Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid

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.

Introduction mediated recruitment of the adapter protein NDP52 towards bacteria containing vacuoles. NDP52 in turn establishes the link to the autophagic machinery for cargo degradation exemplifying how the initial sensing via galectins leads to an active antimicrobial response [15,17].
Autophagy, or selective autophagy when the cargo is sequestered via receptors, is an evolutionary-conserved cytosolic, lysosome-dependent, degradation process [18]. It serves as a cellular survival pathway to maintain homeostasis e.g. by providing the cell with nutrients upon starvation. Autophagy is initiated through formation of an isolated membrane structure, the phagophore, via the concerted action of several factors belonging to the AuTophaGy-related genes (ATG) [19,20]. After initiation the cytoplasmic protein LC3 (Microtubule-associated protein 1A/1B-light chain 3) becomes lipidated and incorporated into the elongating membrane, which forms a double-membrane vesicular structure, the autophagosome, and closes around the cargo destined for degradation. In contrast autophagosome formation and maturation into autolysosomes is still not fully understood but may involve LC3 mediated transport from the site of origin towards the perinuclear region by using a retrograde, microtubule dependent and dynein-dynactin motor complex mediated transport. This active transport facilitates efficient fusion of closed autophagosomes with perinuclear lysosomes [21][22][23].
Selective autophagy is part of the intrinsic immune response to attenuate intracellular pathogen infections via lysosomal degradation [24]. Next to galectins, selective autophagy involves additional marker molecules such as ubiquitin and further adapter molecules [25]. We recently demonstrated that Gal3 also marks membrane damage caused by AdV and observed that AdV traffics in Gal3 positive membranes prior to endosomal escape [5,26] although a role in selective autophagy was not addressed. Still, several studies have reported that autophagy can promote or restrict viral infections including AdV [27][28][29]. Autophagy also indirectly restricts viral infection by increasing viral peptide presentation on MHC-II molecules to mount an adaptive immune response as recently shown for AdV [28] and other viruses [30,31]. Accordingly several viruses have developed efficient strategies to temper with the autophagic machinery [32].
Within this study we use AdV to show that selective autophagy mediated by Gal8 also targets endosomolytic viruses. Moreover we show that AdV escape from and limit the autophagic response through a conserved PPxY peptide motif encoded in the membrane lytic capsid protein VI. We show that this is achieved by restricting the formation of autolysosomes via a mechanism involving the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Furthermore we provide evidence that AdV hijacks the autophagic response to achieve efficient endosomal escape and accelerated nuclear transport and genome delivery showing a multilayered viral control of the autophagic machinery during virus entry.

Microtubules and a conserved PPxY motif in capsid protein VI are required for AdV efficient endosomal escape
We showed previously that E1/E3 deleted GFP expressing adenoviral vectors with the PPxYmotif (WT) in capsid protein VI mutated to PGAA (M1) have reduced transduction efficiency and altered nuclear transport [11]. Both vectors were used throughout this study and are referred to as "WT" and "M1" respectively. To verify the phenotype we infected cells with WT and M1 and quantified the transduction levels at different multiplicities of infection (MOI). The results confirmed the M1 phenotype in a variety of cell types suggesting a virion-associated mechanism (Figs 1A, 1B and S1). We then asked if the defect was linked to the role of PVI in membrane penetration. We infected cells for different times with WT and M1 and used Gal3 as a marker for AdV induced membrane damage. The results showed that both viruses efficiently induce Gal3 punctae (Fig 1C). We quantified both the number of Gal3 punctae per cell as a measure of membrane rupture and the colocalization of virus particles with Gal3 as a measure for endosomal escape. We observed no apparent difference in the kinetics of membrane lysis between WT and M1 ( Fig 1D) and only minor differences in the absolute number of cell-associated virus over time (Fig 1E). In contrast, the M1 colocalized with Gal3 punctae to a much larger extend than WT (Fig 1F), suggesting a post-lysis endosomal escape defect. Previous in vivo imaging of AdV endosomal escape suggested a propelled and microtubule dependent escape mechanism [5,26] and both dynein and microtubule transport have been implicated in AdV entry [33,34]. To confirm that AdV utilize dynein to escape from ruptured endosomes we used Ciliobrevin D (CilioD), a reversible chemical inhibitor of the dynein AAA+ ATPase motor to inhibit dynein activity during viral transduction (Fig 2). At non-toxic CilioD concentrations we observed decreased GFP transgene expression compared to vehicle control treated cells (Fig 2A). Time course infection experiments with fluorescently labeled WT revealed no change in the initial number of Gal3 punctae upon CilioD treatment. However, Gal3 punctae increased over time (Fig 2B) and the WT remained associated with Gal3 ( Fig 2C) in drug-treated cells suggesting involvement of dynein in virus escape and membrane damage removal but not in the initial lysis. Sequestration of the WT after CilioD treatment also prevented efficient virus translocation to the perinuclear area ( Fig 2D) consistent with previous observations made for the M1 virus [11]. Taken together, these data show that the PVI PPxY-motif and dynein are critical for efficient escape from ruptured endosomes and subsequent transport to the nucleus.

Adenovirus induces selective autophagy upon endosomal membrane penetration
We next asked if AdV, similar to invasive bacteria, would activate selective autophagy upon membrane rupture. To address this question we used three different AdV's all based on genotype HAdV-C5, which share receptor-mediated endocytosis but differ upon endosomal sorting. Next to WT and M1 we infected cells with an E1/E3 deleted GFP expressing ts1 adenoviral vector grown at the non-permissive temperature [10]. This hyperstable ts1 mutant virus ("TS1" in this study) does not release PVI and does not lyse the endosome upon uptake. We then performed an infection time course (shown in Fig 3A) and quantified appearance of Gal8 punctae to mark membrane damage. We used Gal8 because of its functional role in selective autophagy [15]. Gal8 punctae formation occurred in WT and M1 but not TS1 infected cells (S2 Fig), was often linked to particles also positive for PVI ( Fig 3B) and punctae formation and particle association was rapid ( Fig 3C) and followed similar kinetics as Gal3 punctae formation. To show that AdV induced membrane damage activates autophagy we next stained for LC3 and quantified LC3 punctae reminiscent of autophagosome formation [35]. We observed LC3 punctae formation for WT and M1 but not for TS1 supporting that membrane lysis was required to initiate autophagy ( Fig 3C). We confirmed the results in a different cell type (S2B Fig) and by showing the membrane damage-dependent accumulation of phosphatidyl-ethanolamin (PE) conjugated LC3-II by western blot in WT and M1 but not in TS1 infected cells ( Fig  3E). LC3 punctae formation occurred rapidly within 15-30 min after virus uptake ( Fig 3F) resembling PVI release kinetics [10]. We then asked if AdV induced autophagy was selective and mediated by adapter proteins. We repeated the time course experiment and analyzed recruitment of autophagy adapter p62, NDP52 and optineurin. Accumulation of p62 and NDP52 punctae was dependent on virus induced membrane damage (S2C and S2D Fig).
Punctae appearance and association with WT and M1 particles followed similar kinetics ( Fig  4A for NDP52 and 4C for p62) as previously observed for galectins and LC3. In contrast optineurin, was not detected in association with viral particles under our conditions. Co-staining for cellular marker molecules and viruses showed large overlap in colocalization suggesting functional links. E.g. several viruses positive for NDP52 also stained positive for Galectins ( Fig  4B) and p62 positive viruses stained also positive for ubiquitin ( Fig 4D) reminiscent of their recruitment towards invasive bacteria.
In summary our analysis suggested that PVI released from entering viral particles caused membrane damage to which the cell responds with selective autophagy. Interestingly we observed higher levels and more prolonged accumulation of LC3 punctae for the escape defective mutant M1 vs. WT (Fig 4F). We hypothesized that M1 virions maybe degraded by autophagy, which could explain their strongly reduced infectivity.
AdV with mutated PPxY motif in capsid protein VI are subject to autophagic degradation To determine if M1 viruses are degraded via autophagy we first visualized AdV association with LC3 in living cells. We infected cells stably expressing LC3-GFP [36] with Alexa594 We observed that within~15-30 min several cell associated viruses turned LC3 positive, mostly occurring at the cellular periphery ( Fig 3A, S1 Movie). At >1hpi most WT viruses showed no more association with LC3 and accumulated in the vicinity of the nucleus (Fig 3B  and 3C, top). In contrast, M1 viruses lacked nuclear accumulation and were often found inside large LC3 positive structures reminiscent of autophagosomes ( Fig 3B and 3C, bottom). This difference in dynamic LC3 association between WT and M1 viruses was confirmed using quantitative time resolved fluorescence analysis ( Fig 3D). Autophagosomes are characterized by forming a double membrane around their substrate. To confirm that M1 particles associate with autophagosomes we next performed transmission electron microscopy (TEM). Cells were infected with fluorescently labeled WT or M1 virus, fixed at 30 min post infection and processed as detailed in the material and methods section. WT viruses were observed in the cytoplasm (Fig 5Ea and 5Eb) or at the nuclear pore complex (NPC , Fig 5Ec and 5Ed). In contrast M1 viruses could be observed inside vesicular structures (Fig 5Ee and 5Eg). At higher magnification we could observe that M1 particles seemed to be entrapped in partially ruptured vesicles resembling endosomes (Fig 5Ef and 5Eh). These virus containing vesicles were partially (f) or fully (h) engulfed by a double membrane vesicle resembling autophagosomes. Interestingly we observe a cleft between the endosome and the inner autophagosomal membrane filled with electron dense material, which would be the putative location for the linking autophagy receptors (marked with asterisk in Fig 5Ef and 5Eh). Our analysis thus strongly supports that M1 particles still inside the ruptured vesicle are sequestered by autophagy whereas WT viruses escape from the endosomal compartment prior to autophagic sequestration.
To investigate a potential autophagic degradation for the M1 virus we treated cells with 3-methyladenin (3-MA), which blocks autophagosome formation via the inhibition of class III PI3K [35]. Pretreated and vehicle control cells were transduced with WT or M1 viral vector expressing GFP and relative transduction levels were determined using fluorescence activated cell sorting (FACS). 3-MA treatment did not affect WT virus infectivity but significantly increased M1 virus infectivity restoring WT infectivity to nearly 75% ( Fig 6A). In addition we found that the M1 virus located per se more than the WT virus with Pi3P positive compartments from which autophagosome formation originates (S3A Fig) [37] and that upon M1 infection Beclin-1, a subunit of the class III PI3K kinase complex, was enriched on cellular membranes (S3B and S3C Fig) suggesting differences in class III PI3K kinase complex activity between both viruses. We next inhibited the autophagic flux by blocking lysosome acidification with chloroquine (CQ, Fig 6B) [35]. CQ treatment increased M1 virus infectivity without affecting WT infectivity although the effects were more moderate (Fig 6B). Next we depleted cells of the LC3 conjugation factor ATG5 using SH-RNAs [35]. Again we observed a specific but moderate increase in M1 infectivity ( Fig 6C). Because of the reduced impact in restoring M1 infectivity and because our depletion efforts yielded only partial removal of ATG5 we next infected ATG5 -/-MEFs (Mouse Embryonic Fibroblasts) with WT and M1. As shown in   Together our results showed that M1 virus infectivity could be fully or partially restored by blocking autophagy at different steps, showing that the M1 virus but not the WT virus becomes an autophagic substrate. To exclude that the increase in M1 infectivity was not due to pleiotropic effects of autophagy inhibition we depleted cells of galectins or adapter molecules that link the virus induced membrane damage to the autophagic machinery (S4A Fig). M1 infectivity increased only after depleting Gal8 ( Fig 7A) but not when depleting either Gal3 ( Fig  7B) or Gal9 ( Fig 7C). The rescue of M1 infectivity was strongly correlated to the depletion level of Gal8 ( Fig 7A) and we were able to observe almost complete restoration of WT infectivity in our best depletion experiments. In contrast M1 infectivity was neither influenced by Gal3 depletion levels nor by Gal9 depletion levels. We next determined WT vs. M1 virus association with LC3 and lysosomes in control-and Gal8-depleted cells over time. Gal8 knockdown had no effect on LC3 colocalization with the WT, but colocalization of the M1 with LC3 decreased to WT levels ( Fig 7D, top panel). Additionally, M1 colocalization with the lysosome marker Lamp1 significantly decreased in Gal8 knockdown cells strongly suggesting that Gal8 depletion prevented efficient targeting of the M1 virus for autophagic degradation (Fig 7D, bottom panel). Taken together our results identify Gal8 as an essential restriction factor for the M1 virus and show that Gal8 recruitment towards AdV-ruptured membranes is necessary to target virus-containing membranes for autophagosomal degradation.
We next depleted cells from the autophagy receptors p62, NDP52 and optineurin. We observed no rescue of M1 infectivity when depleting optineurin in agreement with the absence of optineurin recruitment to virus particles. M1 infectivity was only slightly but specifically increased upon depletion of the Gal8 specific autophagy adapter NDP52 ( Fig 7E). In contrast, depletion of the autophagy adapter p62 slightly increased infectivity for both viruses implying a somewhat different role in AdV entry ( Fig 7E). Thus we next asked if co-depletion of autophagy receptors would have a more pronounced effect and would overcome the M1 restriction. Efficient double depletion of NDP52 and p62 gave similar results as depletion of p62 alone excluding an additive effect ( Fig 7F). Taken together this implied that none of the autophagy adapters tested plays a major role in M1 restriction, suggesting that Gal8 mediated restriction of bacteria and adenoviruses work at least in part via different pathways.

AdV limit autophagy and prevent antigenic presentation
Autophagy inhibition had little impact on WT infectivity suggesting that AdV might actively target autophagic processes. To address this question we infected cells with WT and M1 and performed a time course western blot analysis to determine the levels of LC3-II. LC3-I conversion initiated within 15-30 min pi for both viruses and continued at least up to 2 hpi for the M1 virus ( Fig 8A). In contrast LC3 conjugation induced by the WT virus was comparable in the beginning but declined at~1hpi and returned to basal levels at 2hpi (Fig 8A). The same 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.
doi:10.1371/journal.ppat.1006217.g005 We next determined the ratio of autophagosomes to autolysosomes in WT and M1 infected cells at 1hpi. Our analysis showed that at 1hpi~60% of LC3 positive structures in M1 infected cells were also positive for the lysosome marker Lamp2 indicating conversion into autolysosomes. In contrast in WT infected cells only~40% were Lamp2 positive (Fig 8B and 8C) indicating a defect in autolysosome formation. To confirm that the WT impairs autolysosome formation we transduced cells with LC3 fused to GFP and RFP (LC3-GFP-RFP). Due to the pH-sensitivity of GFP, neutral autophagosomal membranes appear yellow while acidic autophagolysosomes appear red [35]. Quantification in WT and M1 infected cells at 1hpi confirmed a reduced level of autolysosome for the WT (S5B and S5C Fig). To understand how the WT prevents autolysosome formation we first ask if the virus controls the onset of autophagy. We induced non-selective autophagy through overnight starvation followed by infection with WT or M1 and quantified number and maturation state of LC3-positive structures at 1hpi (Fig 8D  and 8E). Both viruses induced comparable levels of autophagy marked by an increase in LC3 positive punctae in starved cells (Fig 8D) while the ratio of lamp2 positive vs. lamp2 negative LC3-positive structures was reduced in WT infected cells vs. M1 (Fig 8E) showing that the WT controls autolysosome formation without impairing the onset of autophagy.
Autophagy promotes antigen presentation through fusion with MHC-II containing compartments including antigens incorporated into the AdV capsid [28,31]. Thus we asked if limiting autolysosome formation restricts AdV antigenic presentation. For this we infected mice with equal particle numbers of WT, M1 or PBS control. Ten days later, the mice were sacrificed and purified splenocytes were stimulated with either AdV capsids or GFP purified from E.coli. T-cell activation was measured by IFNγ ELISPOT (Fig 8F). We saw more IFNγ spot forming units (SFU) in splenocytes stimulated with AdV capsids from the M1 infected mice vs. WT infected mice, suggesting an increased display of AdV capsid antigens during M1 infection. An inverse effect was observed when the splenocytes were stimulated with purified GFP protein and compared to WT infected mice. Because the GFP is only expressed after adenovirus transduction of cells, this observation suggests that there is a defect in M1 infectivity in vivo. Additionally, we generated a human CD4 + T cell clone that responds to a conserved hexon epitope. We incubated these with syngenic APCs transduced with equivalent viral particles of either WT or M1, and measured T cell activation by IFNγ ELISA (Fig 8G). Again, we saw increased IFNγ levels from the T cells exposed to M1 vs. WT transduced APCs suggesting increased antigen presentation on MHC-II. No significant difference in MHC-II or CD86 expression was observed in human monocyte derived DCs between WT and M1 treated  groups, suggesting that differences in the expression of these molecules between treatments did not account for differences in T-cell activation (S5D Fig). Taken together we show that AdV use the PVI PPxY motif to limit capsid epitope presentation on MHC-II presumably by restricting autolysosome formation.

Nedd4.2 regulates autolysosome formation and AdV nuclear transport
While several Nedd4 ligases bind the PVI PPxY motif, we previously showed that only depletion of Nedd4.2 caused a transport defect and reduced infectivity [11]. To test if Nedd4.2 was also involved in AdV control of autophagy we infected Nedd4.2 depleted cells (S6A Fig) with WT virus and analyzed LC3-I conversion into LC3-II by western blot (Fig 9A). We observed increased and prolonged levels of LC3-II in infected cells following Nedd4.2 depletion, however, basal levels of LC3-II were also elevated. In individual cells (including non-infected cells) LC3 positive structures appeared more abundant, were larger in size and showed differences in subcellular distribution compared to control cells (Fig 9B and 9D). We next analyzed the percentage of autolysosomes by costaining LC3 with Lamp2 in control vs. Nedd4.2 depleted cells after AdV infection. In control depleted cells the proportion of autolysosomes in WT infected cells was reduced compared to M1 infected or non-infected control cells similar to our previous observation. In contrast upon depletion of Nedd4.2 all cells showed similar reduced levels of autolysosomes including M1 infected and non-infected control cells, without any further reduction in WT infected cells (Fig 9C). These results identified an important physiological role for Nedd4.2 in autolysosome formation and suggested that the WT virus interferes with Nedd4.2 to limit autolysosome formation. We next addressed the physiological role for Nedd4.2 in autophagy regulation in more detail. We analyzed autophagosome formation in Nedd4.  (Fig 9D) a distribution that is typical for AdV and other incoming viruses [7,38]. Interestingly, upon infection of Nedd4.2 depleted cells LC3 positive structures lost the MTOC accumulation and displayed a more perinuclear distribution (Fig 9D). We confirmed this observation by quantifying the proximity of LC3 positive structures to the MTOC (marked by pericentrin stain) in control and Nedd4.2 depleted cells at 1 hpi (Fig 9E).  Because we previously observed a similar MTOC accumulation defect in Nedd4.2 depleted cells with WT viruses [11], we next asked if autophagy contributes to nuclear transport of AdV.

Autophagy is required for efficient nuclear transport of AdV
To address a possible role of autophagy in AdV nuclear transport we first infected cells with WT and M1 and analyzed the subcellular distribution of LC3 positive structures at 1hpi. This experiment should distinguish if MTOC accumulation of LC3 positive structures required recruitment of Nedd4.2 towards WT viruses or, in the case of the M1 virus, if availability of Nedd4.2 without capsid recruitment via protein VI was sufficient for MTOC targeting of LC3 (Fig 10A). WT infected cells showed increased MTOC accumulation of LC3 positive structures compared to M1 infected cells, which we confirmed by MTOC proximity quantification ( Fig  10B). Because in vivo imaging of fluorescent viruses in LC3-GFP expressing cells often displayed virus mobility in association with LC3 ( S7 Fig and S2 Movie) we asked if the autophagic machinery plays a more direct role in WT MTOC targeting. We repeated the above assay in ATG5 depleted cells and quantified the virus distribution around the MTOC. Most cells showed reduced MTOC accumulation for WT viruses in ATG5 depleted cells (Fig 10C), which we confirmed by MTOC proximity quantification. In contrast the more random distribution of M1 was not affected by ATG5 depletion (Fig 10D). Because the WT virus distribution in ATG5 depleted cells was very similar to the distribution of the M1 virus we asked if the endosomal escape was still functional. For this we compared PVI release between WT and M1 in ATG5 depleted cells vs. control cells as a measure of endosomal escape (Fig 10E). The analysis showed that ATG5 depletion did not impact on the initial release of PVI from either WT or M1 but delayed the separation of PVI from the WT (Fig 10F) showing that efficient endosomal escape requires ATG5.
Nuclear genome delivery can be considered as endpoint of AdV entry, which can be quantified by accumulation of genome associated protein VII dots in the nucleus [39] (Fig 10G). We quantified nuclear import of adenoviral genomes over time in ATG5 depleted and control cells for both viruses. Our analysis showed an initial delay for WT genomes in ATG5 depleted cells at 1 hpi that was fully compensated at 2 hpi confirming that fast nuclear genome transport relies on ATG5 while compensatory transport mechanisms exist (Fig 10G, left panel). It also confirmed that the M1 mutant virus was subject to autophagic degradation because ATG5 depletion increased nuclear genomes compared to control depleted cells suggesting that more virion particles survive the entry process and are able to deliver their genome. Nevertheless, nuclear genome arrival in ATG5 depleted cells showed the same delay as WT genomes ( Fig  10G, right panel).
Taken together our study demonstrates how upon entry adenoviruses use a capsid encoded PPxY-peptide motif to exploit and control the autophagic machinery at multiple levels to secure efficient genome delivery.

Discussion
Many invasive pathogens challenge the cellular membrane integrity to access the cytosol. Cells are able to sense pathogen invoked membrane damage via out-of-place detection of intralumenal glycans exposed to the cytosol. Thurston and co-workers showed that several galectins as well as poly-ubiquitin serve as danger sensor for ruptured membranes but only Gal8 via its specific adapter NDP52 restricts bacterial proliferation targeting the damaged vesicles for autophagic degradation [15]. Here we show that cells respond via a similar principle to incoming AdV demonstrating that non-enveloped endosomolytic viruses also activate selective autophagy through membrane damage. Under our assay conditions we show that release of the membrane lytic PVI from internalized viruses induced the transient association with galectins (3,8 and 9), ubiquitin, adapter molecules (p62 and NDP52) and LC3 within minutes of virus uptake. Using the control TS1 mutant virus, which lacks PVI release, we were able to pinpoint the membrane damage as the initiating event that causes this response. While WT viruses rapidly escaped from the ruptured endosome, and accumulate at the MTOC, the mutant M1 virus lacking a conserved PPxY motif in PVI remained associated with the ruptured (Gal3, Gal8 and PVI positive) vesicle and was subject to autophagic degradation. EM analysis revealed that during this process autophagosomal membranes engulf M1 viruses still associated with ruptured endosomes. Accordingly degradation through autophagy was able to explain the M1 infectivity defect and autophagy inhibition restored M1 infectivity. Thus we identified the capsid encoded PPxY motif as molecular determinant that allows the WT virus to subvert the cellular antiviral response providing the first example of a virus encoded motif to combat cellular antimicrobial autophagy. M1 virus removal occurred through a pathway involving Gal8 detection of virus induced membrane damage corroborating that autophagy recruitment through Gal8 is part of a conserved cellular pathway for the detection and removal of membrane damage evoked by microbes as previously suggested [15]. Gal3, which was also recruited to AdV ruptured membranes was not restricting the M1 mutant virus despite its recently described link to autophagy [40]. Depletion of the Gal8 binding autophagy receptor NDP52 as well as p62, which are both involved in restricting invasive bacteria was much less effective than autophagy inhibition or Gal8 depletion in protecting M1 from degradation. This observation suggests the existence of additional and/or alternative pathways to link AdV induced membrane damage via Gal8 towards autophagy clearance. These pathways may include the use of alternative autophagy receptors such as TAXBP1 recently shown to be involved in Salmonella clearance although no Gal8 binding was reported [41].
Our work with the M1 virus underpins the power of selective autophagy as antimicrobial mechanisms and shows how AdV have evolved capsid encoded evasive mechanism. This may not be true for all endosomolytic viruses. A recent report showed that picornavirus induced membrane damage also activates Gal8 mediated autophagy. In this case however the virus uses a cellular factor, the lipid-modifying enzyme PLA2G16, to counteract selective autophagy [42]. Remarkably, escaping endosomes would suffice for AdV to evade autophagic degradation.  Nevertheless we observe that the WT virus uses the PPxY motif in PVI also to prevent efficient formation of autolysosomes. This effect is not linked to the onset of autophagy because LC3 punctae are induced and LC3-II conversion takes place upon WT infection. However, unlike with the M1 virus, we see much less large autophagosomal structures in WT infected cells and LC3-II levels rapidly return to basal levels. One possible explanation is that the WT virus interferes with the elongation process of the autophagosomal membrane to prevent autophagosome formation. This would make sense because only fully formed autophagosmes can fuse with lysosomes [43]. Depletion of Nedd4.2 removes the ability of the WT to interfere with autolysosome formation showing that the recruitment of Nedd4.2 through the PPxY motif in capsid protein PVI is central to AdV autophagy evasion. Murine Nedd4.2 was recently identified as factor that promotes autophagy using knockdown approaches [44]. In our hands Nedd4.2 depletion increases basal autophagy levels and also inhibits autolysosome formation independently of infection suggesting a key role for Nedd4.2 in physiological autophagy regulation. This observation is supported by recent work showing that Nedd4.2 is involved in controlling ULK1 levels under stress conditions to limit autophagy [45]. Diverting Nedd4.2 from its physiological role could therefore be a major function of the PVI PPxY motif. Another Nedd4 ligase, Nedd4.1, was recently shown to also regulate autophagy [46]. In addition, Nedd4.1 (but not Nedd4.2) was shown to control cellular Beclin-1 levels, a subunit of the class III phosphatidylinositol 3-kinase complex, which is crucial for the phagophore formation and membrane elongation [47,48]. We observe a stronger association of the M1 virus with PI3P positive membranes coinciding with enrichment of Beclin-1 on cellular membranes, suggesting that the WT selectively alters the function of the class III PI3K complex during entry although we were unable to functionally link this observation to Nedd4.2. Still, this seems to be a common viral strategy as several enveloped viruses target Beclin-1 and the class III PI3K complex to interfere with autophagic processes [49] including autophagosome maturation [50].
One attractive reason for viruses to control autolysosome formation is to limit antigenic presentation via the MHC-II pathway, which can be fed through autophagy [51]. E.g. Herpes simplex virus (HSV-1) employs the viral γ34.5 protein to bind and inactivate Beclin-1 to limit antigenic presentation [52]. AdV down regulates the antigen presenting machinery (MHC-I) During replication via regulatory E3 proteins [53,54]. Currently there is not a known mechanism underlying control of MHC-II presentation once transcription has begun although it might be an important obstacle. Our observations would suggest that using a capsid-encoded determinant, the PPxY motif, allows viral control of MHC-II antigen presentation upon entry preceding viral gene expression, which gives the virus an advantage over its host. In turn it identifies M1 capsids as potential tools for enhanced MHC-II exposure, which maybe of relevance for vaccination or other therapeutic approaches.
Another important observation in our study is the role of autophagy in virus transport towards the nucleus. We show that MTOC targeting for the WT requires dynein motors, Nedd4.2, the PVI PPxY motif and the LC3 conjugation system (ATG5). Removal of either component prevents MTOC accumulation, including a delay in genome delivery presumably because the WT becomes entrapped at the site of membrane rupture similar to the M1. WT and M1 virus do not differ in the kinetics with which they release PVI or acquire membrane damage sensors (Gal3, Gal8) nor does the turnover of membrane damage appear accelerated in either case. An attractive explanation for our observations would be that the LC3 conjugation system aids in motor recruitment towards the entrapped WT virus permitting efficient endosomal escape while Nedd4.2 recruitment via the PPxY motif in PVI interferes with the autophagosome maturation process initiated by the detection of damaged endosomes. A direct role for LC3 in microtubule transport during autophagosomal maturation and trafficking was recently established [21][22][23]. Thus PVI exposure could provide a higher affinity-binding site for Nedd4.2, which might redirect LC3 acquisition by molecular motor or motor scaffolding proteins for autophagic transport and maturation in favor of viral escape. Escape of the virus would not end the autophagosome maturation defect because (some) PVI remains within the ruptured membrane although the exact fate of the ruptured membrane including PVI after viral escape remains unclear at this stage.
Our preliminary assessment using in vivo imaging analysis suggest that WT virus may move from the membrane penetration site to the MTOC in association with LC3. Furthermore the depletion of LC3 conjugation impairs the MTOC accumulation of viral particles. It is thus possible that AdV acquire LC3 for its own transport to the nucleus. While recently autophagosomes where suggested to support AdV endosomal escape by fusing with endosomes [27] future work has to clarify if LC3 plays a more active role during endosomal escape e.g. as part of the motor complex, which extracts the virus from the endosome and/or as part of the complex that drives cytosolic transport of escaped viruses. Such a tentative model as detailed in Fig  11 would accommodate our observation that the WT on one hand limits autolysosome formation upon membrane damage while at the same time depletion of ATG5 prevents efficient endosomal escape and nuclear transport suggesting a reprogramming of autophagy to benefit the virus. It would also be in agreement with the observation that subsequent transport and genome delivery is delayed (but not abolished) in ATG5 depleted cells without affecting the overall accumulated infectivity measured at 24hpi. Delayed nuclear transport could also (in Fig 11. Model for adenovirus control of autophagic processes upon entry. 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).
In summary our work has highlighted how a minimum of genetic conservation through preserving a short capsid encoded peptide motif allows incoming viruses (and presumably other pathogens) to subvert host cell defense mechanisms to profit at multiple layers. In this specific case the accumulated benefits for AdV include limited antigenic presentation paired with accelerated genome delivery showing that autophagy may act as a pro-viral mechanism upon entry of non-enveloped viruses. This is also the first direct demonstration how a viral pathogen overcomes the cellular response to membrane breach. Given the high prevalence of capsid encoded PPxY motifs in other viral systems (including HSV-1) [56][57][58] and a common association of viruses with the MTOC in entry it may be worthwhile to investigate if diverting Nedd4.2 during entry is part of a broader viral strategy for immune evasion and transport, which would make for an excellent drug target.

Autophagy assays
All autophagy related assays were done in accordance to the "guidelines for the use and interpretation of assays for monitoring autophagy" (3 rd Edition) [34] Cell culture and virus production All cells were grown under standard conditions (detailed in SI). ATG5 -/-and control MEFs were a kind gift from R. Duran, Institute Bergonié, Bordeaux, France. Amplification of human recombinant Ad5-VI-WT, Ad5-VI-M1 viruses and their E1-deleted GFP-transgene expressing vector counterparts (including Ad5-ts1-GFP) was done in HEK293 (Human Embryonic Kidney 293 cells, ATCC CRL-1573, kindly provided by G Nemerow, Scrips research institute, La Jolla, USA) cells and purified using double CsCl 2 -banding [10,11]. Virus particle to cell ratios were calculated based on the estimated copy numbers of viral genomes. Copy numbers were calculated according to the OD 260 method (1 OD 260 = 1.16×10 12 particles/ml) [59]. Viruses were labeled by using the Alexa Fluor Microscale labeling kit (LifeTechnologies) as detailed in [26].

Transfection, RNA interference and lentiviral transduction
Plasmid transfections was done in 12-well dishes using 1×10 5 U2OS (Human bone Osteosarcoma Epithelial cells, ATCC HTB-96, kindly provided by M. Piechaczyk, IGMM, Montpellier, France) cells using Lipofectamine 2000 and OptiMEM (LifeTechnologies) according to the manufacturer's instructions. RNAi mediated depletions were performed in 12-well dishes using 5×10 4 U2OS cells. Following optimization cells were transfected after 24 and 48 h with 20 to 50 pmol of each siRNA constructs using Lipofectamine RNAimax and OptiMEM (Life-Technologies). Depletion using shRNA-encoding lentiviruses was done by overnight incubation of 1×10 5 U2OS cells (in 12-well dishes) with lentiviral particles at MOI of 5. The following day lentiviruses were removed and replaced by fresh complete DMEM and transduced cells were selected 24h later by adding puromycin (2μg/ml, Invitrogen). Sequences are listed in the SI. 50μM of Chloroquine for 3h or DMSO and DMF as vehicle control. Cells were transduced with 50 physical particles per cell of either GFP expressing WT or M1 in the presence of drugs followed by medium replacement with drug-free medium after 3h. Cells were analyzed 24h later by flow cytometry for GFP expression. Acquisitions were done on a FACSCantoII cytometer (BD Biosciences) and the data were processed and analyzed by the FACSDIVA software (BD Biosciences). U2OS depleted cells (siGAL3/8/9, siCTRL, shATG5/p62/NDP52 and shCTRL) were transduced following the same procedure.

Western blot and antibodies
For western blot analysis cell lysates were separated on size-resolution adapted SDS-PAGE and transferred to nitrocellulose membranes (cut off of 0.2μm). Extraction of membrane proteins was done using the Mem-PER™ plus membrane protein extraction kit (ThermoFisher). Membranes were blocked in TBS containing 10% of dry-milk and 0.01% of Tween 20 (Sigma) during one hour at room temperature, followed by over-night incubation at 4˚C with primary antibodies and with HRP-conjugated secondary antibodies against rabbit, goat or mouse (Sigma) at a dilution of 1∶10 000 for 1 one hour at room temperature. Specific signals were revealed using the enhanced chemiluminescence detection system (Super signal West femto, Thermoscientific) and signals were acquired using an ImageQuant LAS 4010 system (GE Healthcare life Sciences). Antibodies are listed in the SI.

Time course of infection and immunofluorescence
For immunofluorescence analysis 1×10 5 U2OS were grown on coverslips in a 12-well dish. Coverslips were incubated with 150μl of complete DMEM containing viruses (250 physical particles per cells) during 30 min at 37˚C. Then viruses were removed and replaced by complete DMEM. Coverslips were fixed with 4% PFA at each time point (or in Methanol during 20 minutes at -20˚C, for LC3 staining). Cells were blocked/permeabilized with IF-buffer (10% FCS in PBS and 0.5% Saponin). Primary antibody and secondary antibodies where applied to the coverslip in IF-buffer for 1 h at 37˚C. Cells were mounted in DAKO mounting media containing DAPI and analyzed by confocal microscopy. Antibodies are listed in the SI.

Confocal microscopy, live-cell imaging and image analysis
Confocal images were taken on a Leica SP5 confocal microscope equipped with Leica software and analyzed as detailed in SI. For quantitative image analysis we analyzed for each condition/ time point at least 10 cells with >50 virus particles each (n>500). Live cell imaging was performed as described previously [39]. Briefly, cells were seeded in ibidi μ-slide VI 0.4 (Ibidi), and images were acquired using a Leica spinning-disk microscopy system equipped with an environmental chamber heating the whole optical system to 37˚C. Frames were taken every second (x100 objective) for each color channel and recorded using MetaMorph software and assembled in ImageJ.

Transmission Electron Microscopy (TEM)
U2OS cells were grown to 70% confluency in 35mm glass bottom Grid-500 μ-Dish (Ibidi, Cat. No. 81168) and infected with Alexa594 fluorescently labeled WT or M1 viruses (250 physical particles per cell) at 37˚C for 30 min. At 30 min post infection cells were fixed with 2.5% glutaraldehyde (GA) in PBS overnight at 4˚C. Subsequently, the cells were washed with PBS, postfixed for 30 minutes with 1% OsO4 in PBS, washed with ddH2O, and stained with 1% uranyl acetate in water. The samples were gradually dehydrated with ethanol and embedded in Epon resin (Carl Roth, Germany) for sectioning. Ultrathin 50 nm sections were prepared using Ultracut Microtome (Leica Microsystems, Germany). The sections were poststained with 2% uranyl acetate. Electron micrographs were obtained with a 2K wide angle CCD camera (Veleta, Olympus Soft Imaging Solutions GmbH, Münster, Germany) attached to a FEI Tecnai G 20 Twin transmission electron microscope (FEI, Eindhoven, The Netherlands) at 80kv.
ELIspot and ELISA assays 0.5-10x10 6 monocyte-derived dendritic cells were derived from healthy anonymous donors and transduced with virus or media alone for 2 hours. Cells were incubated with CD4+ Tcell clones that had been cultured for 4 weeks to recognize a conserved AdV hexon epitope at a 3:1 ratio overnight. Supernatants were collected and ELISA was performed according to manufacturer's instructions (eBioscience). For ELISpot assays, B6 mice (The Jackson Laboratory, Barr Harbor, ME) were infected with 10 10 viral particles, or PBS control, intramuscularly. Mice were sacrificed 10 days after infection, and whole spenocytes were collected. Splenocytes were plated into ELIispot plates (Millipore) and stimulated with Ad5-luc capsids or GFP purified from E. coli for 48 hours. ELIspot was performed per manufacturer's instructions (Millipore) and spots were reported as the number of spot forming colonies (SFC)/10^5 PBMCs.
Human monocyte-derived dendritic cells were cultured from peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. Briefly, PBMCs were isolated from fresh whole blood by centrifugation on Histopaque cell separation medium. Monocytes were selected by adherence to culture dishes for 1 h, washed, and cultured in Iscove's modified Eagle's medium plus 10% FBS plus 25 ng/ml recombinant human GM-CSF (PeproTech) for 7 days to obtain differentiated dendritic cells. Human CD4+ T-cells recognizing a conserved epitope in adenovirus hexon were cloned as described previously [60]. Briefly, PBMC were suspended in 200 μl R10 media and incubated with 100 μM peptide for 1 h. Cells were diluted to 2 ml with R10 and incubated in 24-well plate at 37 C in a 5% CO2 atmosphere. After 7 days, human recombinant IL-2 (Becton Dickinson, Bedford, MA, USA) was added to a final concentration of 20 U/ml and then every 3-4 days. T-cell clones (1x10(6) per well) were restimulated with peptide-loaded, irradiated autologous PBMC (3000 rad) or (x10(6) per well) every 7-10 days. CD4+ T-cells were further isolated by negative selection magnetic cell sorting using a commercially available kit (Miltenyi Biotec, cat# 130-096-533).  bly Gal3-mCherry expressing cell were depleted with specific or control siRNA transfection as indicated above each lane. Depletion levels were detected with specific antibodies against mCherry and GAPDH as loading control as shown to the right. Bottom panel: U2OS cells were depleted with specific or control siRNAs as indicated above each lane and detected with Gal8 or Gal9 specific antibodies shown to the right. Tubulin specific antibodies were used as loading control. (B) Left panel: U2OS cells were depleted using lentiviral SH-RNA transduction as indicated above each lane followed by selection as detailed in the material and methods section. Depletion levels were detected by western blot with NDP52 or p62 specific antibodies as shown to the right. GAPDH specific antibodies were used as loading control. Right panel: U2OS cells were control-or NDP52-depleted as indicated using lentiviral SH-RNA transduction followed by si RNA transfection to deplete p62 where indicated. Depletion levels were detected with specific antibodies shown to the right.