26 research outputs found

    Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 Îłâ€“Ïƒ1 and AP-3 Ύ–σ3 hemicomplexes

    Get PDF
    The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the Îł and σ1 subunits of AP-1 and the ÎŽ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the Îł/ÎŽ and σ subunits of AP-1 and AP-3

    Rab7A Is Required for Efficient Production of Infectious HIV-1

    Get PDF
    Retroviruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Rab proteins regulate specific steps in intracellular membrane trafficking by recruiting tethering, docking and fusion factors, as well as the actin- and microtubule-based motor proteins that facilitate vesicle traffic. Using virological tests and RNA interference targeting Rab proteins, we demonstrate that the late endosome-associated Rab7A is required for HIV-1 propagation. Analysis of the late steps of the HIV infection cycle shows that Rab7A regulates Env processing, the incorporation of mature Env glycoproteins into viral particles and HIV-1 infectivity. We also show that siRNA-mediated Rab7A depletion induces a BST2/Tetherin phenotype on HIV-1 release. BST2/Tetherin is a restriction factor that impedes HIV-1 release by tethering mature virus particles to the plasma membrane. Our results suggest that Rab7A contributes to the mechanism by which Vpu counteracts the restriction factor BST2/Tetherin and rescues HIV-1 release. Altogether, our results highlight new roles for a major regulator of the late endocytic pathway, Rab7A, in the late stages of the HIV-1 replication cycle

    Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

    Get PDF
    BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≄18 years) with S aureus bacteraemia who had received ≀96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

    The ESCRT-0 Component HRS is Required for HIV-1 Vpu-Mediated BST-2/Tetherin Down-Regulation

    Get PDF
    The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, a highly conserved set of four hetero-oligomeric protein complexes, is required for multivesicular body formation, sorting ubiquitinylated membrane proteins for lysosomal degradation, cytokinesis and the final stages of assembly of a number of enveloped viruses, including the human immunodeficiency viruses. Here, we show an additional role for the ESCRT machinery in HIV-1 release. BST-2/tetherin is a restriction factor that impedes HIV release by tethering mature virus particles to the plasma membrane. We found that HRS, a key component of the ESCRT-0 complex, promotes efficient release of HIV-1 and that siRNA-mediated HRS depletion induces a BST-2/tetherin phenotype. This activity is related to the ability of the HIV-1 Vpu protein to down-regulate BST-2/tetherin. We found that BST-2/tetherin undergoes constitutive ESCRT-dependent sorting for lysosomal degradation and that this degradation is enhanced by Vpu expression. We demonstrate that Vpu-mediated BST-2/tetherin down-modulation and degradation require HRS (ESCRT-0) function and that knock down of HRS increases cellular levels of BST-2/tetherin and restricts virus release. Furthermore, HRS co-precipitates with Vpu and BST-2. Our results provide further insight into the mechanism by which Vpu counteracts BST-2/tetherin and promotes HIV-1 dissemination, and they highlight an additional role for the ESCRT machinery in virus release

    Perturbations du transport vésiculaire des protéines au sein de la voie d'endocytose par la protéine Nef du VIH-1

    No full text
    La protéine Nef du VIH joue un rÎle primordial in vivo dans la réplication virale et l'induction SIDA. In vitro, Nef module l'expression à la surface cellulaire de nombreuses protéines membranaires telles que CD4, les molécules du CMH-I et du CMH-II, de CD28, de DC-SIGN et des cytokines membranaires TNF et LIGHT. Ces perturbations du trafic des protéines provoquées par Nef au cours de l'infection, résultent de sa capacité à interagir avec les constituants de la machinerie impliquée dans le transport vésiculaire des protéines au sein de la voie d'endocytose. Nef est en effet capable d'interagir avec les complexes AP-1, AP-2 et AP-3, ainsi qu'avec les complexes COP-I. Nous avons entrepris de caractériser les mécanismes moléculaires des interactions de Nef avec les complexes AP-1, AP-3 et COP-I et leurs contributions dans les perturbations du trafic intracellulaire induites par Nef. L'interaction de Nef avec les complexes AP-1 et AP-3 se fait essentiellement grùce à un motif de type di-leucine(LL165) localisé dans la boucle carboxy-terminale de la protéine. Un résidu acide glutamique (E160) situé en position -4 du motif di-leucine participe spécifiquement à l'interaction de Nef avec les complexes AP-3. L'association de Nef aux complexes AP-1 fait également intervenir deux sous-domaines localisés de part et d'autre de cette boucle. De plus, contrairement aux données de la littérature, le motif di-acide (EE155) de Nef ne semble pas requis pour son association avec les complexes COP-I. Nef induit une stabilisation de l'association des complexes AP-1 et AP-3 aux membranes endosomales en favorisant leur recrutement suivant un mécanisme indépendant de la petite protéine G ARF1, normalement impliquée dans le recrutement membranaire de ces complexes. De plus, Nef induit une expansion des endosomes précoces au sein duquel sont séquestrés CD4 et CMH-I, mais également d'autres marqueurs tels que le récepteur à la transferrine (Tf-R). Sur le plan fonctionnel, ces altérations se traduisent par des perturbations du transport de CD4, du Tf-R et du récepteur au mannose-6-phosphate. L'altération du trafic du Tf-R par Nef résulte d'une diminution du recyclage du récepteur des endosomes précoces vers la membrane plasmique. L'ensemble de ces résultats indique que la stabilisation par Nef des complexes AP-1 et AP-3 au niveau des membranes endosomales, provoque une altération générale des voies de transport vers la membrane plasmique, se traduisant par une rétention des protéines au niveau des endosomes précoces de tri.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

    Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

    No full text

    Mechanisms underlying HIV-1 Vpu-mediated viral egress.

    Get PDF
    International audienceViruses such as lentiviruses that are responsible for long lasting infections have to evade several levels of cellular immune mechanisms to persist and efficiently disseminate in the host. Over the past decades, much evidence has emerged regarding the major role of accessory proteins of primate lentiviruses, human immunodeficiency virus and simian immunodeficiency virus, in viral evasion from the host immune defense. This short review will provide an overview of the mechanism whereby the accessory protein Vpu contributes to this escape. Vpu is a multifunctional protein that was shown to contribute to viral egress by down-regulating several mediators of the immune system such as CD4, CD1d, NTB-A and the restriction factor BST2. The mechanisms underlying its activity are not fully characterized but rely on its ability to interfere with the host machinery regulating protein turnover and vesicular trafficking. This review will focus on our current understanding of the mechanisms whereby Vpu down-regulates CD4 and BST2 expression levels to favor viral egress
    corecore