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1.
Med Image Anal ; 73: 102168, 2021 10.
Article in English | MEDLINE | ID: mdl-34340105

ABSTRACT

Automatic tracking of viral structures displayed as small spots in fluorescence microscopy images is an important task to determine quantitative information about cellular processes. We introduce a novel probabilistic approach for tracking multiple particles based on multi-sensor data fusion and Bayesian smoothing methods. The approach exploits multiple measurements as in a particle filter, both detection-based measurements and prediction-based measurements from a Kalman filter using probabilistic data association with elliptical sampling. Compared to previous probabilistic tracking methods, our approach exploits separate uncertainties for the detection-based and prediction-based measurements, and integrates them by a sequential multi-sensor data fusion method. In addition, information from both past and future time points is taken into account by a Bayesian smoothing method in conjunction with the covariance intersection algorithm for data fusion. Also, motion information based on displacements is used to improve correspondence finding. Our approach has been evaluated on data of the Particle Tracking Challenge and yielded state-of-the-art results or outperformed previous approaches. We also applied our approach to challenging time-lapse fluorescence microscopy data of human immunodeficiency virus type 1 and hepatitis C virus proteins acquired with different types of microscopes and spatial-temporal resolutions. It turned out, that our approach outperforms existing methods.


Subject(s)
Algorithms , Viral Structures , Bayes Theorem , Humans , Microscopy, Fluorescence
2.
Sci Immunol ; 4(31)2019 01 04.
Article in English | MEDLINE | ID: mdl-30610013

ABSTRACT

T cell antigen receptor (TCR) signaling triggers selective cytokine expression to drive T cell proliferation and differentiation required for immune defense and surveillance. The nuclear signaling events responsible for specificity in cytokine gene expression upon T cell activation are largely unknown. Here, we uncover formation of a dynamic actin filament network in the nucleus that regulates cytokine expression for effector functions of CD4+ T lymphocytes. TCR engagement triggers the rapid and transient formation of a nuclear actin filament network via nuclear Arp2/3 complex, induced by elevated nuclear Ca2+ levels and regulated via N-Wasp and NIK. Specific interference with TCR-induced formation of nuclear actin filaments impairs production of effector cytokines and prevents generation of antigen-specific antibodies but does not interfere with immune synapse formation and cell proliferation. Ca2+-regulated actin polymerization in the nucleus allows CD4+ T cells the rapid conversion of TCR signals into effector functions required for T cell help.


Subject(s)
Actins/immunology , Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Receptors, Antigen, T-Cell/immunology , Actin-Related Protein 2-3 Complex/immunology , Adoptive Transfer , Animals , Cell Line , Humans , Mice
3.
Curr Biol ; 11(11): 875-9, 2001 Jun 05.
Article in English | MEDLINE | ID: mdl-11516650

ABSTRACT

Lipid rafts, also known as detergent-resistant membranes (DRM), are microdomains in the plasma membrane enriched in sphingolipids and cholesterol (reviewed in [1, 2]). Human immunodeficiency virus 1 (HIV) buds via lipid rafts [3, 4]. However, the targeting of viral structural components to DRM and its consequences for viral replication are not understood. Moreover, the negative factor Nef from HIV increases viral infectivity (reviewed in [5, 6]). With no apparent differences in structural components and morphology between wild-type and DeltaNef virons, the latter viruses display less efficient reverse transcription in target cells. As Nef is expressed abundantly early in the viral replicative cycle [7], we hypothesized that Nef could affect viral morphogenesis and budding to render viruses more infectious. In this report, we demonstrated first that Nef increases viral budding from lipid rafts. Second, in the presence of Nef, viral envelopes contain more ganglioside (GM1), which is a major component of lipid rafts. This finding correlated directly with the increased infectivity of HIV. Finally, the depletion of exogenous and endogenous cholesterol biochemically and genetically, which disrupted lipid rafts, decreased viral infectivity only in the presence of Nef. Importantly, HIV lacking the nef gene remained unaffected by these manipulations. We conclude that lipids in virions are essential for viral infectivity. Thus, HIV becomes more infectious when it buds from lipid rafts, and Nef plays a major role in this process.


Subject(s)
Gene Products, nef/biosynthesis , HIV-1/growth & development , HIV-1/pathogenicity , Membrane Microdomains/virology , Cholesterol/analysis , Membrane Microdomains/chemistry , Virus Replication , nef Gene Products, Human Immunodeficiency Virus
4.
Curr Biol ; 11(16): 1294-9, 2001 Aug 21.
Article in English | MEDLINE | ID: mdl-11525746

ABSTRACT

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.


Subject(s)
Gene Products, nef/genetics , HIV-1/physiology , T-Lymphocytes/physiology , Amino Acid Motifs , CD4 Antigens/metabolism , CD8 Antigens/genetics , CD8 Antigens/metabolism , Cell Line , Dendritic Cells/physiology , Gene Products, nef/chemistry , Gene Products, nef/metabolism , HIV-1/pathogenicity , Humans , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Models, Molecular , Precipitin Tests , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , T-Lymphocytes/virology , nef Gene Products, Human Immunodeficiency Virus
5.
EMBO Rep ; 2(7): 580-5, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11463741

ABSTRACT

The accessory Nef protein of HIV and SIV is essential for viral pathogenesis, yet it is perplexing in its multitude of molecular functions. In this review we analyse the structure-function relationships of motifs recently proposed to play roles in aspects of Nef modification, signalling and trafficking, and thereby to impinge on the ability of the virus to survive in, and to manipulate, its cellular host. Based on the full-length structure assembly of HIV Nef, we correlate surface accessibility with secondary structure elements and sequence conservation. Motifs involved in Nef-mediated CD4 and MHC I downregulation are located in flexible regions of Nef, suggesting that the formation of the transient trafficking complexes involved in these processes depends on the recognition of primary sequences. In contrast, the interaction sites for signalling molecules that contain SH3 domains or the p21-activated kinases are associated with the well folded core domain, suggesting the recognition of highly structured protein surfaces.


Subject(s)
Gene Products, nef/chemistry , Gene Products, nef/metabolism , HIV-1/chemistry , Protein Structure, Tertiary , Signal Transduction , Amino Acid Motifs , Binding Sites , HIV-1/metabolism , Humans , Models, Molecular , Receptors, HIV/chemistry , Receptors, HIV/metabolism , Structure-Activity Relationship , nef Gene Products, Human Immunodeficiency Virus
6.
J Virol ; 75(14): 6601-8, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11413327

ABSTRACT

Human immunodeficiency virus type 1(F12) (HIV-1(F12)) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55(Gag) precursor in the presence of Nef from HIV-1(F12). Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1(NL4-3) also created a dominant-negative Nef protein. Effects of Nef from HIV-1(F12) on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.


Subject(s)
Gene Products, nef/physiology , HIV Infections/virology , HIV-1/physiology , CD8 Antigens/metabolism , Cell Line , Down-Regulation , Gene Products, gag/metabolism , Gene Products, nef/genetics , HIV-1/pathogenicity , Humans , Mutation , Protein Binding , Protein Precursors/metabolism , Proteins/metabolism , Transfection , Virulence , Virus Replication , nef Gene Products, Human Immunodeficiency Virus
7.
Mol Biol Cell ; 12(2): 463-73, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11179428

ABSTRACT

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIV(mac239) for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.


Subject(s)
CD4 Antigens/metabolism , Proton-Translocating ATPases/metabolism , Simian Immunodeficiency Virus/metabolism , Simian Immunodeficiency Virus/pathogenicity , Vacuolar Proton-Translocating ATPases , Viral Regulatory and Accessory Proteins/metabolism , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Catalytic Domain , Cells, Cultured/virology , Down-Regulation , Humans , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/metabolism , Phosphoproteins/metabolism , Viral Regulatory and Accessory Proteins/genetics
8.
Curr Mol Med ; 1(1): 1-7, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11899237

ABSTRACT

Molecular biological investigations of HIV have made fundamental contributions to our understanding of eukaryotic biology. These studies elucidated new paradigms in transcription, RNA and protein export from the nucleus to the cytoplasm, cellular activation, morphology and vesicular trafficking.


Subject(s)
Active Transport, Cell Nucleus/physiology , HIV/metabolism , Retroviridae Proteins/metabolism , Endocytosis/physiology , Gene Products, rev/metabolism , Gene Products, tat/metabolism , HIV/genetics , Humans , Models, Biological , Retroviridae Proteins/genetics , Transcription, Genetic/physiology , rev Gene Products, Human Immunodeficiency Virus , tat Gene Products, Human Immunodeficiency Virus
9.
Curr Biol ; 10(16): 1005-8, 2000 Aug 24.
Article in English | MEDLINE | ID: mdl-10985390

ABSTRACT

Enveloped viruses enter target cells by membrane fusion or endocytosis. In the latter case, fusion of the viral envelope is induced by the acidic pH of the endocytic vesicle [1]. As with most other retroviruses, entry of the human immunodeficiency virus (HIV) is thought to be exclusively by pH-independent membrane fusion after interaction of its envelope with CD4 and a chemokine co-receptor on the target cell [2,3]. Expression of CD4 on the virus-producing cell impairs the release and infectivity of HIV-1(NL4-3) particles [4-6]. In sharp contrast, we found that the infectivity of another HIV isolate, HIV-1SF2, was enhanced by expression of CD4 on the producer cells, which correlated with significantly increased amounts of viral proteins in the vesicular fraction of target cells. Endocytic inhibitors decreased infectivity of HIV-1SF2 but enhanced that of HIV-1 NL4-3. Expression of CD4 in the producer cell did not remove gp41 from HIV-1SF2 virions. With these cells, the formation of syncytia could be induced by acidic medium. Thus, HIV-1SF2 can enter the cytoplasm by an endocytic route after activation of gp41 by the acidic pH of endocytic vesicles. Endocytic entry might expand the range of cells that HIV could infect and should be considered in antiviral strategies against AIDS.


Subject(s)
Endocytosis , Endosomes/virology , HIV-1/physiology , CD4 Antigens/metabolism , Cell Line , Giant Cells , HIV Envelope Protein gp41/metabolism , HeLa Cells , Humans , Hydrogen-Ion Concentration , Jurkat Cells , Membrane Fusion , Virus Replication
10.
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
11.
Mol Cell Biol ; 20(7): 2619-27, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10713183

ABSTRACT

The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef.


Subject(s)
Gene Products, nef/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , COS Cells , Cell Line , Cytoskeleton/metabolism , Fluorescent Antibody Technique , Green Fluorescent Proteins , HIV-1/metabolism , Humans , JNK Mitogen-Activated Protein Kinases , Luminescent Proteins , Mitogen-Activated Protein Kinases/metabolism , Peptide Fragments/pharmacology , Protein Binding , Signal Transduction , Transfection , Viral Proteins/biosynthesis , nef Gene Products, Human Immunodeficiency Virus , p21-Activated Kinases
12.
Mol Cell ; 3(6): 729-39, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10394361

ABSTRACT

Nef of primate lentiviruses is critical for high levels of viremia and the progression to AIDS. Nef associates with and activates a serine/threonine kinase (Nef-associated kinase [NAK]) via the small GTPases Rac1 and Cdc42. We identified the protooncogene and guanine nucleotide exchange factor Vav as the specific binding partner of Nef proteins from HIV-1 and SIV. The interaction between Nef and Vav led to increased activity of Vav and its downstream effectors. Both cytoskeletal changes and the activation of c-Jun N-terminal kinase (JNK) were observed. Furthermore, a dominant-negative Vav protein inhibited NAK activation and viral replication. Thus, the interaction between Nef and Vav initiates a signaling cascade that changes structural and physiological parameters in the infected cell.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cytoskeleton/metabolism , Gene Products, nef/metabolism , HIV-1 , Mitogen-Activated Protein Kinases , Proto-Oncogene Proteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Cycle Proteins/metabolism , Cell Line , Cell Size , Enzyme Activation , GTP-Binding Proteins/metabolism , Gene Products, nef/chemistry , HIV-1/growth & development , HIV-1/metabolism , Humans , JNK Mitogen-Activated Protein Kinases , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-vav , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Signal Transduction , Virus Replication , cdc42 GTP-Binding Protein , nef Gene Products, Human Immunodeficiency Virus , p21-Activated Kinases , src Homology Domains
13.
AIDS ; 12(2): 139-46, 1998 Jan 22.
Article in English | MEDLINE | ID: mdl-9468362

ABSTRACT

OBJECTIVE: To investigate differences in viral and proviral load between the peripheral blood and the intestinal mucosal immune system in HIV-infected patients. DESIGN: HIV-1 p24 and HIV DNA content were compared in blood samples and intestinal biopsies from HIV-infected patients. METHODS: Intestinal biopsies and peripheral blood were simultaneously obtained from 27 HIV-infected patients undergoing diagnostic endoscopy. The p24 concentrations were measured in serum and homogenized intestinal biopsies by enzyme-linked immunosorbent assay after acid-dissociation of immune complexes. Proviral load was determined in blood and intestinal biopsies by a quantitative competitive polymerase chain reaction amplifying the HIV-1 nef gene from genomic DNA. RESULTS: No significant differences were found in proviral load comparing HIV copies per 1.5 x 10(5) cell equivalents in blood [2650 (600-44000)] and intestinal biopsies [4200 (1325-19 625)]. Paired analysis revealed a strong positive correlation between serum and mucosal proviral load. In contrast, HIV core protein p24 was detected in intestinal biopsies from 18 patients in much higher concentrations than in serum [858 (262-4111) pg/g versus 34 (9-242) pg/g; P < 0.005]. The p24 concentrations in serum and intestinal biopsies did not correlate and no significant correlation was observed in serum or intestinal biopsies between proviral load and p24 concentrations. No clear correlations were observed between clinical parameters and HIV DNA or HIV p24 levels in blood or biopsies. CONCLUSIONS: Our findings demonstrate a homogenous distribution of HIV proviral load in the peripheral blood and the intestinal mucosal immune system. The high viral antigen load in the intestine therefore indicates that mucosal HIV production is upregulated at the transcriptional and/or translational level. The intestinal mucosa is a major reservoir for HIV in HIV-infected patients.


Subject(s)
HIV Core Protein p24/blood , HIV Infections/virology , HIV-1/physiology , Intestinal Mucosa/immunology , Intestinal Mucosa/virology , Proviruses/physiology , Adult , Biopsy , Blotting, Southern , DNA, Viral/blood , Endoscopy, Gastrointestinal , Female , Genes, nef , HIV Core Protein p24/analysis , HIV Infections/immunology , HIV-1/genetics , HIV-1/immunology , Humans , Male , Middle Aged , Viral Load
15.
Eur J Biochem ; 247(3): 843-51, 1997 Aug 01.
Article in English | MEDLINE | ID: mdl-9288906

ABSTRACT

Human immunodeficiency virus (HIV) Nef functions are thought to be mediated via interactions with cellular proteins. Utilizing zone velocity sedimentation in glycerol gradients we found that recombinant HIV-1 Nef non-covalently associates with actin forming a high-molecular-mass complex of 150-300 kDa. This Nef/actin complex was present in human B and T lymphocytes but not in insect cells and was dependent on the N-terminal myristoylation of Nef, whereas the SH3-binding proline motif of Nef was not involved. Despite being myristoylated, HIV-2 Nef did not associate with actin. This might reflect differences in the subcellular localization of Nef since cell-fractionation experiments revealed that HIV-1 Nef was virtually exclusively localized in the cytoskeletal (detergent-insoluble) fraction whereas HIV-2 Nef had significantly reduced affinity for the cytoskeleton. Colocalization experiments in HIV-1-infected CD4+ fibroblasts revealed that Nef/actin complexes may also exist in HIV-infected cells. This novel interaction of HIV-1 Nef with actin provides insight into the association of Nef with cellular structures and reveals general differences in the interactions of the Nef proteins from HIV-1 and HIV-2.


Subject(s)
Actins/metabolism , Gene Products, nef/metabolism , HIV-1/metabolism , HIV-2/metabolism , Subcellular Fractions/metabolism , Animals , Cell Line , Gene Products, nef/chemistry , Molecular Weight , Myristic Acid , Myristic Acids/metabolism , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spodoptera , T-Lymphocytes/metabolism , nef Gene Products, Human Immunodeficiency Virus
16.
Virus Res ; 46(1-2): 105-10, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9029783

ABSTRACT

HIV-Nef protein supports viral infectivity prior to proviral integration. This requires Nef to be present before the expression of viral genes and suggests its delivery as part of the virion. We report here that the Nef proteins of HIV-2-HOM and HIV-2-ROD are associated with the virion. After the separation of pelleted virus in a 20-60% sucrose density gradient, both proteins cosedimented with the virion-associated reverse transcriptase (RT) activity at a density characteristic of retroviral particles. Whereas Nef-2-ROD was present in the virion only as the full-length protein, HIV-2-HOM appeared as 32 and 35 kDa isoforms. The smaller isoform is identical in molecular weight to the protein expected from proteolytic cleavage of full-length Nef-2-HOM by the virion-based protease. Virion-association of Nef helps to explain the recently redefined biological function of this regulatory protein.


Subject(s)
Gene Products, nef/physiology , HIV-2/physiology , Virion/physiology , Gene Products, nef/chemistry , Gene Products, nef/genetics , HIV-2/chemistry , HIV-2/genetics , Humans , Immunoblotting , Jurkat Cells , Recombinant Proteins/chemistry , Virion/chemistry , Virion/genetics , nef Gene Products, Human Immunodeficiency Virus
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