphorylation and activation of IRF-5 by TBK1 and IKKepsilon kinases. Although IRF-5 is phosphorylated by IKKepsilon and TBK1 in co-transfected cells, the phosphorylation of IRF-5 did not lead to IRF-5 nuclear localization or activation.     

Regulation of the ATM-activator protein Aven by CRM1-dependent nuclear export

Aven is a regulator of apoptosis whose overexpression is associated with poor prognosis in several cancers, including childhood acute lymphoblastic leukemia and acute myeloid leukemia. We have recently shown that Aven serves as an activator and substrate of ATM, thereby modulating the DNA-damage response and G2/M cell cycle progression. Under physiological conditions, the cellular localization of Aven is mainly cytosolic, but a small fraction of the protein is present in the nucleus. Here, we show that treatment of cells with leptomycin B, an inhibitor of Exportin-1/CRM (chromosomal region maintenance) 1, resulted in nuclear accumulation of Aven. Furthermore, we identified a functional LR-NES between amino acid residues 282-292 of the human Aven protein, a sequence that is evolutionary conserved among a range of vertebrate species. Disruption of this LR-NES by site-directed mutagenesis resulted in enhanced nuclear localization of Aven, but did not alter the ability of the protein to induce G2/M cell cycle arrest in interphase Xenopus laevis extracts. However, elimination of the LR-NES sequence led to a reduction in the capacity of Aven to arrest Xenopus oocytes containing intact nuclei. Our results suggest that the regulation of nucleocytoplasmatic traffic of Aven could modulate its ability to influence cell cycle progression.     

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.     

Borna disease virus phosphoprotein binds a neurite outgrowth factor, amphoterin/HMG-1

The Borna disease virus (BDV) p24 phosphoprotein is an abundant protein in BDV-infected cultured cells and animal brains. Therefore, there is a possibility that binding of the p24 protein to cellular factor(s) induces functional alterations of infected neural cells in the brain. To identify a cellular protein(s) that interacts with BDV p24 protein, we performed far-Western blotting with extracts from various cell lines. Using recombinant p24 protein as a probe, we detected a 30-kDa protein in all cell lines examined. Binding between the 30-kDa and BDV p24 proteins was also demonstrated using BDV p24 affinity and ion-exchange chromatography columns. Microsequence analysis of the purified 30-kDa protein revealed that its N terminus showed complete homology with rat amphoterin protein, which is a neurite outgrowth factor abundant in the brain during development. Mammalian two-hybrid and immunoprecipitation analyses also confirmed that amphoterin is a specific target for the p24 protein in vivo. Furthermore, we showed that infection by BDV, as well as purified p24 protein in the medium, significantly decreased cell process outgrowth of cells grown on laminin, indicating the functional inhibition of amphoterin by interaction with the p24 protein. Immunohistochemical analysis revealed decreased levels of amphoterin protein at the leading edges of BDV-infected cells. Moreover, the expression of the receptor for advanced glycation end products, of which the extracellular moiety is a receptor for amphoterin, was not significantly activated in BDV-infected cells during the process of extension, suggesting that the secretion of amphoterin from the cell surface is inhibited by the binding of the p24 protein. These results suggested that BDV infection may cause direct damage in the developing brain by inhibiting the function of amphoterin due to binding by the p24 phosphoprotein.