Mécanismes d'interaction de la protéine Tat du VIH-1 avec les membranes plasmique et endosomale

La protéine transactivatrice Tat joue un rôle crucial dans la multiplication du VIH-1. Elle a un rôle établi dans la transcription virale. Elle possède de plus la propriété d'être sécrétée par les cellules infectées. Elle peut ensuite pénétrer par endocytose dans les cellules non-infectées, où elle va provoquer divers dysfonctionnements. Dans ce travail, nous avons mis en évidence que Tat est sécrétée directement à travers la membrane plasmique en utilisant un mécanisme de sécrétion non conventionnel. Nous avons montré que le phosphatidylinositol-4,5-bisphosphate, PI(4,5)P2, est responsable du recrutement de Tat sur le feuillet interne de la membrane plasmique. En effet, Tat se fixe au PI(4,5)P2 avec une grande spécificité et une forte affinité. Cette interaction de stochiométrie 1:1 implique un triplet de résidus basiques de Tat (résidus 49-51). Elle est stabilisée par l'insertion membranaire de la chaîne latérale du Trp11. Ce mécanisme de fixation original restreint la fixation de Tat au PI(4,5)P2 membranaire mais permet aussi une très haute affinité. Celle-ci va permettre à Tat d'être sécrétée mais aussi de déplacer des protéines cellulaires du PI(4,5)P2. D'autre part, nous avons élucidé le mécanisme moléculaire permettant l'insertion de Tat dans la membrane endosomale. Elle est induite par le pH acide (pH~5.3) de ces compartiments. Ce pH acide est détecté par un système "sensor" impliquant Tat-Glu2, résidu acide qui est relié par un pont salin avec un triplet d'Arg (55-57). La protonation de Glu2 rompt les ponts salins, permettant l'exposition du Trp11 et l'insertion membranaire de Tat, préalable à sa translocation vers le cytosolThe HIV-1 transactivating factor Tat, plays a critical role in HIV-1 pathogenesis. It is strictly required for viral transcription. It can also be secreted by infected cells. Once in the bloodstream, it can enter the cytosol of uninfected cells by endocytosis and dramatically affect their biological activity. Here, we provide evidence that Tat is directly secreted through the plasma membrane using an unconventional secretion pathway. We found that phosphatidylinositol-4,5-bisphosphate, PI(4,5)P2, is responsible for Tat recruitment to the inner leaflet of the plasma membrane, thereby allowing its membrane insertion and translocation. We observed, using a variety of biophysical techniques, that Tat binds specifically and with a very high affinity to a single molecule of PI(4,5)P2. This interaction is mediated by a triplet of basic residues (residues 49-51), and is stabilized by the concomitant membrane insertion of the side chain of Tat-Trp11. This original mechanism restricts Tat binding to membrane embedded PI(4,5)P2, but also enables very tight binding. This high affinity allows Tat to be secreted and to displace cell proteins?n?nfrom the plasma membrane. We also elucidate the molecular mechanism responsible for Tat insertion into the endosome membrane. This insertion is triggered by the low pH (pH ~5.3) present within the endosome lumen. Acidic pH is detected by a sensor made of Glu2 and an Arg triplet (55-57) that are connected by a salt bridge. Glu2 protonation at low pH will destabilize the salt bridge, thereby allowing Trp11 exposure and enabling membrane penetration. This is the first step of the membrane translocation process that will allow Tat delivery to cytosolMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

The Ins and Outs of HIV-1 Tat

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Bovine herpesvirus 1 (BHV-1) envelope protein gE subcellular trafficking is contributed by two separate YXXL/Φ motifs within the cytoplasmic tail which together promote efficient virus cell-to-cell spread

Bovine herpesvirus envelope glycoprotein E (gE) and, in particular, the gE cytoplasmic tail (CT) is a virulence determinant in cattle. Also, the gE CT contributes to virus cell-to-cell spread and anterograde neuronal transport. In this study, our goal was to map the gE CT sub-domains that contribute to virus cell-to-cell spread property. A panel of gE-CT specific mutant viruses was constructed and characterized, in vitro, with respect to their plaque phenotypes, gE recycling and gE basolateral membrane targeting. The results revealed that disruption of the tyrosine-based motifs, YTSL and YTVV, individually produced smaller plaque phenotypes than the wild type. However, they were slightly larger than the gE CT-null virus plaques. The Y467A mutation affected the gE endocytosis, gE trans-Golgi network (TGN) recycling, and gE virion incorporation properties. However, the Y563A mutation affected only the gE basolateral cell-surface redistribution function. Notably, the simultaneous Y467A/Y563A mutations produced gE CT-null virus-like plaque phenotypes

Two Separate Tyrosine-Based YXXL/Φ Motifs within the Glycoprotein E Cytoplasmic Tail of Bovine Herpesvirus 1 Contribute in Virus Anterograde Neuronal Transport

Bovine herpesvirus 1 (BHV-1) causes respiratory infection and abortion in cattle. Following a primary infection, BHV-1 establishes lifelong latency in the trigeminal ganglia (TG). Periodic reactivation of the latent virus in TG neurons results in anterograde virus transport to nerve endings in the nasal mucosa and nasal virus shedding. The BHV-1 glycoprotein E cytoplasmic tail (gE-CT) is necessary for virus cell-to-cell spread in epithelial cells and neuronal anterograde transport. Recently, we identified two tyrosine residues, Y467 and Y563, within the tyrosine-based motifs YTSL and YTVV, which, together, account for the gE CT-mediated efficient cell-to-cell spread of BHV-1 in epithelial cells. Here, we determined that in primary neuron cultures in vitro, the individual alanine exchange Y467A or Y563A mutants had significantly diminished anterograde axonal spread. Remarkably, the double-alanine-exchanged Y467A/Y563A mutant virus was not transported anterogradely. Following intranasal infection of rabbits, both wild-type (wt) and the Y467A/Y563A mutant viruses established latency in the TG. Upon dexamethasone-induced reactivation, both wt and the mutant viruses reactivated and replicated equally efficiently in the TG. However, upon reactivation, only the wt, not the mutant, was isolated from nasal swabs. Therefore, the gE-CT tyrosine residues Y467 and Y563 together are required for gE CT-mediated anterograde neuronal transport

Two Separate Tyrosine-Based YXXL/Φ Motifs within the Glycoprotein E Cytoplasmic Tail of Bovine Herpesvirus 1 Contribute in Virus Anterograde Neuronal Transport

Bovine herpesvirus 1 (BHV-1) causes respiratory infection and abortion in cattle. Following a primary infection, BHV-1 establishes lifelong latency in the trigeminal ganglia (TG). Periodic reactivation of the latent virus in TG neurons results in anterograde virus transport to nerve endings in the nasal mucosa and nasal virus shedding. The BHV-1 glycoprotein E cytoplasmic tail (gE-CT) is necessary for virus cell-to-cell spread in epithelial cells and neuronal anterograde transport. Recently, we identified two tyrosine residues, Y467 and Y563, within the tyrosine-based motifs 467YTSL470 and 563YTVV566, which, together, account for the gE CT-mediated efficient cell-to-cell spread of BHV-1 in epithelial cells. Here, we determined that in primary neuron cultures in vitro, the individual alanine exchange Y467A or Y563A mutants had significantly diminished anterograde axonal spread. Remarkably, the double-alanine-exchanged Y467A/Y563A mutant virus was not transported anterogradely. Following intranasal infection of rabbits, both wild-type (wt) and the Y467A/Y563A mutant viruses established latency in the TG. Upon dexamethasone-induced reactivation, both wt and the mutant viruses reactivated and replicated equally efficiently in the TG. However, upon reactivation, only the wt, not the mutant, was isolated from nasal swabs. Therefore, the gE-CT tyrosine residues Y467 and Y563 together are required for gE CT-mediated anterograde neuronal transport

Bovine Herpesvirus 1 U49.5 Interacts with gM and VP22 To Ensure Virus Cell-to-Cell Spread and Virion Incorporation: Novel Role for VP22 in gM-Independent U49.5 Virion Incorporation

Alphaherpesvirus envelope glycoprotein N (gN) and gM form a covalently linked complex. Bovine herpesvirus type 1 (BHV-1) U49.5 (a gN homolog) contains two predicted cysteine residues, C42 and C78. The C42 is highly conserved among the alphaherpesvirus gN homologs (e.g., herpes simplex virus 1 and pseudorabies virus). To identify which cysteine residue is required for the formation of the U49.5/gM complex and to characterize the functional significance of the U49.5/gM complex, we constructed and analyzed C42S and C78S substitution mutants in either a BHV-1 wild type (wt) or BHV-1 U49.5 cytoplasmic tail-null (CT-null) virus background. The results demonstrated that BHV-1 U49.5 residue C42 but not C78 was essential for the formation of the covalently linked functional U49.5/gM complex, gM maturation in the Golgi compartment, and efficient cell-to-cell spread of the virus. Interestingly, the C42S and CT-null mutations separately did not affect mutant U49.5 virion incorporation. However, when both of the mutations were introduced simultaneously, the U49.5 C42S/CT-null protein virion incorporation was severely reduced. Incidentally, the anti-VP22 antibody coimmunoprecipitated the U49.5 C42S/CT-null mutant protein at a noticeably reduced level compared to that of the individual U49.5 C42S and CT-null mutant proteins. As expected, in a dual U49.5 C42S/VP22Δ virus with deletion of VP22 (VP22Δ), the U49.5 C42S virion incorporation was also severely reduced while in a gMΔ virus, U49.5 virion incorporation was affected only slightly. Together, these results suggested that U49.5 virion incorporation is mediated redundantly, by both U49.5/gM functional complex and VP22, through a putative gM-independent novel U49.5 and VP22 interaction. Bovine herpesvirus 1 (BHV-1) envelope protein U49.5 is an important virulence determinant because it downregulates major histocompatibility complex class I (MHC-I). U49.5 also forms a covalently linked complex with gM. The results of this study demonstrate that U49.5 regulates gM maturation and virus cell-to-cell spread since gM maturation in the Golgi compartment depends on covalently linked U49.5/gM complex. The results also show that the U49.5 residue cysteine 42 (C42) mediates the formation of the covalently linked U49.5-gM interaction. Furthermore, a C42S mutant virus in which U49.5 cannot interact with gM has defective cell-to-cell spread. Interestingly, U49.5 also interacts with the tegument protein VP22 via its cytoplasmic tail (CT). The putative U49.5 CT-VP22 interaction is essential for a gM-independent U49.5 virion incorporation and is revealed when U49.5 and gM are not linked. Therefore, U49.5 virion incorporation is mediated by U49.5-gM complex interaction and through a gM-independent interaction between U49.5 and VP22

HIV-1 Tat inhibits phagocytosis by preventing the recruitment of Cdc42 to the phagocytic cup

International audienceMost macrophages remain uninfected in HIV-1-infected patients. Nevertheless, the phagocytic capacity of phagocytes from these patients is impaired, favouring the multiplication of opportunistic pathogens. The basis for this phagocytic defect is not known. HIV-1 Tat protein is efficiently secreted by infected cells. Secreted Tat can enter uninfected cells and reach their cytosol. Here we found that extracellular Tat, at the subnanomolar concentration present in the sera of HIV-1-infected patients, inhibits the phagocytosis of Mycobacterium avium or opsonized Toxoplasma gondii by human primary macrophages. This inhibition results from a defect in mannose- and Fcγ-receptor-mediated phagocytosis, respectively. Inhibition relies on the interaction of Tat with phosphatidylinositol (4,5)bisphosphate that interferes with the recruitment of Cdc42 to the phagocytic cup, thereby preventing Cdc42 activation and pseudopod elongation. Tat also inhibits FcγR-mediated phagocytosis in neutrophils and monocytes. This study provides a molecular basis for the phagocytic defects observed in uninfected phagocytes following HIV-1 infection

Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells

International audienceHuman immunodeficiency virus type 1 (HIV-1) transcription relies on its transactivating Tat protein. Although devoid of a signal sequence, Tat is released by infected cells and secreted Tat can affect uninfected cells, thereby contributing to HIV-1 pathogenesis. The mechanism and the efficiency of Tat export remained to be documented. Here, we show that, in HIV-1-infected primary CD4(+) T-cells that are the main targets of the virus, Tat accumulates at the plasma membrane because of its specific binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P(2) molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane-embedded PI(4,5)P(2) only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P(2)-mediated recruitment of cellular proteins. Tat-PI(4,5)P(2) interaction is strictly required for Tat secretion, a process that is very efficient, as approximately 2/3 of Tat are exported by HIV-1-infected cells during their lifespan. The function of extracellular Tat in HIV-1 infection might thus be more significant than earlier thought