The Ebola virus interferon antagonist VP24 undergoes active 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.

The Ebola Virus Interferon Antagonist VP24 Undergoes Active Nucleocytoplasmic Trafficking

Viruses ◽

10.3390/v13081650 ◽

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 ◽

Cytoplasmic Localization ◽

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.

Nucleocytoplasmic Trafficking of G2/M Regulators in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e08-03-0286 ◽

2008 ◽

Vol 19 (9) ◽

pp. 4006-4018 ◽

Cited By ~ 25

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 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.

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

10.1101/2021.07.11.451941 ◽

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.

Antagonism of STAT3 signalling by Ebola virus

10.1101/2020.08.10.245464 ◽

2020 ◽

Author(s):

Angela R. Harrison ◽

Megan Dearnley ◽

Shawn Todd ◽

Diane Green ◽

Glenn A. Marsh ◽

...

Keyword(s):

Molecular Mechanisms ◽

Ebola Virus ◽

Significant Risk ◽

Disease Outbreaks ◽

Case Fatality ◽

Cell Physiology ◽

Haemorrhagic Fever ◽

Nuclear Trafficking ◽

Ebov Infection ◽

Stat3 Signalling

AbstractMany viruses target signal transducers and activators of transcription (STAT) 1 and 2 to antagonise antiviral interferon (IFN) signalling, but targeting of signalling by other STATs/cytokines, including STAT3/interleukin (IL-) 6 that regulate processes important to Ebola virus (EBOV) haemorrhagic fever, is poorly defined. We report that EBOV potently inhibits STAT3 responses to IL-6 family cytokines, and that this is mediated by the IFN-antagonist VP24. Mechanistic analysis indicates that VP24 effects a unique strategy combining distinct karyopherin-dependent and karyopherin-independent mechanisms to antagonise STAT3-STAT1 heterodimers and STAT3 homodimers, respectively. This appears to reflect distinct mechanisms of nuclear trafficking of the STAT3 complexes, revealed for the first time by our analysis of VP24 function. These findings are consistent with major roles for global inhibition of STAT3 signalling in EBOV infection, and provide new insights into the molecular mechanisms of STAT3 nuclear trafficking, significant to pathogen-host interactions, cell physiology and pathologies such as cancer.Author summaryEbola virus (EBOV) continues to pose a significant risk to human health globally, causing ongoing disease outbreaks with case-fatality rates between 40 and 60%. Suppression of immune responses is a critical component of EBOV haemorrhagic fever, but understanding of EBOV impact on signalling by cytokines other than interferon is limited. We find that infectious EBOV inhibits interleukin-6 cytokine signalling via antagonism of STAT3. The antagonistic strategy uniquely combines two distinct mechanisms, which appear to reflect differing nuclear trafficking mechanisms of critical STAT3 complexes. This provides fundamental insights into the mechanisms of pathogenesis of a lethal virus, and biology of STAT3, a critical player in immunity, development, growth and cancer.

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

The Journal of General Physiology ◽

10.1085/jgp.113.2.239 ◽

1999 ◽

Vol 113 (2) ◽

pp. 239-248 ◽

Cited By ~ 35

Author(s):

Carsten Strübing ◽

David E. Clapham

Keyword(s):

Active Transport ◽

Nuclear Import ◽

Nuclear Export ◽

Mammalian Cells ◽

Protein Transport ◽

Fluorescent Protein ◽

Nuclear Pore ◽

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.

Histone Deacetylase 4 Possesses Intrinsic Nuclear Import and Export Signals

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.5992-6005.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 5992-6005 ◽

Cited By ~ 118

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 ◽

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.

The Ran GTPase Gradient Protects the Nucleolus from Aging-Associated Morphological Changes

10.1101/2020.07.24.220079 ◽

2020 ◽

Author(s):

Bartlomiej Remlein ◽

Bryce M. Paschal

Keyword(s):

Molecular Mass ◽

Nuclear Import ◽

Nuclear Export ◽

Cultured Cells ◽

High Molecular Mass ◽

Regulatory Function ◽

Chemical Induction ◽

Ran Gtpase ◽

Passage Number

ABSTRACTIn the context of its regulatory function for nucleocytoplasmic transport, the Ran GTPase undergoes cycles of nuclear import, GTP loading, nuclear export, and GTP hydrolysis. These reactions give rise to a nuclear:cytoplasmic (N:C) Ran gradient. In Hutchinson-Gilford Progeria Syndrome, disruption of the Ran gradient suppresses nuclear import of high molecular mass complexes by reducing the nuclear concentration of Ran. Here, we report that cells undergoing senescence, as a consequence of passage number, chemical induction, and altered nuclear lamina structure, all display a Ran gradient disruption quantitatively similar to that observed in Progeria patient cells. We found that the Ran gradient is critical for maintenance of nucleolar structure, as its disruption increases the size and decreases the average number of nucleoli per cell. Nucleolar number and size are biomarkers of longevity in diverse organisms, thus the nuclear level of Ran may be important for the nucleolar morphology in aging. The contribution of the Ran gradient includes regulating import of nucleolin and nucleophosmin, nucleolar proteins that assemble into high molecular mass complexes. The steepness of the Ran gradient is highly dependent on nuclear heterochromatin, which is reduced by passage number and chemical induction of senescence in cultured cells, and is known to decline during normal aging. Our data suggest that the Ran gradient senses nuclear heterochromatin, and through its function as a transport regulator, helps maintain the protein composition and structure of the nucleolus.

Involvement of Nuclear Import and Export Factors in U8 Box C/D snoRNP Biogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.00516-07 ◽

2007 ◽

Vol 27 (20) ◽

pp. 7018-7027 ◽

Cited By ~ 28

Author(s):

Nicholas J. Watkins ◽

Ira Lemm ◽

Reinhard Lührmann

Keyword(s):

Rna Interference ◽

Nuclear Import ◽

Nuclear Export ◽

Ribosome Biogenesis ◽

Differential Association ◽

Import And Export ◽

Snornp Biogenesis ◽

Cellular Compartments ◽

Distinct Distribution

ABSTRACT Box C/D snoRNPs, factors essential for ribosome biogenesis, are proposed to be assembled in the nucleoplasm before localizing to the nucleolus. However, recent work demonstrated the involvement of nuclear export factors in this process, suggesting that export may take place. Here we show that there are distinct distributions of U8 pre-snoRNAs and pre-snoRNP complexes in HeLa cell nuclear and cytoplasmic extracts. We observed differential association of nuclear export (PHAX, CRM1, and Ran) factors with complexes in the two extracts, consistent with nucleocytoplasmic transport. Furthermore, we show that the U8 pre-snoRNA in one of the cytoplasmic complexes contains an m3G cap and is associated with the nuclear import factor Snurportin1. Using RNA interference, we show that loss of either PHAX or Snurportin1 results in the incorrect localization of the U8 snoRNA. Our data therefore show that nuclear export and import factors are directly involved in U8 box C/D snoRNP biogenesis. The distinct distribution of U8 pre-snoRNP complexes between the two cellular compartments together with the association of both nuclear import and export factors with the precursor complex suggests that the mammalian U8 snoRNP is exported during biogenesis.

Mutations in theYRB1Gene Encoding Yeast Ran-Binding-Protein-1 That Impair Nucleocytoplasmic Transport and Suppress Yeast Mating Defects

Genetics ◽

10.1093/genetics/157.3.1089 ◽

2001 ◽

Vol 157 (3) ◽

pp. 1089-1105 ◽

Cited By ~ 1

Author(s):

Markus Künzler ◽

Joshua Trueheart ◽

Claudio Sette ◽

Eduard Hurt ◽

Jeremy Thorner

Keyword(s):

Nuclear Export ◽

Protein Import ◽

Essential Gene ◽

Binding Protein ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Primary Defect

AbstractWe identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (α-importin) and several β-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Δ and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.

Epigenetic Small Molecules Rescue Nucleocytoplasmic Transport and DNA Damage Phenotypes in C9ORF72 ALS/FTD

Brain Sciences ◽

10.3390/brainsci11111543 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1543

Author(s):

Melina Ramic ◽

Nadja S. Andrade ◽

Matthew J. Rybin ◽

Rustam Esanov ◽

Claes Wahlestedt ◽

...

Keyword(s):

Dna Damage ◽

Small Molecules ◽

Nuclear Import ◽

Nuclear Export ◽

Hdac Inhibitors ◽

Therapeutic Effects ◽

Nuclear Localization Signals ◽

Compound Libraries ◽

Expansion Mutation

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport “biosensors”, composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype—persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects—resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.