J Virol

Autophagy is an essential metabolic program that is also used for clearing intracellular pathogens. This mechanism, also termed selective autophagy, is well characterized for invasive bacteria but remains poorly documented for viral infections. Here we highlight our recent work showing that endosomolytic adenoviruses trigger autophagy when entering cells. Our study revealed how adenoviruses exploit a capsid-associated small PPxY peptide motif to manipulate the autophagic machinery to prevent autophagic degradation and to promote endosomal escape and nuclear trafficking

PLoS Pathog

Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry

Adenovirus control autophagy during cell entry

L’adénovirus (AdV) est un virus non enveloppé à ADN double brin qui entre dans la cellule par endocytose. Dans l’endosome un désassemblage partiel de la capside permet la libération d’une protéine interne de la capside, la protéine VI (PVI). Cette protéine code une hélice amphipathique qui va permettre la rupture de l’endosome. Des travaux antérieurs du laboratoire ont montré que le transport des particules virales vers le noyau nécessite la présence du motif conservé PPxY dans la PVI qui permet le recrutement d’ubiquitines ligases de la famille des Nedd4 (telles que Nedd4.1 et Nedd4.2). Il a précédemment été montré que la rupture des membranes induite lors d’infections bactériennes activait l’autophagie afin d’éliminer le pathogène intracellulaire via une dégradation lysosomale. Nos résultats démontrent que l’AdV induit également l’autophagie lors de son entrée dans la cellule. L’utilisation de différents AdV mutants nous a permis de démontrer que la rupture de l’endosome était responsable de l’induction de l’autophagie. De plus nos résultats montrent que le virus sauvage est capable d’éviter sa dégradation en contrôlant l’autophagie grâce au recrutement de la ligase Nedd4.2 via le motif PPxY de la PVI. Au contraire, un virus mutant dépourvu du motif PPxY et donc incapable de recruter la Nedd4.2 est séquestré dans les vésicules autophagiques puis dégradé par la fusion de ces vésicules avec les lysosomes. Ainsi le motif PPxY constitue un déterminant moléculaire permettant au virus de contourner les défenses cellulaires antivirales.Adenoviruses (AdV) are linear ds-DNA containing, non-enveloped viruses that enter cells by receptor-mediated endocytosis. Once in the endosome it occurs a partial disassembly of the capsid allowing the releases of the membrane lytic capsid protein VI, which encodes an N-terminal amphipathic helix responsible for the endosome rupture. Our previous work showed that transport to the nucleus requires a conserved PPxY motif in PVI, which recruits ubiquitin ligases of the Nedd4 family (e.g. Nedd4.1 and 4.2). Previous work has shown that membrane damage induced by invasive bacteria elicits selective cellular autophagy to eliminate the pathogen via lysosomal degradation. In our current work we show that Adv also induce autophagy upon entry. Using a set of mutant AdV’s attenuated at each step of the membrane penetration process we show that indeed the membrane damage induced by the virus is causative for autophagy induction. Moreover the data show that wildtype AdV limit the level of autophagy induction and evade autophagic degradation by using a Nedd4.2 dependent process. In contrast, mutant viruses mutated for its PPxY and that fail to recruit Nedd4.2 are subject to autophagic degradation. Our data suggest that the presence of the PPxY motif in the virus subverts the autophagic process and thus identify the PPxY motif as an integral part of the virus to undermine cellular antiviral mechanism

Contrôle de l’autophagie lors des phases précoces de l’infection par l’adénovirus

Adenoviruses (AdV) are linear ds-DNA containing, non-enveloped viruses that enter cells by receptor-mediated endocytosis. Once in the endosome it occurs a partial disassembly of the capsid allowing the releases of the membrane lytic capsid protein VI, which encodes an N-terminal amphipathic helix responsible for the endosome rupture. Our previous work showed that transport to the nucleus requires a conserved PPxY motif in PVI, which recruits ubiquitin ligases of the Nedd4 family (e.g. Nedd4.1 and 4.2). Previous work has shown that membrane damage induced by invasive bacteria elicits selective cellular autophagy to eliminate the pathogen via lysosomal degradation. In our current work we show that Adv also induce autophagy upon entry. Using a set of mutant AdV’s attenuated at each step of the membrane penetration process we show that indeed the membrane damage induced by the virus is causative for autophagy induction. Moreover the data show that wildtype AdV limit the level of autophagy induction and evade autophagic degradation by using a Nedd4.2 dependent process. In contrast, mutant viruses mutated for its PPxY and that fail to recruit Nedd4.2 are subject to autophagic degradation. Our data suggest that the presence of the PPxY motif in the virus subverts the autophagic process and thus identify the PPxY motif as an integral part of the virus to undermine cellular antiviral mechanism.L’adénovirus (AdV) est un virus non enveloppé à ADN double brin qui entre dans la cellule par endocytose. Dans l’endosome un désassemblage partiel de la capside permet la libération d’une protéine interne de la capside, la protéine VI (PVI). Cette protéine code une hélice amphipathique qui va permettre la rupture de l’endosome. Des travaux antérieurs du laboratoire ont montré que le transport des particules virales vers le noyau nécessite la présence du motif conservé PPxY dans la PVI qui permet le recrutement d’ubiquitines ligases de la famille des Nedd4 (telles que Nedd4.1 et Nedd4.2). Il a précédemment été montré que la rupture des membranes induite lors d’infections bactériennes activait l’autophagie afin d’éliminer le pathogène intracellulaire via une dégradation lysosomale. Nos résultats démontrent que l’AdV induit également l’autophagie lors de son entrée dans la cellule. L’utilisation de différents AdV mutants nous a permis de démontrer que la rupture de l’endosome était responsable de l’induction de l’autophagie. De plus nos résultats montrent que le virus sauvage est capable d’éviter sa dégradation en contrôlant l’autophagie grâce au recrutement de la ligase Nedd4.2 via le motif PPxY de la PVI. Au contraire, un virus mutant dépourvu du motif PPxY et donc incapable de recruter la Nedd4.2 est séquestré dans les vésicules autophagiques puis dégradé par la fusion de ces vésicules avec les lysosomes. Ainsi le motif PPxY constitue un déterminant moléculaire permettant au virus de contourner les défenses cellulaires antivirales

The PPxY-mutant M1 associates with autophagosomes.

<p>(A) Live-cell imaging showing LC3 acquisition of WT upon entry. Stable expressing U2OS-LC3-GFP cells were infected with Alexa594 coupled WT and imaged using spinning-disk confocal microscopy. The top two images show individual frames separated by ~45 seconds from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006217#ppat.1006217.s008" target="_blank">S1 Movie</a>. The arrow points to an LC3 negative virus (left panel) becoming LC3-positive (right panel). The bottom panel shows a higher magnification and frame resolution of the same event. (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006217#ppat.1006217.s008" target="_blank">S1 Movie</a>). (B) The panel shows single frames of cells infected as in (A) either with WT (top) or M1 virus (bottom) at 1 hpi. The arrow at the top panel points to the microtubule organizing center where WT viruses accumulate (shown at higher magnification to the right). The arrows at the bottom panel point to autophagosomes engulfing mutant M1 viruses (shown at higher magnification to the right). (C) Representative confocal images of cells at 1hpi infected with WT (top row) and M1 (bottom row) and stained for AdV (red signal) and LC3 (green signal). Virus association with LC3 appears as yellow signal (see detail). (D) Experiment as in (C). The percentage of each AdV colocalizing with LC3 was quantified over time and is given as percentage of total virus. Error bars show cell to cell variation (n>10 cells; *: P<0.05; **: P<0.01). (E) TEM analysis of U2OS cells infected for 30 minutes with WT (a-d, top row) or M1 (e-h, bottom row). The overview images show cytosolic WT viruses (a, c) and vesicle associated M1 viruses (e, g) depicted by a black arrow. At higher magnification WT (b, d) and M1 (f, h) particles (AdV) are depicted by white arrowheads. The nuclear pore complex (NPC in d) and the endosomal membrane (EM in f, g) are indicated with grey arrowheads and autophagosomes (AP) by black arrowheads (f, h). The * indicates the putative location for autophagy receptors between EM and AP. Error bars are 100 nm.</p

PPxY-mediated endosomal escape prevents autophagic degradation of incoming virions.

<p>(A) Left panel: U2OS cells expressing the Pi3P <i>in cellulo</i> binding probe PX-GFP were treated with vehicle (top) or with 5mM of the Pi3K inhibitor 3’MA (bottom). Middle panel: U2OS cells pre-treated with vehicle alone (black bars) or 3’MA (red bars) were transduced with WT or M1. Transgene expression was determined and normalized to vehicle treated controls to show the fold induction of infectivity upon treatment. Right panel: The same data as in the middle panel showing the level of M1 infectivity rescue compared to the normalized WT infectivity upon treatment. (B) Left panel: U2OS cells were treated with vehicle (top) or with chloroquine (CQ, 50μM, bottom) to block the autophagic flux, fixed and stained for LC3. Middle panel: Cells were treated with vehicle alone (black bars) or chloroquine (red bars) and transduced with WT or M1. Transgene expression was determined and normalized to vehicle treated controls to show the fold induction of infectivity. Right panel: The same data as in the middle panel showing the level of M1 infectivity rescue compared to normalized WT infectivity. (C) Left panel: U2OS cells depleted for ATG5 (SH-ATG5) or control depleted cells (SH-CTRL) and starved using HBSS during 4h, fixed and stained for LC3. Middle panel: Cells were transduced with WT or M1 and the relative transduction efficiency in SH-CTRL cells (black bars) and SH-ATG5 cells (red bars) was determined. Right panel: The same data as in the middle panel showing the level of M1 infectivity rescue compared to normalized WT infectivity. ATG5 expression levels were determined by western blot. (D) Left panel: Control MEFs (ATG5 +/+) and KO MEFs (ATG5 -/-) were transduced with WT or M1 as indicated and the relative transduction efficiency for the M1 (red bars) compared to the WT (black bars) was determined. Right panel: The panel shows the absolute number of transduced cells at indicated amounts of physical particles added to the cell (pp/c) for the WT (black bars) and the M1 (red bars) in ATG5 control (left) and KO (right) MEFs. ATG5 expression levels were determined by western blot.</p

M1 restriction is mediated by Gal8.

<p>(A) Left panel: U2OS cells were depleted with siRNAs specific for galectin8. Cells were transduced with WT or M1 as indicated and the relative transduction efficiency was calculated for control depleted cells (black bars) or galectin depleted cells (red bars). Middle panel: Relative transduction efficiency of the M1 virus following two rounds of siRNA depletion. Right panel: The same data as in the middle panel showing the level of M1 infectivity rescue compared to the normalized WT infectivity upon treatment. Each experiment was done in triplicate (B) Experiment essentially done as in (A) except that galectin 3 was depleted. (C) Experiment essentially done as in (A) except that galectin 9 was depleted. (D) HeLa cells were transfected twice with control or galectin8 specific siRNAs followed by infection with WT or M1 viruses and fixed at different time points after infection. Quantification of AdV colocalizing with LC3 (top panel.) and Lamp1 (bottom panel) was performed and is shown as percentage of colocalization for each virus and condition according to the legend. (E) Left panel: Cells were depleted with SH-RNA specific for p62. Specific or control depleted cells were transduced with WT or M1 as indicated and the relative transduction efficiency was calculated for control depleted cells (black bars) or p62 depleted cells (red bars). Middle panel: Experiment essentially done as for p62 except that SH-RNA was directed against NDP52. Right panel: Experiment essentially done as for p62 except that SH-RNA was directed against optineurin (OPTN). (F) Left panel: Cells were depleted with SH-RNA specific for NDP52 followed by transfection with siRNA against p62. Double depleted or control depleted cells were transduced with WT or M1 as indicated and the relative transduction efficiency was calculated for control depleted cells (black bars) or p62 depleted cells (red bars). Middle panel: The same data as in the left panel showing the level of M1 infectivity rescue compared to the normalized WT infectivity upon treatment.</p

Model for adenovirus control of autophagic processes upon entry.

<p>AdV enter cells by receptor-mediated endocytosis (1) followed by partial disassembly to release the internal membrane lytic capsid protein PVI (2). PVI release initiates membrane rupture and intralumenal glycans are recognized via galectins and the autophagic machinery is recruited through Gal8 and LC3 to the damaged endosome mediated by yet to clarify adapter molecules (3). The recruitment of Nedd4.2 via the PPxY motif in PVI prevents formation of autophagosomes via an unknown mechanisms and facilitates endosomal escape (4). Endosomal escape involves the autophagic machinery because ATG5 depletion affects dissociation of virus from damaged vesicle. Dynein motor complexes are also required to access cytosolic microtubule mediated transport towards the MTOC which may occur in association with LC3 (5). Subsequent genome release occurs at the nuclear pore complex (6). Genome delivery is delayed upon ATG5 depletion. If PVI is not released (AdV-TS1), no membrane damage occurs and viruses are degraded via lysosomal sorting (7). If PVI is released and membrane damage occurs but the virus does not escape (AdV-M1), capsids are degraded via autophagy (8). This degradation is limited in absence of functional autophagy (e.g. upon ATG5 depletion).</p

AdV endosomal escape is dynein dependent.

<p>(A) Cells were pretreated with 100 μM of Ciliobrevin D for 30 minutes or DMSO and transduced with GFP expressing WT vector in presence of drugs. Twenty four hours later transduction levels were determined by FACS and normalized for the vehicle control (B) Cells were infected with fluorescent viruses in presence and absence of 100μM CilioD and fixed at different time points. The number of Gal3 punctae per cell was determined (C) Cells were infected as in B. Colocalization between Gal3 and AdV signals was quantified and is displayed as percentage of colocalization. (D) Cells were infected as in B. Fluorescent viruses in control or drug treated or control cells were quantified at indicated time points in the perinuclear region vs. the rest of the cytosol. Error bars (SE) show cell-to-cell variations.</p

Nuclear transport of AdV involves the autophagic machinery.

<p>(A) Representative panel of WT or M1 infected cells at 1hpi stained with LC3 (red signal) and pericentrin (green signal) specific antibodies. (B) Quantification of the relative distribution of autophagosomes for experiment shown in (A) essentially analyzed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006217#ppat.1006217.g009" target="_blank">Fig 9E</a>. (n>12 cells; NS: no significant; *: P<0.05; **: P<0.01) (C) Representative panel of WT infected cells at 1hpi depleted for ATG5 or control depleted (as indicated) and stained with AdV (red signal) and pericentrin (green signal) specific antibodies. (D) Quantification of the relative virus distribution as in (B) for the experiment shown in (C) including the distribution of M1 and WT viruses. (n>12 cells; NS: no significant, **: P<0.01) (E) Representative panel of WT infected cells at 1hpi depleted for ATG5 or control depleted (as indicated) and stained with AdV (red signal) and PVI (green signal) specific antibodies to mark PVI separation from the virus. (F) Infection time course analysis of PVI release from M1 (red line) vs. WT (black line) viruses in ATG5 depleted (dotted line) vs. control depleted cells (solid line). Shown is the percentage of PVI positive AdV at indicated time points. The errors bars are cell-to-cell variation (10 cells were analyzed for each conditions). (G) Representative panel of WT infected cells at 1hpi and stained with specific antibodies against AdV (red signal) and specific antibodies against PVII (green signal) to mark nuclear genomes. (H) Quantification of nuclear genome delivery. ATG5 and control depleted cells were infected with WT and M1 and fixed at 1 and 2hpi and stained for AdV and PVII. The number of nuclear PVII dots was calculated and normalized for virus particles at each condition as indicated below the graph (n>16 cells; **: P<0.01; ***: P<0.0001). (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006217#ppat.1006217.s007" target="_blank">S7 Fig</a>).</p