scholarly journals The Ebola Virus Interferon Antagonist VP24 Undergoes Active Nucleocytoplasmic Trafficking

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1650
Author(s):  
Angela R. Harrison ◽  
Cassandra T. David ◽  
Stephen M. Rawlinson ◽  
Gregory W. Moseley
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Ebola Virus ◽  
Molecular Mapping ◽  
Nucleocytoplasmic Transport ◽  
Cytoplasmic Localization ◽  
Nucleocytoplasmic Trafficking ◽  
Nuclear Trafficking ◽  
C Terminus ◽  
Import Receptors

Viral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, with distinct roles in viral replication. The Ebola virus IFN antagonist VP24 mediates nucleocapsid assembly, and inhibits IFN-activated signaling by preventing nuclear import of STAT1 via competitive binding to nuclear import receptors (karyopherins). Proteins of many viruses, including viruses with cytoplasmic replication cycles, interact with nuclear trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis; however, despite established karyopherin interaction, potential nuclear trafficking of VP24 has not been investigated. We find that inhibition of nuclear export pathways or overexpression of VP24-binding karyopherin results in nuclear localization of VP24. Molecular mapping indicates that cytoplasmic localization of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism, while export mediates return of nuclear VP24 to the cytoplasm where replication/nucleocapsid assembly occurs.

scholarly journals The Ebola virus interferon antagonist VP24 undergoes active nucleocytoplasmic trafficking

2020 ◽  
Author(s):  
Angela R. Harrison ◽  
Gregory W. Moseley
Keyword(s):  
Viral Replication ◽  
Immune Evasion ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Ebola Virus ◽  
Molecular Mapping ◽  
Case Fatality ◽  
Nucleocytoplasmic Transport ◽  
Nucleocytoplasmic Trafficking ◽  
Nuclear Trafficking

AbstractViral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, having distinct roles in viral replication processes. Functions of the Ebola virus (EBOV) IFN antagonist VP24 include nucleocapsid assembly during cytoplasmic replication and inhibition of IFN-activated signalling by STAT1. For the latter, VP24 prevents STAT1 nuclear import via competitive binding to nuclear import receptors (karyopherins). Many viral proteins, including proteins from viruses with cytoplasmic replication cycles, interact with the trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis. Despite established karyopherin interaction, the nuclear trafficking profile of VP24 has not been investigated. We find that VP24 becomes strongly nuclear following overexpression of karyopherin or inhibition of nuclear export pathways. Molecular mapping indicates that cytoplasmic localisation of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism while export mediates return of nuclear VP24 to the cytoplasm for replication functions. Thus, nuclear export of VP24 might provide novel targets for antiviral approaches.ImportanceEbola virus (EBOV) is the causative agent of ongoing outbreaks of severe haemorrhagic fever with case-fatality rates between 40 and 60%. Proteins of many viruses with cytoplasmic replication cycles similar to EBOV interact with the nuclear trafficking machinery, resulting in active nucleocytoplasmic shuttling important to immune evasion and other intranuclear functions. However, exploitation of host trafficking machinery for nucleocytoplasmic transport by EBOV has not been directly examined. We find that the EBOV protein VP24 is actively trafficked between the nucleus and cytoplasm, and identify the specific pathways and sequences involved. The data indicate that nucleocytoplasmic trafficking is important for the multifunctional nature of VP24, which has critical roles in immune evasion and viral replication, identifying a new mechanism in infection by this highly lethal pathogen, and potential target for antivirals.

scholarly journals Nucleocytoplasmic Trafficking of G2/M Regulators in Yeast

2008 ◽  
Vol 19 (9) ◽  
pp. 4006-4018 ◽  
Author(s):  
Mignon A. Keaton ◽  
Lee Szkotnicki ◽  
Aron R. Marquitz ◽  
Jake Harrison ◽  
Trevin R. Zyla ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Cell Cycle Regulators ◽  
Cyclin Dependent Kinase ◽  
Nucleocytoplasmic Trafficking ◽  
Nucleocytoplasmic Shuttling ◽  
Upstream Regulator ◽  
Cellular Compartments ◽  
Cdk Regulation

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

scholarly journals Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2

2021 ◽  
Author(s):  
Chris Y. Cheung ◽  
Ting-Ting Huang ◽  
Ning Chow ◽  
Shuqi Zhang ◽  
Yanxiang Zhao ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Proteomics Analysis ◽  
Nuclear Trafficking ◽  
Localization Signal ◽  
Underlying Mechanisms ◽  
Sirna Screening

NFAT5 is the only known mammalian tonicity-responsive transcription factor functionally implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity but the underlying mechanisms remain elusive. We demonstrated that NFAT5 enters the nucleus via the nuclear pore complex. We also found that NFAT5 utilizes a non-canonical nuclear localization signal (NFAT5-NLS) for nuclear imports. siRNA screening revealed that karyopherin beta-1 (KPNB1) drives nuclear import of NFAT5 via directly interacting with NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is mediated by Exportin-T, and that it requires RuvB-Like AAA type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified KPNB1 and RUVBL2 as key molecules responsible for the unconventional tonicity-regulated nucleocytoplasmic shuttling of NFAT5. These findings offer an opportunity for developing novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

scholarly journals Active Nuclear Import and Export Is Independent of Lumenal Ca2+ Stores in Intact Mammalian Cells

1999 ◽  
Vol 113 (2) ◽  
pp. 239-248 ◽  
Author(s):  
Carsten Strübing ◽  
David E. Clapham
Keyword(s):  
Active Transport ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
Acetoxymethyl Ester ◽  
Store Depletion

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (≈10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca2+ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca2+ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M1 receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca2+ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.

scholarly journals The hsp90-FKBP52 Complex Links the Mineralocorticoid Receptor to Motor Proteins and Persists Bound to the Receptor in Early Nuclear Events

2009 ◽  
Vol 30 (5) ◽  
pp. 1285-1298 ◽  
Author(s):  
Mario D. Galigniana ◽  
Alejandra G. Erlejman ◽  
Martín Monte ◽  
Celso Gomez-Sanchez ◽  
Graciela Piwien-Pilipuk
Keyword(s):  
Nuclear Export ◽  
Mineralocorticoid Receptor ◽  
Soluble Fraction ◽  
Molecular System ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Cytoplasmic Localization ◽  
Nucleocytoplasmic Trafficking ◽  
Cytoplasmic Transport ◽  
Nuclear Events

ABSTRACT In this study, we demonstrate that the subcellular localization of the mineralocorticoid receptor (MR) is regulated by tetratricopeptide domain (TPR) proteins. The high-molecular-weight immunophilin (IMM) FKBP52 links the MR-hsp90 complex to dynein/dynactin motors favoring the cytoplasmic transport of MR to the nucleus. Replacement of this hsp90-binding IMM by FKBP51 or the TPR peptide favored the cytoplasmic localization of MR. The complete movement machinery, including dynein and tubulin, could be recovered from paclitaxel/GTP-stabilized cytosol and was fully reassembled on stripped MR immune pellets. The whole MR-hsp90-based heterocomplex was transiently recovered in the soluble fraction of the nucleus after 10 min of incubation with aldosterone. Moreover, cross-linked MR-hsp90 heterocomplexes accumulated in the nucleus in a hormone-dependent manner, demonstrating that the heterocomplex can pass undissociated through the nuclear pore. On the other hand, a peptide that comprises the DNA-binding domain of MR impaired the nuclear export of MR, suggesting the involvement of this domain in the process. This study represents the first report describing the entire molecular system that commands MR nucleocytoplasmic trafficking and proposes that the MR-hsp90-TPR protein heterocomplex is dissociated in the nucleus rather than in the cytoplasm.

scholarly journals Calcium-calmodulin kinase I cooperatively regulates nucleocytoplasmic shuttling of CCTα by accessing a nuclear export signal

2012 ◽  
Vol 23 (14) ◽  
pp. 2755-2769 ◽  
Author(s):  
Marianna Agassandian ◽  
Bill B. Chen ◽  
Roopa Pulijala ◽  
Leah Kaercher ◽  
Jennifer R. Glasser ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Membrane Phospholipids ◽  
Nuclear Entry ◽  
Nuclear Trafficking ◽  
C Terminus ◽  
Calmodulin Kinase ◽  
Calcium Stimulation ◽  
Export Signal ◽  
In Cells

We identified a new calmodulin kinase I (CaMKI) substrate, cytidyltransferase (CCTα), a crucial enzyme required for maintenance of cell membranes. CCTα becomes activated with translocation from the cytoplasm to the nuclear membrane, resulting in increased membrane phospholipids. Calcium-activated CCTα nuclear import is mediated by binding of its C-terminus to 14-3-3 ζ, a regulator of nuclear trafficking. Here CaMK1 phosphorylates residues within this C-terminus that signals association of CCTα with 14-3-3 ζ to initiate calcium-induced nuclear entry. CaMKI docks within the CCTα membrane-binding domain (residues 290–299), a sequence that displays similarities to a canonical nuclear export signal (NES) that also binds CRM1/exportin 1. Expression of a CFP-CCTα mutant lacking residues 290–299 in cells results in cytosolically retained enzyme. CRM1/exportin 1 was required for CCTα nuclear export, and its overexpression in cells was partially sufficient to trigger CCTα nuclear export despite calcium stimulation. An isolated CFP-290-299 peptide remained in the nucleus in the presence of leptomycin B but was able to target to the cytoplasm with farnesol. Thus CaMKI vies with CRM1/exportin 1 for access to a NES, and assembly of a CaMKI–14-3-3 ζ–CCTα complex is a key effector mechanism that drives nuclear CCTα translocation.

scholarly journals Destabilization of Ran C-terminus promotes GTP loading and occurs in multiple Ran cancer mutations

2018 ◽  
Author(s):  
Yuqing Zhang ◽  
Jinhan Zhou ◽  
Yuping Tan ◽  
Qiao Zhou ◽  
Aiping Tong ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Mitotic Spindle ◽  
Nuclear Export ◽  
Cellular Transformation ◽  
Nuclear Pore ◽  
Ras Proteins ◽  
Transformation Mechanism ◽  
C Terminus ◽  
Cytoplasmic Transport ◽  
Cancer Mutations

AbstractRan (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other diverse functions in the cytoplasm, as well as in cellular transformation when activated. Unlike other Ras superfamily proteins, Ran contains an auto-inhibitory C-terminal tail, which packs against its G domain and bias Ran towards binding GDP over GTP. The biological importance of this C-terminal tail is not well understood. By disrupting the interaction between the C-terminus and the G domain, we were able to generate Ran mutants that are innately active and potently bind to RanBP1 (Ran Binding Protein 1), nuclear export factor CRM1 and nuclear import factor KPNB1. In contrast to previously reported activated Ran mutants, the C-terminus destabilized mutants are hydrolysis competent in cells, support nuclear transport, and do not form nuclear rim staining. Crystal structures show that one of these C-terminal mutations slightly changes its mode of binding to RanBP1. Finally, a high percentage of Ran C-terminus mutations from cancer patients were found to be destabilizing and hyperactivating, suggesting that Ran C-destabilization might be an unprecedented cellular transformation mechanism in affected cancers. This study also highlights a new drug design strategy towards treating patients with hyperactivated Ras proteins including K-Ras.

scholarly journals Histone Deacetylase 4 Possesses Intrinsic Nuclear Import and Export Signals

2001 ◽  
Vol 21 (17) ◽  
pp. 5992-6005 ◽  
Author(s):  
Audrey H. Wang ◽  
Xiang-Jiao Yang
Keyword(s):  
Histone Deacetylase ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Mutational Analysis ◽  
Nuclear Export Signal ◽  
Middle Part ◽  
Nucleocytoplasmic Trafficking ◽  
Histone Deacetylase 4 ◽  
Import And Export

ABSTRACT Nucleocytoplasmic trafficking of histone deacetylase 4 (HDAC4) plays an important role in regulating its function, and binding of 14-3-3 proteins is necessary for its cytoplasmic retention. Here, we report the identification of nuclear import and export sequences of HDAC4. While its N-terminal 118 residues modulate the nuclear localization, residues 244 to 279 constitute an authentic, strong nuclear localization signal. Mutational analysis of this signal revealed that three arginine-lysine clusters are necessary for its nuclear import activity. As for nuclear export, leucine-rich sequences located in the middle part of HDAC4 do not function as nuclear export signals. By contrast, a hydrophobic motif (MXXLXVXV) located at the C-terminal end serves as a nuclear export signal that is necessary for cytoplasmic retention of HDAC4. This motif is required for CRM1-mediated nuclear export of HDAC4. Furthermore, binding of 14-3-3 proteins promotes cytoplasmic localization of HDAC4 by both inhibiting its nuclear import and stimulating its nuclear export. Unlike wild-type HDAC4, a point mutant with abrogated MEF2-binding ability remains cytoplasmic upon exogenous expression of MEF2C, supporting the notion that direct MEF2 binding targets HDAC4 to the nucleus. Therefore, HDAC4 possesses intrinsic nuclear import and export signals for its dynamic nucleocytoplasmic shuttling, and association with 14-3-3 and MEF2 proteins affects such shuttling and thus directs HDAC4 to the cytoplasm and the nucleus, respectively.

scholarly journals The L2 Minor Capsid Protein of Human Papillomavirus Type 16 Interacts with a Network of Nuclear Import Receptors

2004 ◽  
Vol 78 (22) ◽  
pp. 12179-12188 ◽  
Author(s):  
Medha S. Darshan ◽  
John Lucchi ◽  
Emily Harding ◽  
Junona Moroianu
Keyword(s):  
Human Papillomavirus ◽  
Nuclear Import ◽  
Capsid Proteins ◽  
Permeabilized Cells ◽  
Nuclear Localization Signals ◽  
Human Papillomavirus Type 16 ◽  
C Terminus ◽  
Binding Data ◽  
Import Receptors ◽  
Productive Phase

ABSTRACT The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapβ2 and Kapβ3. Moreover, binding of Ran-GTP to either Kapβ2 or Kapβ3 inhibits its interaction with L2, suggesting that the Kapβ/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapα2β1 heterodimer via interaction with the Kapα2 adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapα2β1 heterodimers, Kapβ2, or Kapβ3. Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapα2β1 heterodimers, Kapβ2, and Kapβ3.

scholarly journals Defective nuclear import of Tpr in Progeria reflects the Ran sensitivity of large cargo transport

2013 ◽  
Vol 201 (4) ◽  
pp. 541-557 ◽  
Author(s):  
Chelsi J. Snow ◽  
Ashraf Dar ◽  
Anindya Dutta ◽  
Ralph H. Kehlenbach ◽  
Bryce M. Paschal
Keyword(s):  
Nuclear Import ◽  
Protein Complexes ◽  
Nucleocytoplasmic Transport ◽  
Large Protein ◽  
Localization Signal ◽  
Import Receptors ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome ◽  
Major Defect ◽  
Import Receptor

The RanGTPase acts as a master regulator of nucleocytoplasmic transport by controlling assembly and disassembly of nuclear transport complexes. RanGTP is required in the nucleus to release nuclear localization signal (NLS)–containing cargo from import receptors, and, under steady-state conditions, Ran is highly concentrated in the nucleus. We previously showed the nuclear/cytoplasmic Ran distribution is disrupted in Hutchinson-Gilford Progeria syndrome (HGPS) fibroblasts that express the Progerin form of lamin A, causing a major defect in nuclear import of the protein, translocated promoter region (Tpr). In this paper, we show that Tpr import was mediated by the most abundant import receptor, KPNA2, which binds the bipartite NLS in Tpr with nanomolar affinity. Analyses including NLS swapping revealed Progerin did not cause global inhibition of nuclear import. Rather, Progerin inhibited Tpr import because transport of large protein cargoes was sensitive to changes in the Ran nuclear/cytoplasmic distribution that occurred in HGPS. We propose that defective import of large protein complexes with important roles in nuclear function may contribute to disease-associated phenotypes in Progeria.

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