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1.
Front Microbiol ; 14: 1201640, 2023.
Article in English | MEDLINE | ID: mdl-37779700

ABSTRACT

Flaviviruses have emerged as major arthropod-transmitted pathogens and represent an increasing public health problem worldwide. High-throughput screening can be facilitated using viruses that easily express detectable marker proteins. Therefore, developing molecular tools, such as reporter-carrying versions of flaviviruses, for studying viral replication and screening antiviral compounds represents a top priority. However, the engineering of flaviviruses carrying either fluorescent or luminescent reporters remains challenging due to the genetic instability caused by marker insertion; therefore, new approaches to overcome these limitations are needed. Here, we describe reverse genetic methods that include the design and validation of infectious clones of Zika, Kunjin, and Dengue viruses harboring different reporter genes for infection, rescue, imaging, and morphology using super-resolution microscopy. It was observed that different flavivirus constructs with identical designs displayed strikingly different genetic stabilities, and corresponding virions resembled wild-type virus particles in shape and size. A successful strategy was assessed to increase the stability of rescued reporter virus and permit antiviral drug screening based on quantitative automated fluorescence microscopy and replication studies.

2.
iScience ; 25(10): 105066, 2022 Oct 21.
Article in English | MEDLINE | ID: mdl-36093378

ABSTRACT

Patients with severe COVID-19 show an altered immune response that fails to control the viral spread and suffer from exacerbated inflammatory response, which eventually can lead to death. A major challenge is to develop an effective treatment for COVID-19. NF-κB is a major player in innate immunity and inflammatory process. By a high-throughput screening approach, we identified FDA-approved compounds that inhibit the NF-κB pathway and thus dampen inflammation. Among these, we show that Auranofin prevents post-translational modifications of NF-κB effectors and their recruitment into activating complexes in response to SARS-CoV-2 infection or cytokine stimulation. In addition, we demonstrate that Auranofin counteracts several steps of SARS-CoV-2 infection. First, it inhibits a raft-dependent endocytic pathway involved in SARS-CoV-2 entry into host cells; Second, Auranofin alters the ACE2 mobility at the plasma membrane. Overall, Auranofin should prevent SARS-CoV-2 infection and inflammatory damages, offering new opportunities as a repurposable drug candidate to treat COVID-19.

4.
Front Immunol ; 12: 697329, 2021.
Article in English | MEDLINE | ID: mdl-34386007

ABSTRACT

Various neurological symptoms have been associated to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection including headache, fever, anosmia, ageusia, but also, encephalitis, Guillain-Barre syndrome and ischemic stroke. Responsible for the current coronavirus disease (COVID-19) pandemic, SARS-CoV-2 may access and affect the central nervous system (CNS) by several pathways such as axonal retrograde transport or through interaction with the blood-brain barrier (BBB) or blood-cerebrospinal fluid (CSF) barrier. Here, we explored the molecular and cellular effects of direct SARS-CoV-2 infection of human BBB cells. We observed low replication of SARS-CoV-2 that was accompanied by very moderate inflammatory response. Using a human in vitro BBB model, we also described low replication levels without strong inflammatory response or modulation of endothelium integrity. Finally, using serum samples from COVID-19 patients, we highlighted strong concentrations of pro-inflammatory factors that did not perturb BBB integrity after short term exposure. Altogether, our results show that the main mechanism of brain access following SARS-CoV-2 infection does not seem to be directed by brain infection through endothelial cells.


Subject(s)
Blood-Brain Barrier/virology , Brain/virology , Endothelial Cells/virology , SARS-CoV-2/growth & development , Virus Replication/physiology , Animals , COVID-19/pathology , Cell Line, Tumor , Chlorocebus aethiops , Humans , Vero Cells
5.
Sci Rep ; 11(1): 11885, 2021 06 04.
Article in English | MEDLINE | ID: mdl-34088957

ABSTRACT

SARS-CoV-2 is an enveloped virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Here, single viruses were analyzed by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess at the nanoscale level and in 3D infectious virus morphology in its native conformation, or upon inactivation treatments. AFM imaging reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol combining AFM and plaque assays allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-AFM is a remarkable toolbox for rapid and direct virus analysis based on nanoscale morphology.


Subject(s)
COVID-19/virology , Microscopy, Atomic Force , SARS-CoV-2/ultrastructure , Virion/ultrastructure , Animals , Chlorocebus aethiops , Humans , SARS-CoV-2/physiology , Vero Cells , Virion/physiology , Virus Inactivation
6.
Retrovirology ; 9: 87, 2012 Oct 23.
Article in English | MEDLINE | ID: mdl-23092122

ABSTRACT

BACKGROUND: Quiescent CD4+ T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription. RESULTS: Examination of SAMHD1 expression in peripheral blood lymphocytes shows that SAMHD1 is expressed in both CD4+ and CD8+ T cells at levels comparable to those found in myeloid cells. Treatment of CD4+ T cells with Virus-Like Particles (VLP) containing Vpx results in the loss of SAMHD1 expression that correlates with an increased permissiveness to HIV-1 infection and accumulation of reverse transcribed viral DNA without promoting transcription from the viral LTR. Importantly, CD4+ T-cells from patients with Aicardi-Goutières Syndrome harboring mutation in the SAMHD1 gene display an increased susceptibility to HIV-1 infection that is not further enhanced by VLP-Vpx-treatment. CONCLUSION: Here, we identified SAMHD1 as the restriction factor preventing efficient viral DNA synthesis in non-cycling resting CD4+ T-cells. These results highlight the crucial role of SAMHD1 in mediating restriction of HIV-1 infection in quiescent CD4+ T-cells and could impact our understanding of HIV-1 mediated CD4+ T-cell depletion and establishment of the viral reservoir, two of the HIV/AIDS hallmarks.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , HIV-1/immunology , HIV-1/pathogenicity , Host-Pathogen Interactions , Monomeric GTP-Binding Proteins/metabolism , Reverse Transcription , DNA, Viral/metabolism , Humans , Monomeric GTP-Binding Proteins/immunology , SAM Domain and HD Domain-Containing Protein 1 , Viral Regulatory and Accessory Proteins/immunology , Viral Regulatory and Accessory Proteins/metabolism
7.
Cell Host Microbe ; 11(2): 205-17, 2012 Feb 16.
Article in English | MEDLINE | ID: mdl-22305291

ABSTRACT

SAMHD1 has recently been identified as an HIV-1 restriction factor operating in myeloid cells. As a countermeasure, the Vpx accessory protein from HIV-2 and certain lineages of SIV have evolved to antagonize SAMHD1 by inducing its ubiquitin-proteasome-dependent degradation. Here, we show that SAMHD1 experienced strong positive selection episodes during primate evolution that occurred in the Catarrhini ancestral branch prior to the separation between hominoids (gibbons and great apes) and Old World monkeys. The identification of SAMHD1 residues under positive selection led to mapping the Vpx-interaction domain of SAMHD1 to its C-terminal region. Importantly, we found that while SAMHD1 restriction activity toward HIV-1 is evolutionarily maintained, antagonism of SAMHD1 by Vpx is species-specific. The distinct evolutionary signature of SAMHD1 sheds light on the development of its antiviral specificity.


Subject(s)
Evolution, Molecular , HIV-2/pathogenicity , Host-Pathogen Interactions , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Animals , Binding Sites , Cluster Analysis , HIV-1/immunology , HIV-1/pathogenicity , HIV-2/immunology , Humans , Phylogeny , Primates , Protein Binding , Protein Interaction Mapping , Sequence Homology, Amino Acid
8.
Nature ; 474(7353): 654-7, 2011 May 25.
Article in English | MEDLINE | ID: mdl-21613998

ABSTRACT

The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.


Subject(s)
Dendritic Cells/metabolism , HIV-1/physiology , Monomeric GTP-Binding Proteins/metabolism , Myeloid Cells/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Cell Line , DNA, Viral/metabolism , Dendritic Cells/virology , Gene Silencing , HIV Infections/metabolism , HeLa Cells , Humans , Monomeric GTP-Binding Proteins/genetics , Myeloid Cells/virology , Proteasome Endopeptidase Complex/metabolism , SAM Domain and HD Domain-Containing Protein 1 , U937 Cells , Virus Replication
9.
Nat Struct Mol Biol ; 18(3): 323-7, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21297638

ABSTRACT

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function by regulating gene expression post-transcriptionally. Alterations in miRNA expression can strongly influence cellular physiology. Here we demonstrated cross-regulation between two components of the RNA interference (RNAi) machinery in human cells. Inhibition of exportin-5, the karyopherin responsible for pre-miRNA export, downregulated expression of Dicer, the RNase III required for pre-miRNA maturation. This effect was post-transcriptional and resulted from an increased nuclear localization of Dicer mRNA. In vitro assays and cellular RNA immunoprecipitation experiments showed that exportin-5 interacted directly with Dicer mRNA. Titration of exportin-5 by overexpression of either pre-miRNA or the adenoviral VA1 RNA resulted in loss of Dicer mRNA-exportin-5 interaction and reduction of Dicer level. This saturation also occurred during adenoviral infection and enhanced viral replication. Our study reveals an important cross-regulatory mechanism between pre-miRNA or viral small RNAs and Dicer through exportin-5.


Subject(s)
DEAD-box RNA Helicases/genetics , Gene Expression Regulation , Karyopherins/metabolism , MicroRNAs/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Ribonuclease III/genetics , Adenoviridae/genetics , Adenoviridae/physiology , Adenoviridae Infections/virology , DEAD-box RNA Helicases/metabolism , HeLa Cells , Humans , Karyopherins/genetics , MicroRNAs/genetics , Protein Binding , RNA Interference , RNA, Messenger/genetics , RNA, Viral/genetics , Ribonuclease III/metabolism , Virus Replication
10.
Retrovirology ; 6: 26, 2009 Mar 09.
Article in English | MEDLINE | ID: mdl-19272132

ABSTRACT

The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.


Subject(s)
Gene Expression Regulation, Viral , HIV-1/physiology , MicroRNAs/metabolism , RNA, Viral/metabolism , Virus Replication/physiology , Argonaute Proteins , Cell Line , Cells, Cultured , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Eukaryotic Initiation Factor-2/metabolism , Gene Knockdown Techniques , HeLa Cells , Humans , Polyribosomes/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference/physiology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Virus Latency/physiology
11.
Nat Cell Biol ; 9(3): 331-8, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17293853

ABSTRACT

The p300-CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) involved in the reversible acetylation of various transcriptional regulators, including the tumour suppressor p53. It is implicated in many cellular processes, such as transcription, differentiation, proliferation and apoptosis. We observed that knockdown of PCAF expression in HeLa or U2OS cell lines induces stabilization of the oncoprotein Hdm2, a RING finger E3 ligase primarily known for its role in controlling p53 stability. To investigate the molecular basis of this effect, we examined whether PCAF is involved in Hdm2 ubiquitination. Here, we show that PCAF, in addition to its acetyltransferase activity, possesses an intrinsic ubiquitination activity that is critical for controlling Hdm2 expression levels, and thus p53 functions. Our data highlight a regulatory crosstalk between PCAF and Hdm2 activities, which is likely to have a central role in the subtle control of p53 activity after DNA damage.


Subject(s)
Cell Cycle Proteins/metabolism , Histone Acetyltransferases/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis/radiation effects , Binding Sites/genetics , Catalytic Domain/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Damage/drug effects , DNA Damage/radiation effects , Gene Expression/drug effects , Gene Expression/radiation effects , HeLa Cells , Histone Acetyltransferases/genetics , Humans , Mutation , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-mdm2/genetics , RNA, Antisense/genetics , RNA, Small Interfering/genetics , Transcription Factors/genetics , Transfection , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ultraviolet Rays , Zinostatin/pharmacology , p300-CBP Transcription Factors
12.
Mol Cell ; 25(3): 369-83, 2007 Feb 09.
Article in English | MEDLINE | ID: mdl-17289585

ABSTRACT

Although the proteasome facilitates transcription from several yeast promoters, it is unclear if its role is proteolytic or which subunits are involved. We show that the proteasome regulates the HIV-1 promoter in both proteolytic and nonproteolytic modes. In the absence of transcription factor, Tat, proteasome was associated with promoter and coding regions, and its proteolytic activity regulated the level of basal transcription emanating from the promoter. Tat switched the proteasome to a nonproteolytic mode by recruiting a proteasome-associated protein, PAAF1, which favors proteasome dissociation into 19S and 20S particles. Gel filtration chromatography showed that expression of both Tat and PAAF1 enhanced the abundance of a 19S-like complex in nuclear extracts. 19S, but not 20S, subunits were strongly recruited to the promoter in the presence of Tat and PAAF1 and coactivated Tat-dependent transcription. 19S components facilitated transcriptional elongation and may be involved in clearance of paused transcriptional elongation complexes from the promoter.


Subject(s)
Gene Expression Regulation, Viral , HIV-1/genetics , Proteasome Endopeptidase Complex/metabolism , Transcription, Genetic , Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Gene Products, tat/genetics , Gene Products, tat/metabolism , HIV Long Terminal Repeat , HeLa Cells , Humans , Promoter Regions, Genetic , tat Gene Products, Human Immunodeficiency Virus
13.
Science ; 315(5818): 1579-82, 2007 Mar 16.
Article in English | MEDLINE | ID: mdl-17322031

ABSTRACT

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1-infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.


Subject(s)
HIV-1/physiology , Leukocytes, Mononuclear/virology , MicroRNAs/genetics , RNA Interference , Virus Replication , 3' Untranslated Regions , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Gene Expression Regulation , Gene Products, tat/metabolism , HIV-1/genetics , HeLa Cells , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Humans , Jurkat Cells , Leukocytes, Mononuclear/enzymology , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering/genetics , Ribonuclease III/genetics , Ribonuclease III/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection , Virus Latency , p300-CBP Transcription Factors , tat Gene Products, Human Immunodeficiency Virus
14.
EMBO J ; 26(2): 424-35, 2007 Jan 24.
Article in English | MEDLINE | ID: mdl-17245432

ABSTRACT

HIV-1 gene expression is the major determinant regulating the rate of virus replication and, consequently, AIDS progression. Following primary infection, most infected cells produce virus. However, a small population becomes latently infected and constitutes the viral reservoir. This stable viral reservoir seriously challenges the hope of complete viral eradication. Viewed in this context, it is critical to define the molecular mechanisms involved in the establishment of transcriptional latency and the reactivation of viral expression. We show that Suv39H1, HP1gamma and histone H3Lys9 trimethylation play a major role in chromatin-mediated repression of integrated HIV-1 gene expression. Suv39H1, HP1gamma and histone H3Lys9 trimethylation are reversibly associated with HIV-1 in a transcription-dependent manner. Finally, we show in different cellular models, including PBMCs from HIV-1-infected donors, that HIV-1 reactivation could be achieved after HP1gamma RNA interference.


Subject(s)
Chromatin/physiology , Chromosomal Proteins, Non-Histone/physiology , Gene Silencing , HIV-1/physiology , Methyltransferases/physiology , Repressor Proteins/physiology , Virus Integration , Virus Latency , Cell Cycle Proteins/physiology , Cells, Cultured , HIV Long Terminal Repeat , HeLa Cells , Histone Acetyltransferases/physiology , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Humans , Jurkat Cells , Models, Biological , Positive Transcriptional Elongation Factor B/physiology , Protein Methyltransferases , Sp1 Transcription Factor/physiology , Transcription Factors/physiology , Transcription, Genetic , Transcriptional Activation , p300-CBP Transcription Factors
15.
J Immunol ; 177(11): 7607-17, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17114430

ABSTRACT

MAPK cascades play a central role in the cellular response to the environment. The pathway involving the MAPK ERK5 mediates growth factor- and stress-induced intracellular signaling that controls proliferation or survival depending upon the cell context. In this study, we show that reducing ERK5 levels with a specific small hairpin RNA 5 (shERK5) reduced cell viability, sensitized cells to death receptor-induced apoptosis, and blocked the palliative effects of phorbol ester in anti-Fas Ab-treated cells. shERK5 decreased nuclear accumulation of the NF-kappaB p65 subunit, and conversely, ectopic activation of ERK5 led to constitutive nuclear localization of p65 and increased its ability to trans activate specific reporter genes. Finally, the T lymphoma cell line EL-4, upon expression of shERK5, proliferated in vitro, but failed to induce s.c. tumors in mice. Our results suggest that ERK5 is essential for survival of leukemic T cells in vivo, and thus represents a promising target for therapeutic intervention in this type of malignancy.


Subject(s)
Cell Proliferation , Leukemia/enzymology , Mitogen-Activated Protein Kinase 7/metabolism , NF-kappa B/metabolism , T-Lymphocytes/metabolism , Animals , Apoptosis/physiology , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Humans , Immunoprecipitation , Jurkat Cells , Lymphocyte Activation/immunology , Mice , Protein Transport/immunology , RNA, Small Interfering , Transcriptional Activation , Transfection , eIF-2 Kinase/metabolism
16.
EMBO J ; 24(14): 2634-45, 2005 Jul 20.
Article in English | MEDLINE | ID: mdl-16001085

ABSTRACT

HIV-1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP-associated E4-type ubiquitin-ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60-dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV-1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination.


Subject(s)
Acetyltransferases/metabolism , Apoptosis/physiology , DNA Damage/physiology , Gene Products, tat/metabolism , Gene Products, tat/physiology , HIV-1/metabolism , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Histone Acetyltransferases , Humans , Jurkat Cells , Lysine Acetyltransferase 5 , Nuclear Proteins/metabolism , Polyubiquitin/metabolism , Trans-Activators/metabolism , tat Gene Products, Human Immunodeficiency Virus
17.
J Clin Invest ; 112(7): 1108-15, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14523047

ABSTRACT

X-linked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the gene encoding NEMO/IKKgamma, the regulatory subunit of the IkappaB kinase (IKK) complex. IKK normally phosphorylates the IkappaB-inhibitors of NF-kappaB at specific serine residues, thereby promoting their ubiquitination and degradation by the proteasome. This allows NF-kappaB complexes to translocate into the nucleus where they activate their target genes. Here, we describe an autosomal-dominant (AD) form of EDA-ID associated with a heterozygous missense mutation at serine 32 of IkappaBalpha. This mutation is gain-of-function, as it enhances the inhibitory capacity of IkappaBalpha by preventing its phosphorylation and degradation, and results in impaired NF-kappaB activation. The developmental, immunologic, and infectious phenotypes associated with hypomorphic NEMO and hypermorphic IKBA mutations largely overlap and include EDA, impaired cellular responses to ligands of TIR (TLR-ligands, IL-1beta, and IL-18), and TNFR (TNF-alpha, LTalpha1/beta2, and CD154) superfamily members and severe bacterial diseases. However, AD-EDA-ID but not XL-EDA-ID is associated with a severe and unique T cell immunodeficiency. Despite a marked blood lymphocytosis, there are no detectable memory T cells in vivo, and naive T cells do not respond to CD3-TCR activation in vitro. Our report highlights both the diversity of genotypes associated with EDA-ID and the diversity of immunologic phenotypes associated with mutations in different components of the NF-kappaB signaling pathway.


Subject(s)
Ectodermal Dysplasia/genetics , I-kappa B Proteins/genetics , Immunologic Deficiency Syndromes/genetics , Mutation , NF-kappa B/physiology , Protein Serine-Threonine Kinases/genetics , T-Lymphocytes/immunology , Child , Humans , I-kappa B Kinase , Male , Receptor-CD3 Complex, Antigen, T-Cell/physiology , Signal Transduction/physiology , Transcription, Genetic , Tumor Necrosis Factor-alpha/physiology
18.
Nature ; 424(6950): 801-5, 2003 Aug 14.
Article in English | MEDLINE | ID: mdl-12917691

ABSTRACT

NF-kappaB transcription factors have key roles in inflammation, immune response, oncogenesis and protection against apoptosis. In most cells, these factors are kept inactive in the cytoplasm through association with IkappaB inhibitors. After stimulation by various reagents, IkappaB is phosphorylated by the IkappaB kinase (IKK) complex and degraded by the proteasome, allowing NF-kappaB to translocate to the nucleus and activate its target genes. Here we report that CYLD, a tumour suppressor that is mutated in familial cylindromatosis, interacts with NEMO, the regulatory subunit of IKK. CYLD also interacts directly with tumour-necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), an adaptor molecule involved in signalling by members of the family of TNF/nerve growth factor receptors. CYLD has deubiquitinating activity that is directed towards non-K48-linked polyubiquitin chains, and negatively modulates TRAF-mediated activation of IKK, strengthening the notion that ubiquitination is involved in IKK activation by TRAFs and suggesting that CYLD functions in this process. Truncations of CYLD found in cylindromatosis result in reduced enzymatic activity, indicating a link between impaired deubiquitination of CYLD substrates and human pathophysiology.


Subject(s)
NF-kappa B/antagonists & inhibitors , Signal Transduction , Tumor Suppressor Proteins/metabolism , Ubiquitin/metabolism , Amino Acid Sequence , Catalysis , Cell Line , Deubiquitinating Enzyme CYLD , Humans , I-kappa B Kinase , Molecular Sequence Data , Mutation , NF-kappa B/metabolism , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Substrate Specificity , TNF Receptor-Associated Factor 2 , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics
19.
Nat Cell Biol ; 5(8): 754-61, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12883554

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat, which functions through binding to a short leader RNA, called transactivation responsive element (TAR). Recent studies suggest that Tat activates the HIV-1 long terminal repeat (LTR), mainly by adapting co-activator complexes, such as p300, PCAF and the positive transcription elongation factor P-TEFb, to the promoter. Here, we show that the proto-oncoprotein Hdm2 interacts with Tat and mediates its ubiquitination in vitro and in vivo. In addition, Hdm2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination. Fusion of ubiquitin to Tat bypasses the requirement of Hdm2 for efficient transactivation, supporting the notion that ubiquitin has a non-proteolytic function in Tat-mediated transactivation.


Subject(s)
Gene Products, tat/metabolism , HIV-1/genetics , Nuclear Proteins , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , Transcriptional Activation , Ubiquitin/metabolism , Cell Line , Gene Products, tat/genetics , HIV Long Terminal Repeat , HIV-1/metabolism , Humans , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-mdm2 , RNA, Small Interfering , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , tat Gene Products, Human Immunodeficiency Virus
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