A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry

Viruses use cellular machinery to enter and infect cells. In this study we address the cell entry mechanisms of nonenveloped adenoviruses (Ads). We show that protein VI, an internal capsid protein, is rapidly exposed after cell surface attachment and internalization and remains partially associated with the capsid during intracellular transport. We found that a PPxY motif within protein VI recruits Nedd4 E3 ubiquitin ligases to bind and ubiquitylate protein VI. We further show that this PPxY motif is involved in rapid, microtubule-dependent intracellular movement of protein VI. Ads with a mutated PPxY motif can efficiently escape endosomes but are defective in microtubule-dependent trafficking toward the nucleus. Likewise, depletion of Nedd4 ligases attenuates nuclear accumulation of incoming Ad particles and infection. Our data provide the first evidence that virus-encoded PPxY motifs are required during virus entry, which may be of significance for several other pathogens

CAR-associated vesicular transport of an adenovirus in motor neuron axons.

Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2) that preferentially infects neurons. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR) surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis

Biologie des Adenovirus : recepteurs et transport intracellulaire

Les adénovirus ont une double nature, soit comme pathogène omniprésent qui peuvent occasionnellement causer des maladies soit comme vecteurs utilisés de transfert de gène. À nos connaissances, les 30 premières minutes depuis la liaison au récepteur jusqu'à l'arrivée au pore nucléaire sont identiques pour le pathogène comme pour le vecteur. L'objectif de ma thèse était de comprendre les mécanismes impliqués dans la liaison au récepteur, l'internalisation, l'échappement et le trafic endosomal vers le MTOC. J'ai d'abord étudié le mécanisme impliqué dans l'hémagglutination des virus à tropisme pour CAR et à tropisme pour SA. J'ai identifié la présence de CAR sur les érythrocytes humains et montré qu'il était le principal responsable de l'agglutination induite par les virus à tropisme pour CAR. De plus, j'ai montré que la présence de CAR sur les érythrocytes pouvait piéger le virus dans le sang et ainsi empêcher l'infection au niveau du foie. Dans un deuxième temps, j'ai participé à la caractérisation du rôle de la protéine VI et la translocation du virus au MTOC. Nous avons montré que Nedd4 était impliqué dans le ciblage du virus au MTOC via l'ubiquitination de la protéine VI. Enfin, j'ai travaillé sur le neurotropisme de CAV-2 et caractérisé sa localisa tion subcellulaire au niveau des synapses. J'ai montré qu'une partie de CAR était localisée dans des radeaux lipidiques à la synapse et que CAV-2 entrait via la voie de recyclage des vésicules synaptiques.Adenoviruses have a dual nature as ubiquitous pathogens that occasionally cause life-threatening disease and their use as gene transfer vectors. To the best of our current knowledge, the first 30 min from binding to nuclear pore docking of both wild-type virus and vector are identical. The goal of my thesis is to understand different mechanisms involved in receptor binding, internalization, endosomal escape and trafficking to the MTOC. First I studied the mechanism involved in hemagglutination of CAR-tropic and SA-tropic viruses. I identified the presence of CAR on human erythrocytes and showed that it was the main responsible for the agglutination mediated by CAR-tropic viruses. Moreover, I show that CAR on erythrocytes can sequester virus in the bloodstream and block liver infection. In a second part I participated to the characterization of the role of the protein VI and the translocation of HAd to the MTOC. We showed that Nedd4 was involved in the targeting of the virus to MTOC through ubiquitination of this protein VI. Finally, I worked on the neurotropism of CAV-2 and characterize its subcellular localization at the synapse. I showed that a part of CAR was localized in lipid raft at the synapse and enter through the synaptic vesicle-recycling pathway

Adenovirus biology (receptors and intracellular trafficking)

Les adénovirus ont une double nature, soit comme pathogène omniprésent qui peuvent occasionnellement causer des maladies soit comme vecteurs utilisés de transfert de gène. À nos connaissances, les 30 premières minutes depuis la liaison au récepteur jusqu'à l'arrivée au pore nucléaire sont identiques pour le pathogène comme pour le vecteur. L'objectif de ma thèse était de comprendre les mécanismes impliqués dans la liaison au récepteur, l'internalisation, l'échappement et le trafic endosomal vers le MTOC. J'ai d'abord étudié le mécanisme impliqué dans l'hémagglutination des virus à tropisme pour CAR et à tropisme pour SA. J'ai identifié la présence de CAR sur les érythrocytes humains et montré qu'il était le principal responsable de l'agglutination induite par les virus à tropisme pour CAR. De plus, j'ai montré que la présence de CAR sur les érythrocytes pouvait piéger le virus dans le sang et ainsi empêcher l'infection au niveau du foie. Dans un deuxième temps, j'ai participé à la caractérisation du rôle de la protéine VI et la translocation du virus au MTOC. Nous avons montré que Nedd4 était impliqué dans le ciblage du virus au MTOC via l'ubiquitination de la protéine VI. Enfin, j'ai travaillé sur le neurotropisme de CAV-2 et caractérisé sa localisa tion subcellulaire au niveau des synapses. J'ai montré qu'une partie de CAR était localisée dans des radeaux lipidiques à la synapse et que CAV-2 entrait via la voie de recyclage des vésicules synaptiques.Adenoviruses have a dual nature as ubiquitous pathogens that occasionally cause life-threatening disease and their use as gene transfer vectors. To the best of our current knowledge, the first 30 min from binding to nuclear pore docking of both wild-type virus and vector are identical. The goal of my thesis is to understand different mechanisms involved in receptor binding, internalization, endosomal escape and trafficking to the MTOC. First I studied the mechanism involved in hemagglutination of CAR-tropic and SA-tropic viruses. I identified the presence of CAR on human erythrocytes and showed that it was the main responsible for the agglutination mediated by CAR-tropic viruses. Moreover, I show that CAR on erythrocytes can sequester virus in the bloodstream and block liver infection. In a second part I participated to the characterization of the role of the protein VI and the translocation of HAd to the MTOC. We showed that Nedd4 was involved in the targeting of the virus to MTOC through ubiquitination of this protein VI. Finally, I worked on the neurotropism of CAV-2 and characterize its subcellular localization at the synapse. I showed that a part of CAR was localized in lipid raft at the synapse and enter through the synaptic vesicle-recycling pathway.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a RNA interference screen.

Viruses are strictly dependent on cells to propagate and many incorporate host proteins in their viral particles, but the significance of this incorporation is poorly understood. Recently, we performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1) in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify if these cellular factors are relevant for the HSV-1 life cycle. To this end, we performed a small interfering RNA functional screen and found that 15 of these host proteins altered HSV-1 proliferation in cell culture, without any significant effect on cell viability. Moreover, the siRNA used had no negative consequences for Adenovirus type 5 propagation (with one exception) indicating that the modulation was specific for HSV-1 and not merely due to unhealthy cells. The positive host proteins include several Rab GTPases and other intracellular transport components as well as proteins involved in signal transduction, gene regulation and immunity. Remarkably, in most cases when virions were depleted for one of the above proteins, they replicated more poorly in subsequent infections in wild type cells. This highlights for the first time that both the cellular and virion-associated pools of many of these proteins actively contribute to viral propagation. Altogether, these findings underscore the power and biological relevance of combining proteomics and RNA interference to identify novel host-pathogen interactions

Coagulation factor X mediates adenovirus type 5 liver gene transfer in non-human primates (Microcebus murinus).

International audienceCoagulation factor X (FX)-binding ablated adenovirus type 5 (Ad5) vectors have been genetically engineered to ablate the interaction with FX, resulting in substantially reduced hepatocyte transduction following intravenous administration in rodents. Here, we quantify viral genomes and gene transfer mediated by Ad5 and FX-binding-ablated Ad5 vectors in non-human primates. Ad5 vectors accumulated in and mediated gene transfer predominantly to the liver, whereas FX-binding-ablated vectors primarily targeted the spleen but showed negligible liver gene transfer. In addition, we show that Ad5 binding to hepatocytes may be due to the presence of heparan sulfate proteoglycans (HSPGs) on the cell membrane. Therefore, the Ad5-FX-HSPG pathway mediating liver gene transfer in rodents is also the mechanism underlying Ad5 hepatocyte transduction in Microcebus murinus

Virions harvested from siRNA treated cells are efficiently depleted of the siRNA-targeted protein.

<p>143B cells were transfected with Lipofectamine only or siRNAs against the control VP16 or the positive hits Arf1, DDX3X and eIF4H. They were then infected at a MOI of 5 with HSV-1 K26GFP and the supernatants collected at 24 hours. Concentrated supernatants corresponding to equal amounts of VP5 were analyzed and 20 µg of Lipofectamine-treated cell lysates were used as control. The reduced levels of <b>A</b>) VP16, <b>B</b>) ARF1, <b>C</b>) DDX3X and <b>D</b>) eIF4H in newly produced virions in the supernatants were confirmed by Western blotting.</p