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Barbara Wolff - One of the best experts on this subject based on the ideXlab platform.
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Leptomycin B Inhibition of Signal-Mediated Nuclear Export by Direct Binding to CRM1
Experimental cell research, 1998Co-Authors: Nobuaki Kudo, Sueharu Horinouchi, Yoshihiro Yoneda, Barbara Wolff, Toshihiro Sekimoto, Erwin P. Schreiner, Mitsuhiro Yanagida, Minoru YoshidaAbstract:Leptomycin B (LMB) is aStreptomycesmetabolite that inhibits nuclear export of the human immunodeficiency virus type 1 regulatory protein Rev at low nanomolar concentrations. Recently, LMB was shown to inhibit the function of CRM1, a receptor for the nuclear export signal (NES). Here we show evidence that LMB binds directly to CRM1 and that CRM1 is essential for NES-dependent nuclear export of proteins in both yeast and mammalian cells. Binding experiments with a biotinylated derivative of LMB and a HeLa cell extract led to identifying CRM1 as a major protein that bound to the LMB derivative. Microinjection of a purified anti-human CRM1 antibody into the mammalian nucleus specifically inhibited nuclear export of NES-containing proteins, as did LMB. Consistent with this, CRM1 was found to interact with NES, when assayed with immobilized NES and HeLa cell extracts. This association was disrupted by adding LMB or purified anti-human CRM1 antibody. The inhibition of CRM1 by LMB was also observed in fission yeast. The fission yeastcrm1mutant was defective in the nuclear export of NES-fused proteins, but not in the import of nuclear localization signal (NLS)-fused proteins. Interestingly, a protein containing both NES and NLS, which is expected to shuttle between nucleus and cytoplasm, was highly accumulated in the nucleus of thecrm1mutant cells or of cells treated with LMB. These results strongly suggest that CRM1 is the target of LMB and is an essential factor for nuclear export of proteins in eukaryotes.
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Microbial Conversion Products of Leptomycin B
Applied and environmental microbiology, 1998Co-Authors: Michaela Kuhnt, Ying Wang, Monique Ponelle, Jeanjacques Sanglier, Francis Bitsch, Barbara WolffAbstract:Leptomycin B (LMB), a secondary metabolite produced by Streptomyces sp. strain ATS 1287, with known antifungal and antitumor effects, inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 regulatory protein Rev and exhibits significant antiproliferative activity. Since LMB itself turned out to be distinctly cytotoxic, a bioconversion screening with a selected set of 29 bacterial and 72 fungal strains was performed in order to obtain metabolites of LMB with reduced antiproliferative effects. Several derivatives of LMB, more polar than the parent compound and produced in yields of >5%, were detected. Liquid chromatography-mass spectroscopy analysis indicated the type of bioconversion. Fermentations (1-liter scale) of those strains with high rates of transformation were suitable for isolation and characterization of the most prominent metabolites. Thus, bioconversion of LMB with Aspergillus flavus ATCC 9170 and Emericella unguis ATCC 13431 served for isolation of the novel derivatives 26-hydroxy-LMB (30% was the concentration of the metabolite [with respect to LMB] used for bioconversion) and LMB-24-glutaminamide (90%), respectively. Streptomyces rimosus ATCC 28893 converted LMB into 4,11-dihydroxy-LMB (13%) and 2,3-dihydro-LMB (55%). Although the antiproliferative effects of the LMB metabolites could be reduced through microbial conversion, none of these metabolites inhibited the nuclear export of Rev better than LMB itself.
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NOVEL LeptomycinS FROM A STREPTOMYCES STRAIN A92-308902 : INHIBITORS OF THE NUCLEO-CYTOPLASMIC TRANSLOCATION OF THE HIV-1 REGULATORY PROTEIN REV
Helvetica Chimica Acta, 1997Co-Authors: Ying Wang, Monique Ponelle, J.‐j. Sanglier, Barbara WolffAbstract:As one of the regulatory gene products in the HIV-1 genome, Rev protein must be translocated from the nucleus to the cytoplasm to exert its function. Therefore, inhibition of Rev protein translocation could be a useful target for HIV therapy. An extract from the Streptomyces strain A92-308902 with very potent inhibitory activity was found in the course of a high throughput screening with a Rev translocation assay (RTA). Bioassay-guided fractionation with gel filtration, normal-phase and reversed-phase chromatography yielded six RTA-active metabolites belonging to the Leptomycin family, the known Leptomycin A (1), Leptomycin B (2), kazusamycin B (3), and kazusamycin A (4). and the hitherto unknown dilactonmycin (5) and delactonmycin (6), together with an inactive cyclic hexadepsipeptide L-156,620 (7). The structures were established mainly by spectroscopic methods (UV, FT-IR, FAB-MS, 1H-NMR, 13C-NMR(JMOD), DQ-COSY, ROESY, HSQC, and HMBC). The configuration of all CC bonds of 1–6 was unambiguously established by analysis of coupling constants and ROESY spectra. All isolated Leptomycins 1–6 inhibit Rev translocation at nanomolar concentrations. Six derivatives (2a–c and 4a–c) of Leptomycin B (2) and kazusamycin A (4) were also prepared and tested in the RTA for preliminary investigations on structure-activity relationships.
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Leptomycin b is an inhibitor of nuclear export inhibition of nucleo cytoplasmic translocation of the human immunodeficiency virus type 1 hiv 1 rev protein and rev dependent mrna
Chemistry & Biology, 1997Co-Authors: Barbara Wolff, Jeanjacques Sanglier, Ying WangAbstract:Abstract Background: The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev is required for unspliced and incompletely spliced viral mRNAs to appear in the cytoplasm and thus for viral replication. Translocation of Rev from the nucleus to the cytoplasm is essential if Rev is to function. We wanted to identify inhibitors of this transport process because they would be potential antiviral agents. Results: The Streptomyces metabolite, Leptomycin B, and other antibiotics of the Leptomycin/kazusamycin family were identified as inhibitors of the nucleo-cytoplasmic translocation of Rev at nanomolar concentrations. Rev-dependent export of mRNA into the cytoplasm is also blocked by Leptomycin B, which inhibits Rev-dependent, but not Rev-independent gene expression in a short-term transfection assay. In primary human monocytes, Leptomycin B suppresses HIV-1 replication. Conclusions: Leptomycin B is the first low molecular weight inhibitor of nuclear export to be identified. Although it cannot be used therapeutically, it should serve as a valuable tool for dissecting nuclear export pathways.
Thomas Stamminger - One of the best experts on this subject based on the ideXlab platform.
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a novel transferable nuclear export signal mediates crm1 independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pul69
The EMBO Journal, 2001Co-Authors: Peter Lischka, Olaf Rosorius, Erik Trommer, Thomas StammingerAbstract:The best studied nuclear export processes are mediated by classical leucine-rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the β-herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus-encoded cofactors. In contrast to proteins containing a leucine-rich export signal, the shuttling activity of pUL69 was not affected by Leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, Leptomycin B-insensitive export signal that is distinct from other export signals described previously and is required for pUL69-mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.
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A novel transferable nuclear export signal mediates CRM1‐independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69
The EMBO journal, 2001Co-Authors: Peter Lischka, Olaf Rosorius, Erik Trommer, Thomas StammingerAbstract:The best studied nuclear export processes are mediated by classical leucine-rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the β-herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus-encoded cofactors. In contrast to proteins containing a leucine-rich export signal, the shuttling activity of pUL69 was not affected by Leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, Leptomycin B-insensitive export signal that is distinct from other export signals described previously and is required for pUL69-mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.
Minoru Yoshida - One of the best experts on this subject based on the ideXlab platform.
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Chromosomal Region Maintenance 1 (CRM1)-dependent Nuclear Export of Smad Ubiquitin Regulatory Factor 1 (Smurf1) Is Essential for Negative Regulation of Transforming Growth Factor-β Signaling by Smad7
The Journal of biological chemistry, 2003Co-Authors: Yoshitaka Tajima, Minoru Yoshida, Yoshihiro Yoneda, Toshihiro Sekimoto, Kouichiro Goto, Kenichi Shinomiya, Kohei Miyazono, Takeshi ImamuraAbstract:Abstract Smad ubiquitin regulatory factor 1 (Smurf1), a HECT type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces translocation of Smad7 to the cytoplasm. Smurf1 then associates with the transforming growth factor (TGF)-β type I receptor, TβR-I, enhancing turnover. However, the mechanism of nuclear export of Smad7 by Smurf1 has not been elucidated. Here we identified a functional nuclear export signal (NES) in a C-terminal region of Smurf1. In transfected cells, the Smurf1-Smad7 complex was accumulated in the cytoplasm by the nuclear export receptor, CRM1; this action was prevented by treatment with Leptomycin B, a specific inactivator of CRM1 function. A green fluorescence protein fusion protein containing the C-terminal NES motif of Smurf1, located in the cytoplasm, accumulated in the nucleus following treatment with Leptomycin B. Moreover, Smurf1 was shown to bind physically to CRM1 through NES, and nuclear export of the Smurf1-Smad7 complex was prevented by mutations of Smurf1 within the NES. Finally, the Smurf1 NES mutant reduced inhibition by Smad7 of the transcriptional activation induced by TGF-β. These results thus suggest that CRM1-dependent nuclear export of Smurf1 is essential for the negative regulation of TGF-β signaling by Smad7.
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Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis
Journal of Cell Science, 2000Co-Authors: M. Callanan, Minoru Yoshida, Nobuaki Kudo, S. Gout, Marie-paule Brocard, Stefan Dimitrov, Saadi KhochbinAbstract:In this work, we have investigated the role of CRM1/XPO1, a protein involved in specific export of proteins and RNA from the nucleus, in early Xenopus embryogenesis. The cloning of the Xenopus laevis CRM1, XCRM1, revealed remarkable conservation of the protein during evolution (96.7% amino acid identity between Xenopus and human). The protein and mRNA are maternally expressed and are present during early embryogenesis. However, our data show that the activity of the protein is developmentally regulated. Embryonic development is insensitive to Leptomycin B, a specific inhibitor of CRM1, until the neurula stage. Moreover, the nuclear localization of CRM1 changes concomitantly with the appearance of the Leptomycin B sensitivity. These data suggest that CRM1, present initially in an inactive form, becomes functional before the initiation of the neurula stage during gastrula-neurula transition, a period known to correspond to a critical transition in the pattern of gene expression. Finally, we confirmed the gastrula-neurula transition-dependent activation of CRM1 by pull-down experiments as well as by the study of the intracellular localization of a green fluorescent protein tagged with a nuclear export signal motif during early development. This work showed that the regulated activity of CRM1 controls specific transitions during normal development and thus might be a key regulator of early embryogenesis.
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Trichostatin and Leptomycin. Inhibition of histone deacetylation and signal-dependent nuclear export.
Annals of the New York Academy of Sciences, 1999Co-Authors: Minoru Yoshida, Sueharu HorinouchiAbstract:Trichostatin A (TSA), an inhibitor of the eukaryotic cell cycle and an inducer of morphological reversion of transformed cells, inhibits histone deacetylase (HDAC) at nanomolar concentrations. Recently, trapoxin, oxamflatin, and FR901228, antitumor agents structurally unrelated to TSA, were found to be potent HDAC inhibitors. These inhibitors activate expression of p21Waf1 and 16INK4A in a p53-independent manner. Changes in the expression of these cell cycle regulators by an increase in histone acetylation may be responsible for cell cycle arrest and antitumor activity by HDAC inhibitors. The target molecule of Leptomycin B (LMB), a potent antitumor agent, was genetically and biochemically identified as CRM1, a protein reported as being required for chromosome structure control. We showed that CRM1 was a receptor for the nuclear export signal (NES) and that LMB inhibited nuclear export of proteins. Using LMB, we identified a novel NES in fission yeast transcription factor Pap1, the function of which is abolished by oxidative stress in a manner conserved in eukaryotes.
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Leptomycin an antifungal and antitumor streptomyces metabolite is a novel chemical probe for eukaryotic nucleo cytoplasmic transport
Actinomycetologica, 1998Co-Authors: Minoru Yoshida, Nobuaki Kudo, Hiroshi Taoka, Shin Akakura, Sueharu HorinouchiAbstract:Leptomycin B (LMB) is a Streptomyces metabolite that causes specific inhibition of the cell cycle of yeast and mammalian cells. The target molecule of LMB in fission yeast has been shown by genetic analysis to be CRM1, a highly conserved protein in eukaryotes. We isolated a human homolog of CRM1 (hCRM1) which was expressed ubiquitously in human tissues and showed that hCRM1 was essential for nuclear export of proteins. CRM1 was shown to be a receptor for the nuclear export signal (NES) of proteins in both yeast and mammalian cells. LMB binds directly to CRM1, which resulted in dissociation of the NES from the nuclear export machinery containing CRM1. Thus, LMB serves as a potent tool for understanding the molecular mechanisms of nucleo-cytoplasmic transport of proteins.
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Leptomycin B Inhibition of Signal-Mediated Nuclear Export by Direct Binding to CRM1
Experimental cell research, 1998Co-Authors: Nobuaki Kudo, Sueharu Horinouchi, Yoshihiro Yoneda, Barbara Wolff, Toshihiro Sekimoto, Erwin P. Schreiner, Mitsuhiro Yanagida, Minoru YoshidaAbstract:Leptomycin B (LMB) is aStreptomycesmetabolite that inhibits nuclear export of the human immunodeficiency virus type 1 regulatory protein Rev at low nanomolar concentrations. Recently, LMB was shown to inhibit the function of CRM1, a receptor for the nuclear export signal (NES). Here we show evidence that LMB binds directly to CRM1 and that CRM1 is essential for NES-dependent nuclear export of proteins in both yeast and mammalian cells. Binding experiments with a biotinylated derivative of LMB and a HeLa cell extract led to identifying CRM1 as a major protein that bound to the LMB derivative. Microinjection of a purified anti-human CRM1 antibody into the mammalian nucleus specifically inhibited nuclear export of NES-containing proteins, as did LMB. Consistent with this, CRM1 was found to interact with NES, when assayed with immobilized NES and HeLa cell extracts. This association was disrupted by adding LMB or purified anti-human CRM1 antibody. The inhibition of CRM1 by LMB was also observed in fission yeast. The fission yeastcrm1mutant was defective in the nuclear export of NES-fused proteins, but not in the import of nuclear localization signal (NLS)-fused proteins. Interestingly, a protein containing both NES and NLS, which is expected to shuttle between nucleus and cytoplasm, was highly accumulated in the nucleus of thecrm1mutant cells or of cells treated with LMB. These results strongly suggest that CRM1 is the target of LMB and is an essential factor for nuclear export of proteins in eukaryotes.
Ying Wang - One of the best experts on this subject based on the ideXlab platform.
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Microbial Conversion Products of Leptomycin B
Applied and environmental microbiology, 1998Co-Authors: Michaela Kuhnt, Ying Wang, Monique Ponelle, Jeanjacques Sanglier, Francis Bitsch, Barbara WolffAbstract:Leptomycin B (LMB), a secondary metabolite produced by Streptomyces sp. strain ATS 1287, with known antifungal and antitumor effects, inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 regulatory protein Rev and exhibits significant antiproliferative activity. Since LMB itself turned out to be distinctly cytotoxic, a bioconversion screening with a selected set of 29 bacterial and 72 fungal strains was performed in order to obtain metabolites of LMB with reduced antiproliferative effects. Several derivatives of LMB, more polar than the parent compound and produced in yields of >5%, were detected. Liquid chromatography-mass spectroscopy analysis indicated the type of bioconversion. Fermentations (1-liter scale) of those strains with high rates of transformation were suitable for isolation and characterization of the most prominent metabolites. Thus, bioconversion of LMB with Aspergillus flavus ATCC 9170 and Emericella unguis ATCC 13431 served for isolation of the novel derivatives 26-hydroxy-LMB (30% was the concentration of the metabolite [with respect to LMB] used for bioconversion) and LMB-24-glutaminamide (90%), respectively. Streptomyces rimosus ATCC 28893 converted LMB into 4,11-dihydroxy-LMB (13%) and 2,3-dihydro-LMB (55%). Although the antiproliferative effects of the LMB metabolites could be reduced through microbial conversion, none of these metabolites inhibited the nuclear export of Rev better than LMB itself.
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NOVEL LeptomycinS FROM A STREPTOMYCES STRAIN A92-308902 : INHIBITORS OF THE NUCLEO-CYTOPLASMIC TRANSLOCATION OF THE HIV-1 REGULATORY PROTEIN REV
Helvetica Chimica Acta, 1997Co-Authors: Ying Wang, Monique Ponelle, J.‐j. Sanglier, Barbara WolffAbstract:As one of the regulatory gene products in the HIV-1 genome, Rev protein must be translocated from the nucleus to the cytoplasm to exert its function. Therefore, inhibition of Rev protein translocation could be a useful target for HIV therapy. An extract from the Streptomyces strain A92-308902 with very potent inhibitory activity was found in the course of a high throughput screening with a Rev translocation assay (RTA). Bioassay-guided fractionation with gel filtration, normal-phase and reversed-phase chromatography yielded six RTA-active metabolites belonging to the Leptomycin family, the known Leptomycin A (1), Leptomycin B (2), kazusamycin B (3), and kazusamycin A (4). and the hitherto unknown dilactonmycin (5) and delactonmycin (6), together with an inactive cyclic hexadepsipeptide L-156,620 (7). The structures were established mainly by spectroscopic methods (UV, FT-IR, FAB-MS, 1H-NMR, 13C-NMR(JMOD), DQ-COSY, ROESY, HSQC, and HMBC). The configuration of all CC bonds of 1–6 was unambiguously established by analysis of coupling constants and ROESY spectra. All isolated Leptomycins 1–6 inhibit Rev translocation at nanomolar concentrations. Six derivatives (2a–c and 4a–c) of Leptomycin B (2) and kazusamycin A (4) were also prepared and tested in the RTA for preliminary investigations on structure-activity relationships.
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Leptomycin b is an inhibitor of nuclear export inhibition of nucleo cytoplasmic translocation of the human immunodeficiency virus type 1 hiv 1 rev protein and rev dependent mrna
Chemistry & Biology, 1997Co-Authors: Barbara Wolff, Jeanjacques Sanglier, Ying WangAbstract:Abstract Background: The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev is required for unspliced and incompletely spliced viral mRNAs to appear in the cytoplasm and thus for viral replication. Translocation of Rev from the nucleus to the cytoplasm is essential if Rev is to function. We wanted to identify inhibitors of this transport process because they would be potential antiviral agents. Results: The Streptomyces metabolite, Leptomycin B, and other antibiotics of the Leptomycin/kazusamycin family were identified as inhibitors of the nucleo-cytoplasmic translocation of Rev at nanomolar concentrations. Rev-dependent export of mRNA into the cytoplasm is also blocked by Leptomycin B, which inhibits Rev-dependent, but not Rev-independent gene expression in a short-term transfection assay. In primary human monocytes, Leptomycin B suppresses HIV-1 replication. Conclusions: Leptomycin B is the first low molecular weight inhibitor of nuclear export to be identified. Although it cannot be used therapeutically, it should serve as a valuable tool for dissecting nuclear export pathways.
Peter Lischka - One of the best experts on this subject based on the ideXlab platform.
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a novel transferable nuclear export signal mediates crm1 independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pul69
The EMBO Journal, 2001Co-Authors: Peter Lischka, Olaf Rosorius, Erik Trommer, Thomas StammingerAbstract:The best studied nuclear export processes are mediated by classical leucine-rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the β-herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus-encoded cofactors. In contrast to proteins containing a leucine-rich export signal, the shuttling activity of pUL69 was not affected by Leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, Leptomycin B-insensitive export signal that is distinct from other export signals described previously and is required for pUL69-mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.
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A novel transferable nuclear export signal mediates CRM1‐independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69
The EMBO journal, 2001Co-Authors: Peter Lischka, Olaf Rosorius, Erik Trommer, Thomas StammingerAbstract:The best studied nuclear export processes are mediated by classical leucine-rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the β-herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus-encoded cofactors. In contrast to proteins containing a leucine-rich export signal, the shuttling activity of pUL69 was not affected by Leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, Leptomycin B-insensitive export signal that is distinct from other export signals described previously and is required for pUL69-mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.