Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 78
Filter
Add more filters










Publication year range
1.
Parasite ; 14(2): 141-7, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17645186

ABSTRACT

Toxoplasma gondii is an obligate intracellular parasite that infects all types of cells in humans. A family of calcium-dependent protein kinases (CDPKs), previously identified as important in the development of plants and protists, was recently shown to play a role in the infectivity of apicomplexans, and in motility and host cell invasion in particular. We report here the isolation of a new calcium-dependent protein kinase gene from the human toxoplasmosis parasite, Toxoplasma gondii. The gene consists of 12 exons. The encoded protein, TgCDPK4, consists of the four characteristic domains of members of the CDPK family and is most similar to PfCDPK2 from Plasmodium falciparum. We measured TgCDPK4 activity, induced by calcium influx, using a kinase assay. A calcium chelator (EGTA) inhibited this activity. These findings provide evidence of signal transduction involving members of the CDPK family in T. gondii.


Subject(s)
Calcium/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Signal Transduction , Toxoplasma/enzymology , Amino Acid Sequence , Animals , Coccidiostats/pharmacology , Exons , Host-Parasite Interactions , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , Protein Kinases/chemistry , Sequence Alignment , Toxoplasma/genetics , Toxoplasma/pathogenicity
2.
J Biol Chem ; 276(36): 33899-905, 2001 Sep 07.
Article in English | MEDLINE | ID: mdl-11438532

ABSTRACT

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.


Subject(s)
HIV Long Terminal Repeat/genetics , RNA-Binding Proteins/chemistry , eIF-2 Kinase/metabolism , Amino Acids/chemistry , Binding Sites , Dimerization , Gene Deletion , Genes, Reporter , HeLa Cells , Humans , Luciferases/metabolism , Models, Genetic , Mutation , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , RNA/metabolism , Transfection , Two-Hybrid System Techniques
3.
Virology ; 282(1): 176-85, 2001 Mar 30.
Article in English | MEDLINE | ID: mdl-11259200

ABSTRACT

The virion-associated Vpr protein of human immunodeficiency virus type 1 (HIV-1) alters cell cycle progression from the G2 phase, influences the virus in vivo mutation rate, and participates in the nuclear translocation of viral DNA. While many Vpr-interacting proteins have been identified, the functional relevance of these interactions remains to be thoroughly documented. We have explored the contribution of the interaction of HIV-1 Vpr with HHR23A, a cellular protein implicated in DNA repair, to the known phenotypes of Vpr. The association of Vpr with HHR23A required the core region of Vpr, which encompasses the two alpha-helical structures of the protein. No binding of HHR23A was detected with the Vpr and Vpx proteins of other primate lentiviruses. HIV-1 Vpr variants containing single amino acid substitutions in each alpha-helix and deficient for binding to HHR23A were isolated. The functional characterization of these Vpr variants indicated that binding to HHR23A did not correlate with the ability of Vpr to induce cell cycle arrest, even though it was previously proposed that HHR23A is a mediator of the Vpr-induced G2 arrest. Also, the Vpr-HHR23A interaction did not influence the HIV-1 in vivo mutation rate. Finally, Vpr and HHR23A are both localized in the nucleus, but no correlation was observed between the nuclear targeting of Vpr and the interaction with HHR23A. Further analysis is needed to determine the functional role(s) of the Vpr-HHR23A association during the HIV-1 life cycle.


Subject(s)
DNA Repair , DNA-Binding Proteins/metabolism , Gene Products, vpr/metabolism , HIV-1/metabolism , Retroviridae Proteins/metabolism , Amino Acid Substitution , Animals , COS Cells , Cell Cycle , Cell Nucleus/metabolism , DNA Repair Enzymes , Genetic Vectors , HeLa Cells , Humans , Transfection , Virus Replication , vpr Gene Products, Human Immunodeficiency Virus
4.
J Virol ; 75(8): 3971-6, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11264386

ABSTRACT

The Nef protein from the human immunodeficiency virus (HIV) induces CD4 cell surface downregulation by interfering with the endocytic machinery. It has been recently proposed that binding of HIV type 1 Nef to the beta subunit of COPI coatomers participated in the Nef-induced CD4 downregulation through recognition of a novel diacidic motif found in the C-terminal disordered loop of Nef (V. Piguet, F. Gu, M. Foti, N. Demaurex, J. Gruenberg, J. L. Carpentier, and D. Trono, Cell 97:63-73, 1999). We have mutated the glutamate residues which formed this motif in order to document this observation. Surprisingly, mutation of the diacidic sequence of Nef did not significantly affect its ability (i) to interact with beta-COP, (ii) to downregulate CD4 cell surface expression, and (iii) to address an integral resident membrane protein containing Nef as the cytoplasmic domain to the endocytic pathway. Our results indicate that these acidic residues are not involved in the connection of Nef with the endocytic machinery through binding to beta-COP. Additional studies are thus required to characterize the residues of Nef involved in the binding to beta-COP and to evaluate the contribution of this interaction to the Nef-induced perturbations of membrane trafficking.


Subject(s)
CD4 Antigens/metabolism , Coatomer Protein/metabolism , Down-Regulation , Gene Products, nef/chemistry , Gene Products, nef/metabolism , Glutamic Acid/metabolism , HIV-1 , Adaptor Protein Complex gamma Subunits , Amino Acid Motifs , Amino Acid Substitution , Biological Transport , CD4 Antigens/genetics , CD8 Antigens/genetics , CD8 Antigens/metabolism , Endocytosis , Gene Products, nef/genetics , Glutamic Acid/genetics , HIV-1/genetics , HeLa Cells , Humans , Macromolecular Substances , Membrane Proteins/metabolism , Mutation , Protein Binding , Recombinant Fusion Proteins , Reproducibility of Results , Two-Hybrid System Techniques , nef Gene Products, Human Immunodeficiency Virus
5.
Mol Cell Biol ; 21(6): 2192-202, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11238952

ABSTRACT

The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein betaTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IkappaBalpha and beta-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of betaTrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of betaTrCP. The F-box-deleted betaTrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCF(betaTrCP) complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.


Subject(s)
Cell Nucleus/metabolism , Peptide Synthases/metabolism , Transcription Factors/metabolism , Activating Transcription Factor 4 , Amino Acid Motifs , Cells, Cultured , Cyclic AMP/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Humans , Mutation , Phosphorylation , Precipitin Tests , SKP Cullin F-Box Protein Ligases , Serine , Transcription Factors/genetics , Transcription, Genetic , beta-Transducin Repeat-Containing Proteins
6.
J Virol ; 75(6): 2982-92, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11222723

ABSTRACT

Short amino acid sequences in the cytosolic domains of transmembrane proteins are recognized by specialized adaptor [corrected] proteins which are part of coated vesicles utilized to transport membrane proteins between the trans-Golgi network (TGN) and the plasma membrane (forward and backward). Previously, we and others reported that the membrane-proximal tyrosine residues Y712 (human immunodeficiency virus [HIV]) and Y721 (simian immunodeficiency virus [SIV]) in the envelope glycoprotein (Env) of the primate lentiviruses are crucial for the association of Env with clathrin-associated adaptor [corrected] complex AP-2. The same tyrosine-based endocytosis motifs in the cytosolic domains (EnvCD) of transmembrane gp41 of HIV type 1 (HIV-1) and SIV, respectively, were also shown to modulate the interaction with TGN- and endosome-based clathrin-associated complex AP-1. Our findings suggested that EnvCD binding to AP-1, unlike the association of EnvCD with AP-2, is dependent largely on residues other than Y712 and Y721. Here, we tested if motifs downstream of Y712 affect HIV-1 EnvCD-AP-1 binding and Env trafficking. Mutational analysis revealed that the C-terminal leucine-based motif in Env was crucial for the recruitment of AP-1 in vitro and in Env-expressing cells. In addition to affecting Env-AP-1 association, mutations at the C terminus of Env also altered the subcellular localization of Env, suggesting that proper post-Golgi routing of Env depends on its recruitment of AP-1. Finally, the C-terminal dileucine was shown to assist the membrane-proximal Y712 motif in restricting the cell surface expression of Env.


Subject(s)
Gene Products, env/chemistry , Gene Products, env/metabolism , HIV-1/metabolism , Membrane Proteins/metabolism , Adaptor Protein Complex delta Subunits , Amino Acid Motifs , Amino Acid Sequence , Gene Expression Regulation, Viral , Gene Products, env/genetics , Genes, env , HIV-1/chemistry , HeLa Cells , Humans , Leucine/chemistry , Leucine/genetics , Membrane Proteins/genetics , Molecular Sequence Data , Mutation , Signal Transduction , Subcellular Fractions/metabolism , Transfection
7.
J Virol ; 74(15): 7039-47, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10888643

ABSTRACT

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) influences the in vivo mutation rate of the virus. Since Vpr interacts with a cellular protein implicated in the DNA repair process, uracil DNA glycosylase (UNG), we have explored the contribution of this interaction to the mutation rate of HIV-1. Single-amino-acid variants of Vpr were characterized for their differential UNG-binding properties and used to trans complement vpr null mutant HIV-1. A striking correlation was established between the abilities of Vpr to interact with UNG and to influence the HIV-1 mutation rate. We demonstrate that Vpr incorporation into virus particles is required to influence the in vivo mutation rate and to mediate virion packaging of the nuclear form of UNG. The recruitment of UNG into virions indicates a mechanism for how Vpr can influence reverse transcription accuracy. Our data suggest that distinct mechanisms evolved in primate and nonprimate lentiviruses to reconcile uracil misincorporation into lentiviral DNA.


Subject(s)
DNA Glycosylases , Gene Products, vpr/metabolism , HIV-1/genetics , Mutation , N-Glycosyl Hydrolases/metabolism , Animals , Base Sequence , COS Cells , Cell Nucleus/enzymology , Gene Products, vpr/genetics , Genetic Complementation Test , Genetic Vectors , HIV-1/physiology , HeLa Cells , Humans , Molecular Sequence Data , Two-Hybrid System Techniques , Uracil-DNA Glycosidase , Virion/metabolism , Virus Replication , vpr Gene Products, Human Immunodeficiency Virus
8.
J Virol ; 74(11): 5310-9, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10799608

ABSTRACT

Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.


Subject(s)
CD4 Antigens/biosynthesis , Conserved Sequence , Down-Regulation/immunology , Gene Products, nef/immunology , HIV-1/immunology , HLA-A2 Antigen/biosynthesis , Thiolester Hydrolases/immunology , Amino Acid Sequence , Cell Membrane/immunology , Dimerization , Gene Products, nef/chemistry , Gene Products, nef/genetics , HIV-1/physiology , HeLa Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/immunology , Palmitoyl-CoA Hydrolase , Protein Binding , Protein Conformation , nef Gene Products, Human Immunodeficiency Virus
9.
J Biol Chem ; 275(6): 4171-6, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10660579

ABSTRACT

The nef gene is required for optimal viral spread of human and simian immunodeficiency viruses. However, the molecular mechanisms underlying the action of the Nef proteins may not be identical for all viral families. Here we investigate the interaction between the Nef protein of human and simian immunodeficiency viruses and SH3 domains from Src family kinases. Using the yeast two-hybrid system and immunoblotting we show that, in contrast to HIV-1 Nef, SIV and HIV-2 Nef poorly interact with Hck SH3 but bind to Src and Fyn SH3 domains. The molecular basis of these differences in SH3 targeting was revealed by sequence analysis and homology modeling of the putative SH3-Nef structures. Three amino acids (Trp-113, Thr-117, and Gln-118) that localize in a "hydrophobic pocket" implicated in SH3 binding of HIV-1 Nef, are systematically substituted in SIV/HIV-2 alleles (by Tyr, Glu, and Glu, respectively). We demonstrate that site-directed mutagenesis of these residues in SIV(mac239) Nef suffices to restore Hck SH3 binding and co-immunoprecipitation with full-length Hck from transfected cells. Our findings identify fundamental mechanistic differences in targeting of Src family kinases by HIV and SIV Nef. The herein described mechanism of SH3 selection by Nef via a "pocket" proximal to the canonical proline-rich motif may be a common feature for SH3 recognition by their natural ligands.


Subject(s)
Gene Products, nef/metabolism , HIV-1/metabolism , HIV-2/metabolism , Simian Immunodeficiency Virus/metabolism , src Homology Domains , src-Family Kinases/metabolism , Amino Acid Sequence , Animals , COS Cells , Gene Products, nef/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding/genetics , Sequence Alignment , Transfection , Yeasts , nef Gene Products, Human Immunodeficiency Virus
10.
Traffic ; 1(11): 871-83, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11208076

ABSTRACT

The Nef protein from the human immunodeficiency virus (HIV) induces down-regulation of the CD4 and major histocompatibility complex class I molecules from the cell surface by interfering with the endocytic machinery. This work focuses on the interaction of HIV-1 Nef with the mu 1 chain of adaptor protein type 1 (AP1) complex and its contribution to the Nef-induced alterations of membrane trafficking. Two independent regions surrounding a disordered loop located in the C-terminal part of Nef are involved in mu 1 binding. Each region can separately interact with mu 1, and simultaneous point mutations within both regions are needed to abolish binding. We used CD8 chimeras in which the cytoplasmic tail was replaced by Nef mutants to show that these mu 1-binding sites contain determinants required to induce CD4 down-regulation and to target the chimera to the endocytic pathway by promoting AP1 complex recruitment. Ultrastructural analysis revealed that the CD8-Nef chimera provokes morphological alterations of the endosomal compartments and co-localizes with AP1 complexes. These data indicate that the recruitment by Nef of AP1 via binding to mu 1 participates in the connection of Nef with the endocytic pathway.


Subject(s)
Endocytosis/physiology , Genes, nef , HIV-1/genetics , HIV-1/physiology , Membrane Proteins/metabolism , Adaptor Protein Complex 1 , Adaptor Protein Complex alpha Subunits , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Binding Sites/genetics , CD4 Antigens/metabolism , CD8 Antigens/genetics , CD8 Antigens/metabolism , Cell Compartmentation , Cell Nucleus/metabolism , Down-Regulation , Endosomes/metabolism , HeLa Cells , Humans , Microscopy, Immunoelectron , Molecular Sequence Data , Point Mutation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection
11.
J Biol Chem ; 274(46): 32738-43, 1999 Nov 12.
Article in English | MEDLINE | ID: mdl-10551832

ABSTRACT

Mammalian peroxisomal proteins adrenoleukodystrophy protein (ALDP), adrenoleukodystrophy-related protein (ALDRP), and 70-kDa peroxisomal protein (PMP70) belong to the superfamily of ATP-binding cassette (ABC) transporters. Unlike many ABC transporters that are single functional proteins with two related halves, ALDP, ALDRP, and PMP70 have the structure of ABC half-transporters. The dysfunction of ALDP is responsible for X-linked adrenoleukodystrophy (X-ALD), a neurodegenerative disorder in which saturated very long-chain fatty acids accumulate because of their impaired peroxisomal beta-oxidation. No disease has so far been associated with mutations of adrenoleukodystrophy-related or PMP70 genes. It has been proposed that peroxisomal ABC transporters need to dimerize to exert import functions. Using the yeast two-hybrid system, we show that homo- as well as heterodimerization occur between the carboxyl-terminal halves of ALDP, ALDRP, and PMP70. Two X-ALD disease mutations located in the carboxyl-terminal half of ALDP affect both homo- and heterodimerization of ALDP. Co-immunoprecipitation demonstrated the homodimerization of ALDP, the heterodimerization of ALDP with PMP70 or ALDRP, and the heterodimerization of ALDRP with PMP70. These results provide the first evidence of both homo- and heterodimerization of mammalian ABC half-transporters and suggest that the loss of ALDP dimerization plays a role in X-ALD pathogenesis.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , Membrane Proteins/chemistry , Peroxisomes/chemistry , Proteins/chemistry , ATP Binding Cassette Transporter, Subfamily D , ATP Binding Cassette Transporter, Subfamily D, Member 1 , ATP-Binding Cassette Transporters/genetics , Adrenoleukodystrophy/etiology , Adrenoleukodystrophy/genetics , Animals , Dimerization , Humans , Membrane Proteins/genetics , Mice , Mutagenesis , Precipitin Tests , Protein Binding , Proteins/genetics , Yeasts
12.
Oncogene ; 18(29): 4262-8, 1999 Jul 22.
Article in English | MEDLINE | ID: mdl-10435639

ABSTRACT

The failure of signal transduction in the JCaM1 cell line was associated with the presence of an abnormal lck mRNA deleted of the exon 7 encoding for an inactive p56lck kinase. Our study of the lck mRNA from various T cell lines and from peripheral blood lymphocytes of healthy donors has revealed the presence of both complete and exon 7-deleted lck transcripts. Thus the exon 7-deleted lck transcript initially described in the JCaM1 mutant cell line, arises from an alternative splicing event occurring in each cells expressing the lck gene. Genomic DNA sequencing of the lck exons 6-8 portion from both the mutant JCaM1 and its parental Jurkat cell lines revealed as the only difference, the presence of a A to G mutation within the 5' splice site of intron 7 in the JCaM1 cell line DNA. To demonstrate the role of this point mutation in the lck pre-mRNA maturation, COS cells were transfected by lck minigenes from the Jurkat and JCaM1 cell lines. In COS cells transfected with minigene from the Jurkat cell line both lck transcripts (with and without exon 7) were observed whereas only the exon 7-spliced lck transcript was observed in COS cells transfected with minigene from the JCaM1 cell line. Thus the mutation is per se responsible for the deletion of exon 7 and the absence of complete lck mRNA in the JCaM1 cell line. Presence of a restriction site (HphI) in the 5' splice site of lck intron 7 from Jurkat DNA allowed to confirm the presence of the mutation on both alleles in the JCaM1 cell line.


Subject(s)
Introns/genetics , Jurkat Cells/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Neoplasm Proteins/genetics , Point Mutation , RNA Splicing , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Animals , Blotting, Southern , COS Cells , Chlorocebus aethiops , DNA, Neoplasm/genetics , Exons/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/physiology , Neoplasm Proteins/physiology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Transfection
13.
J Biol Chem ; 274(12): 7941-5, 1999 Mar 19.
Article in English | MEDLINE | ID: mdl-10075690

ABSTRACT

Activation of NF-kappaB transcription factors requires phosphorylation and ubiquitin-proteasome-dependent degradation of IkappaB proteins. We provide evidence that a human F-box protein, h-betaTrCP, a component of Skp1-Cullin-F-box protein (SCF) complexes, a new class of E3 ubiquitin ligases, is essential for inducible degradation of IkappaBalpha. betaTrCP associates with Ser32-Ser36 phosphorylated, but not with unmodified IkappaBalpha or Ser32-Ser36 phosphorylation-deficient mutants. Expression of a F-box-deleted betaTrCP inhibits IkappaBalpha degradation, promotes accumulation of phosphorylated Ser32-Ser36 IkappaBalpha, and prevents NF-kappaB-dependent transcription. Our findings indicate that betaTrCP is the adaptor protein required for IkappaBalpha recognition by the SCFbetaTrCP E3 complex that ubiquitinates IkappaBalpha and makes it a substrate for the proteasome.


Subject(s)
Cell Cycle Proteins/metabolism , Cysteine Endopeptidases/metabolism , DNA-Binding Proteins/metabolism , GTP-Binding Proteins/metabolism , I-kappa B Proteins , Multienzyme Complexes/metabolism , Peptide Synthases/metabolism , HeLa Cells , Humans , Models, Chemical , NF-KappaB Inhibitor alpha , NF-kappa B/biosynthesis , NF-kappa B/genetics , Phosphorylation , Proteasome Endopeptidase Complex , S-Phase Kinase-Associated Proteins , SKP Cullin F-Box Protein Ligases , Serine/metabolism , Transcription, Genetic , beta-Transducin Repeat-Containing Proteins
14.
J Virol ; 73(4): 3236-45, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10074177

ABSTRACT

Cell cycle G2 arrest, nuclear localization, and cell death induced by human immunodeficiency virus type 1 Vpr were examined in fission yeast by using a panel of Vpr mutations that have been studied previously in human cells. The effects of the mutations on Vpr functions were highly similar between fission yeast and human cells. Consistent with mammalian cell studies, induction of cell cycle G2 arrest by Vpr was found to be independent of nuclear localization. In addition, G2 arrest was also shown to be independent of cell killing, which only occurred when the mutant Vpr localized to the nucleus. The C-terminal end of Vpr is crucial for G2 arrest, the N-terminal alpha-helix is important for nuclear localization, and a large part of the Vpr protein is responsible for cell killing. It is evident that the overall structure of Vpr is essential for these cellular effects, as N- and C-terminal deletions affected all three cellular functions. Furthermore, two single point mutations (H33R and H71R), both of which reside at the end of each alpha-helix, disrupted all three Vpr functions, indicating that these two mutations may have strong effects on the overall Vpr structure. The similarity of the mutant effects on Vpr function in fission yeast and human cells suggests that fission yeast can be used as a model system to evaluate these Vpr functions in naturally occurring viral isolates.


Subject(s)
Gene Expression Regulation, Fungal , Gene Expression Regulation, Viral , Gene Products, vpr/genetics , HIV-1/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/virology , Cell Death/genetics , Cell Nucleus/genetics , DNA Mutational Analysis , G2 Phase/genetics , Genes, vpr , Humans , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/ultrastructure , vpr Gene Products, Human Immunodeficiency Virus
15.
Curr Biol ; 9(4): 207-10, 1999 Feb 25.
Article in English | MEDLINE | ID: mdl-10074433

ABSTRACT

Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.


Subject(s)
Cytoskeletal Proteins/metabolism , GTP-Binding Proteins/metabolism , Trans-Activators , Animals , Cadherins/metabolism , Carrier Proteins/metabolism , Cell Line , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Drosophila , GTP-Binding Proteins/chemistry , Genes, Reporter , HeLa Cells , Humans , Phosphorylation , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Repetitive Sequences, Amino Acid , Transfection , Ubiquitin-Protein Ligases , beta Catenin , beta-Transducin Repeat-Containing Proteins
16.
J Biol Chem ; 274(3): 1635-45, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9880543

ABSTRACT

heed, the human homolog of mouse eed and Drosophila esc, two members of the trithorax (trx) and Polycomb group (Pc-G) of genes, was isolated by screening an activated lymphocyte cDNA library versus the immunodeficiency virus type 1 (HIV-1) MA protein used as a bait in a two-hybrid system in yeast. The human EED protein (HEED) had 99. 5% identity with the mouse EED protein and contained seven WD repeats. Two heed gene transcripts were identified, with a putative 407-nucleotide-long intron, giving rise to two HEED protein isoforms of 535 and 494 residues in length, respectively. The shorter HEED isoform, originated from the unspliced message, lacked the seventh WD repeat. HEED was found to bind to MA protein in vitro, as efficiently as in vivo in yeast cells. Site-directed mutagenesis and phage biopanning suggested that the interaction between HEED and MA involved the N-terminal region of the MA protein, including the first polybasic signal, in a MA conformation-dependent manner. In the HEED protein, however, two discrete linear MA-binding motifs were identified within residues 388-403, overlapping the origin of the fifth WD repeat. Deletion of the C-terminal 41 residues of HEED, spanning the seventh WD repeat, as in the 494-residue HEED protein, was detrimental to HEED-MA interaction in vivo, suggesting the existence of another C-terminal binding site and/or a conformational role of the HEED C-terminal domain in the MA-HEED interaction. MA and HEED proteins co-localized within the nucleus of co-transfected human cells and of recombinant baculovirus co-infected insect cells. This and the failure of HEED to bind to uncleaved GAG precursor suggested a role of HEED at the early stages of virus infection, rather than late in the virus life cycle.


Subject(s)
Gene Products, gag/metabolism , HIV Antigens/metabolism , HIV-1 , Repressor Proteins/metabolism , Viral Proteins , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Gene Library , Gene Products, gag/genetics , HIV Antigens/genetics , HIV-1/genetics , HIV-1/pathogenicity , Humans , Mice , Microscopy, Electron , Microscopy, Immunoelectron , Molecular Sequence Data , Peptide Mapping , Polycomb Repressive Complex 2 , Transcription, Genetic , gag Gene Products, Human Immunodeficiency Virus
17.
J Virol ; 73(2): 1350-61, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9882340

ABSTRACT

The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXO, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXO motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXO motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXO motifs interact with the micro1 and micro2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the beta2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXO-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.


Subject(s)
Clathrin/metabolism , Gene Products, env/metabolism , HIV Envelope Protein gp41/metabolism , HIV-1/metabolism , Human T-lymphotropic virus 1/metabolism , Membrane Proteins/metabolism , Retroviridae Proteins, Oncogenic/metabolism , Simian Immunodeficiency Virus/metabolism , Viral Fusion Proteins/metabolism , Adaptor Protein Complex alpha Subunits , Adaptor Protein Complex beta Subunits , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , CD8 Antigens/metabolism , Cell Membrane/metabolism , Cytoplasm/metabolism , Gene Products, env/genetics , HIV Envelope Protein gp41/genetics , HIV-1/genetics , Haplorhini , HeLa Cells , Human T-lymphotropic virus 1/genetics , Humans , Intracellular Fluid , Molecular Sequence Data , Retroviridae Proteins, Oncogenic/genetics , Simian Immunodeficiency Virus/genetics , Subcellular Fractions , Tyrosine , Viral Fusion Proteins/genetics , env Gene Products, Human Immunodeficiency Virus
18.
J Virol ; 73(1): 592-600, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9847364

ABSTRACT

Vpr and Vpx proteins from human and simian immunodeficiency viruses (HIV and SIV) are incorporated into virions in quantities equivalent to those of the viral Gag proteins. We demonstrate here that Vpr and Vpx proteins from distinct lineages of primate lentiviruses were able to bind to their respective Gag precursors. The capacity of HIV type 1 (HIV-1) Vpr mutants to bind to Pr55(Gag) was correlated with their incorporation into virions. Molecular analysis of these interactions revealed that they required the C-terminal p6 domain of the Gag precursors. While the signal for HIV-1 Vpr binding lies in the leucine triplet repeat region of the p6 domain reported to be essential for incorporation, SIVsm Gag lacking the equivalent region still bound to SIVsm Vpr and Vpx, indicating that the determinants for Gag binding are located upstream of this region of the p6 domain. Binding to Gag cleavage products showed that HIV-1 Vpr interacted directly with the nucleocapsid protein (NC), whereas SIVsm Vpr and Vpx did not interact with NC but with the p6 protein. These results (i) reveal differences between HIV-1 and SIVsm for the p6 determinants required for Vpr and Vpx binding to Gag and (ii) suggest that HIV-1 Vpr and SIVsm Vpr and Vpx interact with distinct cleavage products of the precursor following proteolytic processing in the virions.


Subject(s)
Gene Products, gag/metabolism , Gene Products, vpr/metabolism , HIV/metabolism , Protein Precursors/metabolism , Simian Immunodeficiency Virus/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Virion/metabolism , Amino Acid Sequence , Humans , Molecular Sequence Data , Repetitive Sequences, Amino Acid , vpr Gene Products, Human Immunodeficiency Virus
19.
Mol Cell ; 1(4): 565-74, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9660940

ABSTRACT

HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Human beta TrCP identified by interaction with Vpu connects CD4 to this proteolytic machinery, and CD4-Vpu-beta TrCP ternary complexes have been detected by coimmunoprecipitation. beta TrCP binding to Vpu and its recruitment to membranes require two phosphoserine residues in Vpu essential for CD4 degradation. In beta TrCP, WD repeats at the C terminus mediate binding to Vpu, and an F box near the N terminus is involved in interaction with Skp1p, a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of beta TrCP had a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that beta TrCP and Skp1p represent components of a novel ER-associated protein degradation pathway that mediates CD4 proteolysis.


Subject(s)
CD4 Antigens/metabolism , Endoplasmic Reticulum/metabolism , GTP-Binding Proteins/genetics , HIV-1 , Viral Regulatory and Accessory Proteins/metabolism , Binding Sites/immunology , Cell Cycle Proteins/metabolism , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/virology , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Human Immunodeficiency Virus Proteins , Humans , Jurkat Cells , Molecular Sequence Data , Mutagenesis/physiology , Repetitive Sequences, Nucleic Acid , S-Phase Kinase-Associated Proteins , Sequence Homology, Amino Acid , Serine/metabolism , Ubiquitins/metabolism , beta-Transducin Repeat-Containing Proteins
20.
Immunity ; 8(4): 483-95, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9586638

ABSTRACT

The surface expression of MHC I is reduced in HIV-infected cells. We show that the Nef protein affects the intracellular sorting of HLA-A and -B molecules. In the presence of Nef, these proteins accumulate in the Golgi and colocalize with clathrin-coated vesicles. MHC I modulation relies on a tyrosine-based sorting signal located in the cytoplasmic domain of HLA-A and -B heavy chains. This cryptic sorting signal becomes operative only in the presence of Nef. Nef interacts with the medium (mu) subunit of AP adaptor complexes involved in the recognition of tyrosine-based sorting signals, likely facilitating the connection between MHC I and the clathrin-dependent sorting machinery.


Subject(s)
Clathrin/metabolism , Gene Products, nef/metabolism , HLA-A Antigens/metabolism , HLA-B Antigens/metabolism , Adaptor Protein Complex alpha Subunits , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Binding Sites , CD4 Antigens/metabolism , Clathrin/chemistry , Down-Regulation , HIV Infections/metabolism , HIV-1/metabolism , HIV-2/metabolism , HLA-A Antigens/chemistry , HLA-A Antigens/genetics , HLA-B Antigens/chemistry , HLA-B Antigens/genetics , HLA-C Antigens/chemistry , HLA-C Antigens/genetics , HLA-C Antigens/metabolism , HeLa Cells , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Models, Biological , Molecular Sequence Data , Protein Conformation , Signal Transduction , Simian Immunodeficiency Virus/metabolism , nef Gene Products, Human Immunodeficiency Virus
SELECTION OF CITATIONS
SEARCH DETAIL
...