Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm

I kappa B alpha tightly regulates the transcriptional activity of NF-kappa B by retaining it in the cytoplasm in an inactive form. In the present work, we report that I kappa B alpha, when expressed in the nuclear compartment, not only abrogates NF-kappa B/DNA interactions and NF-kappa B-dependent transcription, but also transports NF-kappa B back to the cytoplasm. This function of I kappa B alpha is insured by a nuclear export sequence located in the C-terminal domain of I kappa B alpha and homologous to the previously described export signal found in HIV-1 Rev protein as well as in PKI (the inhibitor of the catalytic subunit of protein kinase A). Thus, inhibition of NF-kappa B/DNA binding and the consecutive efficient nuclear export of the transcription factor of I kappa B alpha could represent an important mechanism for the control of the expression of NF-kappa B-dependent genes.

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Location of the HIV-1 Rev protein during mitosis: inactivation of the nuclear export signal alters the pathway for postmitotic reentry into nucleoli

Journal of Cell Science ◽

10.1242/jcs.109.9.2239 ◽

1996 ◽

Vol 109 (9) ◽

pp. 2239-2251 ◽

Cited By ~ 1

Author(s):

M. Dundr ◽

G.H. Leno ◽

N. Lewis ◽

D. Rekosh ◽

M.L. Hammarskjoid ◽

...

Keyword(s):

Nuclear Envelope ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Nucleolar Protein ◽

Early Prophase ◽

Nucleolar Proteins ◽

Late Telophase ◽

Export Signal ◽

Hiv 1 ◽

Rev Protein

The HIV-1 Rev protein localizes predominantly to the nucleolus of HIV-1-infected or Rev-expressing cells. The subcellular location of Rev during mitotic nucleolar disintegration was examined at various stages of mitosis in synchronized Rev-expressing CMT3 cells. During early prophase Rev was predominantly located in disintegrating nucleoli and began to accumulate at the peripheral regions of chromosomes in late prophase, eventually distributing uniformly on all chromosomes in prometaphase. In anaphase Rev remained associated with the perichromosomal regions, but significant amounts of Rev were also seen in numerous nucleolus-derived foci. The movement of Rev from disintegrating nucleoli to perichromosomal regions and foci was similar to that of nonribosomal nucleolar proteins, including fibrillarin, nucleolin, protein B23 and p52 of the granular component. During telophase Rev remained associated with perichromosomal regions and mitotic foci until the nuclear envelope started to reform. When nuclear envelope formation was complete in late telophase, nonribosomal nucleolar proteins were present in prenucleolar bodies (PNBs) which were eventually incorporated into nucleoli; at the same time, Rev was excluded from nuclei. In contrast, a trans-dominant negative Rev protein containing an inactive nuclear export signal reentered nuclei by the nonribosomal nucleolar protein pathway in late telophase, associating with PNBs and reformed nucleoli. Rev protein reentry into postmitotic nuclei was delayed until early G1 phase, but before the arrival of ribosomal protein S6. Thus, Rev behaves like a nonribosomal nucleolar protein through mitosis until early telophase; however, its nuclear reentry seems to require reestablishment of both a nuclear import system and active nucleoli.

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Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

Journal of Virology ◽

10.1128/jvi.02107-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 17

Author(s):

Ryan T. Behrens ◽

Mounavya Aligeti ◽

Ginger M. Pocock ◽

Christina A. Higgins ◽

Nathan M. Sherer

Keyword(s):

Gene Expression ◽

Nuclear Export ◽

Rna Binding ◽

Nuclear Export Signal ◽

Viral Gene Expression ◽

Viral Gene ◽

Virion Production ◽

Export Signal ◽

Hiv 1 ◽

Rev Protein

ABSTRACT HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. IMPORTANCE HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.

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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 ◽

10.1016/s1074-5521(97)90257-x ◽

1997 ◽

Vol 4 (2) ◽

pp. 139-147 ◽

Cited By ~ 474

Author(s):

Barbara Wolff ◽

Jean-Jacques Sanglier ◽

Ying Wang

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Nuclear Export ◽

Leptomycin B ◽

Immunodeficiency Virus ◽

Cytoplasmic Translocation ◽

Hiv 1 ◽

Rev Protein

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Mutational Definition of Functional Domains within the Rev Homolog Encoded by Human Endogenous Retrovirus K

Journal of Virology ◽

10.1128/jvi.74.20.9353-9361.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9353-9361 ◽

Cited By ~ 10

Author(s):

Hal P. Bogerd ◽

Heather L. Wiegand ◽

Jin Yang ◽

Bryan R. Cullen

Keyword(s):

Nuclear Export ◽

Biological Activities ◽

Endogenous Retrovirus ◽

Viral Rna ◽

Human Endogenous Retrovirus ◽

Nuclear Rna ◽

Rna Export ◽

Nuclear Rna Export ◽

Hiv 1 ◽

Rev Protein

ABSTRACT Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a cis-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the ∼25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.

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A New Nucleoporin-like Protein Interacts with Both HIV-1 Rev Nuclear Export Signal and CRM-1

Journal of Biological Chemistry ◽

10.1074/jbc.274.24.17309 ◽

1999 ◽

Vol 274 (24) ◽

pp. 17309-17317 ◽

Cited By ~ 31

Author(s):

Géraldine Farjot ◽

Alain Sergeant ◽

Ivan Mikaélian

Keyword(s):

Nuclear Export ◽

Nuclear Export Signal ◽

Export Signal ◽

Hiv 1

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Nuclear export signal of IkappaBalpha interferes with the Rev-dependent posttranscriptional regulation of human immunodeficiency virus type I

Journal of Cell Science ◽

10.1242/jcs.110.22.2883 ◽

1997 ◽

Vol 110 (22) ◽

pp. 2883-2893

Author(s):

F. Bachelerie ◽

M.S. Rodriguez ◽

C. Dargemont ◽

D. Rousset ◽

D. Thomas ◽

...

Keyword(s):

Dna Binding ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Binding Activity ◽

Nuclear Accumulation ◽

Viral Rnas ◽

Dna Binding Activity ◽

Nf Kappab ◽

Export Signal ◽

Hiv 1

De novo synthesized IkappaBalpha accumulates transiently in the nucleus where it inhibits NF-kappaB-dependent transcription and reduces nuclear NF-kappaB content. A sequence present in the C-terminal domain of IkappaBalpha and homologous to the HIV-1 Rev nuclear export signal (NES) has been recently defined as a functional NES conferring on IkappaBalpha the ability to export IkappaBalpha/NF-kappaB complexes. Rev utilises its RNA-binding activity and NES sequence to promote specifically the transport of unspliced and monospliced viral RNAs to the cytoplasm. The object of this work was to determine if nuclear IkappaBalpha could interfere with Rev-dependent transport of viral RNA from the nucleus to the cytoplasm. We report that accumulation of IkappaBalpha in the cell nucleus blocks viral replication. This effect could be dissociated from the capacity of IkappaBalpha to inhibit NF-kappaB-DNA-binding activity and required a functional IkappaBalpha NES motif. Indeed, mutation of the NES abrogated the capacity of IkappaBalpha to inhibit Rev-dependent mechanisms involved in the replication of either wild-type or NF-kappaB-mutated HIV-1 molecular clones. Nuclear accumulation of a reporter protein tagged with a nuclear localization signal (NLS) and fused to the IkappaBalpha NES motif (NLS-PK-NES) was sufficient to inhibit HIV-1 replication at a post-transcriptional level by specifically blocking the expression of a Rev-dependent gene. Furthermore, in cells pulsed with TNF, a treatment which favors nuclear accumulation of newly synthesized IkappaBalpha, NLS-PK-NES expression promoted sustained accumulation of nuclear NF-kappaB lacking DNA-binding activity. This NES-mediated accumulation of inactive nuclear NF-kappaB is likely the consequence of interference in the IkappaBalpha-mediated export of NF-kappaB. These findings indicate that IkappaBalpha and Rev compete for the same nuclear export pathway and suggest that nuclear accumulation of IkappaBalpha, which would occur during normal physiological cell activation process, may interfere with the Rev-NES-mediated export pathway of viral RNAs, thus inhibiting HIV-1 replication.

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