gtag('config', 'UA-114241270-1');
Your browser is out-of-date!

Update your browser to view this website correctly. Update my browser now

×

Discipline
Biological
Keywords
Damaged Early Endosomes But Not Viruses Are Cleared By Endosomophagy
Autophagy
Cell Biology
Observation Type
Standalone
Nature
Standard Data
Submitted
Jun 15th, 2016
Published
Aug 7th, 2016
  • Abstract

    Enveloped viruses fuse with host membranes without affecting cell integrity. Non-enveloped viruses and bacteria penetrate by rupturing endosomal membranes and thus expose complex-type carbohydrates from the endosome lumen to cytosolic proteins. Here we report on the dynamics and initial marker analyses of Galectin-3 (Gal3)-positive membranes triggered by incoming adenovirus species B/C in HeLa cells. Using mCherry-Gal3 reporter constructs, immunolabeling, confocal and electron microscopy, we detected robust signals from Gal3-containing, early endosomal antigen 1-positive membranes 1 h post-infection (pi). Adenoviruses penetrate from non-acidic endosomes with high efficiency, 15 min pi, and largely outnumbered the Gal3-positive membranes, suggesting that Gal3 recruitment to broken membranes is transient, or Gal3-positive membranes are rapidly turned-over. In support of rapid turn-over, Gal3 was found within single-membrane vesicles and degradative autophagosomes. The Gal3 membranes contained ubiquitin and the poly-ubiquitin binding protein p62/sequestosome-1, but only low amounts of virus, or membrane-lytic protein VI exposed from virions. Remarkably, the Gal3-positive membranes were cleared 3 h pi, slower than protein VI, which was cleared 30 min pi. The data show that broken early endosomes, but not virus particles, are rapidly removed by a process involving autophagy, which we term ‘endosomophagy’. We speculate that endosomophagy is pro-viral and attenuates innate immunity.

  • Figure
  • Introduction

    All viruses and many bacteria that enter eukaryotic cells activate a genetic program for replication and immune evasion. Invariably, host defense against the pathogens or host cell damage antagonizes the intruders. Damage to host cells occurs during invasion of bacteria and non-enveloped viruses, including Salmonella, Shigella, Listeria or Adenovirus, which disrupt phagocytic vacuoles and endosomes. Disrupted vacuoles recruit beta-galactoside binding lectins, such as galectin-3 (Gal3) and Gal8, both widely expressed in epithelial cells. Cytosolic galectins can also be subject to export by leader peptide-independent mechanisms. They can bind to enveloped viruses and bacteria, and act as pattern recognition receptors. Intracellular Gal8 coordinates the destruction of disrupted late endosomes and associated bacteria by autophagy, whereas Gal3 is involved in membrane sorting by clustering glycoproteins and glycolipids, and modulation of cell-cell contacts during organogenesis.

    Recently, Gal3 was reported to be recruited to endosomes disrupted by incoming human adenovirus type 5 (HAdV-C5). Adenoviruses are non-enveloped human pathogens and widely used vectors in clinical gene therapy and vaccination. HAdV-C2 and C5 enter epithelial cells by receptor-mediated endocytosis and a stepwise uncoating program initiated at the plasma membrane by the differential movements of two receptors, integrin and coxsackievirus adenovirus receptor (CAR). Virus penetration into the cytosol occurs from early endosomes in a pH-independent manner. It requires the membrane-lytic viral protein VI, lysosomal secretion, the sphingolipid ceramide, and gates the pathway for viral DNA genome separation from the capsid and nuclear delivery of the viral genome. Here, we describe a novel observation, the recruitment of Gal3, ubiquitin and the poly-ubiquitin binding protein p62/SQSTM1 to ruptured early endosomes, followed by clearance of disrupted membranes without clearance of virus particles from the infected cell. p62/SQSTM1 is an adaptor linking poly-ubiquitination to macroautophagy. We speculate that this mode of clearance is pro-viral, in contrast to the recruitment of Gal8 to ruptured phagosomal vacuoles in Salmonella infected cells, which presents a mode of pathogen restriction.

  • Objective

    Ruptured host membranes are danger signals, and enhance innate immunity against infections. The objective here was to analyze the composition and dynamics of ruptured endosomes induced upon virus entry into cells.

  • Results & Discussion

    Most viruses engage in receptor-mediated endocytosis and extensive membrane trafficking using cellular mechanisms. Enveloped viruses encode membrane proteins, which fuse the viral and endosomal membranes, or, in rare cases, the plasma membrane. Non-enveloped viruses are membrane-free, except for certain picornaviruses which occur in both enveloped and non-enveloped forms. They all encode membrane-interacting proteins, which are typically encased in a capsid, and can be activated or exposed by cues from the host cell during entry. Activation of membrane-active proteins leads to pore formation, piercing or rupture of the host membrane. Rupture of internal membranes by HAdV-C5 exposes sugar epitopes to cytosolic proteins, and recruits cytosolic Gal3 to the broken membranes.

    To track broken membranes, we expressed mCherry-Gal3 fusion protein in HeLa cells, followed by continuous infection with HAdV-C5 or HAdV-B3 for 1h. HAdV-C5 enters epithelial cells through CAR and integrin receptors, and HAdV-B3 through CD46 and desmoglein-2 receptors. Both viruses penetrate HeLa cells independent of endosomal acidification with an efficiency higher than 70% (HAdV-C5) or about 40% (HAdV-B3) within 30 min of cold-synchronized infection. Uninfected cells had diffuse cytoplasmic red fluorescence and variable levels of nuclear red fluorescence, in contrast to the prominent cytoplasmic mCherry-Gal3 foci in both types of infected cells, indicating that membrane rupture was independent of the nature of entry receptors (Fig. 1A, and for additional uninfected cells, see panels 1C, F, H). Most of the mCherry-Gal3 foci were devoid of HAdV-C5 particles labeled with Alexa488 fluorophore, as indicated by quantitative image analyses of single confocal slices (Fig. 1B, shown are maximal projections of confocal stacked images). Note that only two of the overlapping puncta co-localized in any of the single confocal slices (indicated by gray arrow heads; single slices not shown). Poor co-localization of HAdV-C2 and mCherry-Gal3 foci was confirmed by a low overlap of mCherry-Gal3 with the incoming membrane-lytic viral protein VI, detected by an affinity-purified polyclonal antibody (Fig. 1C, two arrow heads point to the only co-localization events in these cells by single slice analyses). In contrast, a large fraction of the mCherry-Gal3 foci was robustly positive for the early endosomal antigen 1 (EEA1), indicating that the broken membranes had features of early endosomes (Fig. 1D). These results were in agreement with earlier notions that HAdV escapes quickly from endosomes and is transported by dynein/dynactin mediated processes toward the nucleus. Protein VI on the other hand likely remains membrane associated, and may be sorted separately for degradation.

    Remarkably, visual inspection of the images suggested that the number of HAdV-C5 particles, or protein VI puncta, exceeded the number of mCherry-Gal3 foci (Fig. 1B,C). The same observation was made with HAdV-B3-infected cells (not shown). Although the resolution of virus structures and Gal3-positive membranes was diffraction-limited in our confocal micrographs, the number of mCherry-Gal3 foci correlated with virus dose (data not shown). We also noted that Gal3 foci robustly emerged with 1 h continuous infection schemes, but were rarely visible with shorter infection pulses or at 20 min post-warming after cold-synchronized infections (data not shown). Since virus penetration from endosomes is asynchronous, and may involve different types of endosomes (clathrin-derived endosomes and macropinosomes), but not late endosomes and lysosomes, we tested if Gal3-positive membranes were turned-over. Ultrastructural analyses of Gal3 by immuno-electron microscopy in cryo-sections of normal HeLa cells (not transfected with mCherry-Gal3 constructs) showed that most Gal3 immuno-gold signal was in the cytosol, as expected, although infected cells tended to have less cytosolic Gal3 than uninfected cells (Fig. 1E). Infected cells, however, had more Gal3 within vesicles and autophagosomes than uninfected cells. We tentatively classified the Gal3-positive autophagosomes as degradative autophagosomes, based on their partly visible double bilayer membranes and on the contents comprising electron-dense organelle-like material (Fig. 1E, structures labeled Aph). The structures described here are different from amphisomes, which are formed when an endosome fuses with an autophagosome. Our Gal3-positive autophagosomes appear to contain internal membrane remnants (Fig. 1E) and are cleared with faster kinetics than calcium-phosphate-damaged endosomes, and silica- or LLOMe-damaged lysosomes, which are cleared by amphisomes.

    Further immuno-fluorescence microscopy analyses indicated that the HAdV-C5 induced mCherry-Gal3 foci were strongly positive for the pan-ubiquitin antibody FK2 (Fig. 1F), and a fraction of these foci was positive for the poly-ubiquitin-binding protein p62/SQSTM1 (Fig. 1G). Interestingly, the autophagic marker LC3B was not enriched on mCherry-Gal3 or EEA1 positive membranes of infected cells, but remained diffuse in the cytoplasm (data not shown). This suggested that ubiquitin coordinates LC3B-independent steps for recruiting the autophagic machinery to clear the broken early endosomes. In agreement with this notion, we found that the mCherry-Gal3 foci were readily cleared from the infected cells within 3 h of infection (Fig. 1H). The clearance kinetics of the broken early endosomes was considerably slower than the disappearance of protein VI, which was nearly undetectable in western blots 30 min pi (Fig. 1I).

    In sum, the data are reminiscent of a previous observation that ruptured phagocytic vacuoles in Shigella-infected cells are poly-ubiquitinated, p62/SQSTM1 positive and targeted for autophagic destruction, with concomitant reduction of early inflammatory and cytokine responses. Our data support the earlier finding that the levels of Gal3 and p62/SQSTM1 are reduced in HAdV-B3- or C5-infected A549 human epithelial cells compared to uninfected cells. We suggest the term ‘endosomophagy’ for the clearance of broken early endosomes, and speculate that endosomophagy suppresses danger signals and cell death pathways, and enhances virus infection. This is distinct from lysophagy, which can be induced by chemicals, such as the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe), which oligomerizes, forms toxic products in the lysosomal lumen, leads to lysosomal lysis, and thus triggers necrosis.

  • Conclusions

    The observation here shows that adenovirus-ruptured early endosomal membranes are targeted for clearance. These membranes are Gal3-positive, contain ubiquitin and the polyubiquitin-binding protein p62/SQSTM1, and are associated with degradative autophagosomes. This endosomal clearance is termed here ‘endosomophagy’. It is distinct from lysosomal rupture, which leads to inflammation and necrotic cell death.

  • Conjectures

    Our observation has implications for innate immunity, inflammation, and infectious disease. Notably, in spite of the robust cross-reacting cellular and humoral immune responses against HAdV infections, latent or persistent adenovirus infections in humans occur, and may last for years. The report here also impinges on gene therapy and vaccination protocols, considering the prominent roles of adenoviruses in clinical gene therapy, and as vaccination adjuvants. Follow-up analyses of this observation can be directed to the identification of host and viral targets for ubiquitin ligases and deubiquitinases, host adaptor proteins, or the role of virus-induced sphingolipids in tuning endosomophagy. Notably, sphingolipids have key roles in the regulation of autophagy at the levels of transcription, translation, and morphogenesis of autophagosomes. Another emerging question is how Gal3 and ubiquitin turnover are orchestrated on the ruptured membranes. Further studies can also be directed toward professional antigen-presenting cells, such as macrophages and dendritic cells, which play a major role in inducing and controlling local and global immune responses. Finally, specific experiments can address the question if the clearance of the membrane-lytic viral protein VI is related to or distinct from the mechanism of endosomophagy.

  • Methods

    Cells and viruses

    HeLa cervical carcinoma cells, subline Ohio (from L. Kaiser, University Hospital, Geneva, Switzerland) were grown at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 7.5% fetal calf serum (FCS; Life Technologies) and 1% nonessential amino acids (Sigma). HAdV-C2, HAdV-C5 and HAdV-B3 were grown in human bronchial epithelial A549 cells (American Type Culture Collection), isolated, and labeled with Alexa Fluor 488 (Alexa488; Life Technologies) as previously described.

    Immunofluorescence analysis of Gal3 foci

    The pmCherry-Gal3 construct was generated by PCR amplification of Gal3 coding sequence from U2OS cDNA flanked by HindIII and EcoRI sites and cloned into pmCherry-C1 (Clontech). HeLa-Ohio cells (0.5×106) were transfected with 8 µg of pmCherry-Gal3 using Neon technology (100 µl tip, 1005 V, 35 ms, 2 pulses; Life Technologies). Cells expressing Gal3 with an N-terminal mCherry tag (mCherry-Gal3) were grown in 24 well glass coverslips for 24 h and then were exposed to continuous infection with 1–5 µg/ml virus for the indicated time points. Cells were subsequently fixed in 4% PFA and stained with an anti-EEA1 antibody (mouse, clone 14; Transduction Laboratories), anti-p62/SQSTM1 antibody (mouse, clone 5F2; MBL) or the anti-ubiquitin antibody FK2 (Life Sensors) that detects K29-, K48-, and K63-linked mono- and poly-ubiquitinated proteins.

    Imaging was performed with a Leica SP5 confocal microscope equipped with a 40x objective (oil immersion, numerical aperture 1.25) and a 63x objective (oil immersion, numerical aperture 1.25). Z-stacks composed of 8×0.5 μm steps were acquired at a frequency of 8000 Hz applying bidirectional scan, line averaging 32x and minimized acquisition time. Maximum projections of Z-stacks were analyzed using a customized Matlab routine (Matlab 2009b, available upon request). Fluorescence intensity of either virus labeling or antibody staining on the position of mCherry-Gal3 foci was determined and mean values per cell are shown. To evaluate background values in uninfected cells without mCherry-Gal3 foci, randomly generated and cytosolic foci of pixel size equivalent to mCherry-Gal3 foci localized were quantified. Further details are described in. Statistical analyses were performed using GraphPad Prism software (Version 5, GraphPad Software, Inc. La Jolla). Single cell-based assays are represented as scatter dot plots, where the horizontal bars indicate the mean value and the vertical bars the standard deviation. Two-tailed p-values were calculated by unpaired t-tests with Welch's correction and confidence interval 95%.

    Co-localization of mCherry-Gal3 and protein VI

    pmCherry-Gal3-transfected HeLa-Ohio cells grown on coverslips in 24 well dish were infected with 0.8 µg atto647-labeled HAdV-C2 (kindly provided by I-Hsuan Wang) for 1 h at 37°C. Control cells did not receive any virus. Cells were stained with affinity-purified rabbit anti-protein VI antibodies and secondary AlexaFluor 488-conjugated anti-rabbit antibodies. Samples were imaged with Leica SP5 confocal laser scanning microscope using a 63x objective (oil immersion, numerical aperture 1.4) and zoom factor 4. Stacks were recorded at 0.5 µm intervals using 4x averaging, between frames sequential method and a frequency of 1000 Hz. Shown are maximal projections of Z-stacks, but co-localization of protein VI and mCherry-Gal3 dots were checked also on individual confocal sections.

    Analysis of incoming protein VI

    HeLa cells (1.5×105) were seeded in a 12 well plate the day before the infection. HAdV-C5 (16.8 µg) was added to the cells and allowed to bind and internalize at 37°C for 30 min in RPMI 1640 medium supplemented with 0.2% bovine serum albumin and 20 mM HEPES-NaOH, pH 7.4. Free virus was removed by washing the cells, which were further incubated at 37°C for the indicated times in DMEM medium supplemented with 10% fetal calf serum, and thereafter lysed in lysis buffer (0.2 ml of 200 mM Tris, pH 8.8, 20% glycerol, 5 mM EDTA, 50 mM DTT, 5% SDS, 0.02% bromophenol blue). Lysates were boiled at 95°C for 5 min and centrifuged at 16,000x g for 5 min. Proteins were resolved by SDS-PAGE and hexon and protein VI were detected by western blotting using anti-hexon (Abcam, ab6982) and anti-protein VI antibodies, and anti-β-tubulin (Amersham) as a loading control.

    Electron microscopy

    Cryo-ultramicrotomy and immunocytochemistry was performed based on the protocol of Tokuyasu. The procedure was similar to one described earlier. About 3×106 subconfluent cells were fixed in PBS containing 2% pFA and 0.2% glutaraldehyde for 1 h, scraped off the dish, pelleted at about 500x g for 10 min, washed several times in PBS, embedded in a small volume of 10% gelatine (Sigma, G6650) in PBS at 37°C, pelleted and solidified in a thin-walled Eppendorf tube on ice o/n. The cells embedded in gelatine were removed by slicing the tube wall and then were infiltrated with 2.3 M sucrose in PBS at 4°C for 2 days. The gelatine cell block was mounted onto a metal plate, snap frozen in liquid nitrogen, and placed into a Leica EM Ultra Cut UC6 / FC6 machine. After trimming to about 0.25 mm2 surface area, ultrathin 80 nm thick cryo-sections were obtained with a diamond knife at -120°C. Frozen sections were collected onto a drop of cold sucrose on a wire loop, brought to room temperature, and transferred from the loop onto a Formvar-coated Ni-EM grid (125 µm mesh size). Grids were washed several times in PBS at room temperature, and at 40°C for 10 min to remove the gelatine. Aldehydes were blocked by 3 short incubations in PBS containing 0.15% glycine, pH 7.5, and 2 washes in PBS. Samples were blocked in PBG buffer consisting of PBS, 0.2% gelatine, 0.5% BSA (AppliChem, A6588, 0050) and 0.01% Tween20 (Thermo Scientific, 28320) for 10 min, and then with the primary rabbit immunoglobulin (IgG) anti-Gal3 antibody (PeproTech) at 1:20 in PBG for 1 h, room temperature, washed 4 times for 3 min in PBG, blocked again for 3 min, incubated with the secondary goat anti-rabbit IgG conjugated to 10 nm gold (BBI Solutions) at 1:50 in PBG, and washed several times in PBG, PBS, and H2O. Samples were fixed in 0.5% glutaraldehyde in H2O for 20 min, washed 5 times in H2O, and stained in 1.8% methylcellulose, 0.3% uranyl acetate on ice for 5 min. Excess liquid was blotted off, the sample dried on ice for several minutes, and analyzed in a Philips CM100 (100 kV at 46000 magnification using a digital CCD camera Gatan Orius 1000, 4kx2.6k pixels) or a ZEISS TEM10 (80 kV, 50000 magnification, using digital CCD camera Gatan Erlangshen ES500W, Model 782).

  • Funding statement

    The work was supported by a research grant from the Swiss National Science Foundation (310030B_160316), and a Medical Research and Development project from the Swiss initiative for systems biology SystemsX.ch (2014/264 Project VirX, evaluated by the Swiss National Science Foundation) to UFG, and DFG grant Me1626/5-1 to HM.

  • Acknowledgements

    The authors would like to thank Gery Barmettler, Center for Microscopy and Image Analysis of the University of Zurich, for advice and support in immunocytochemistry experiments.

  • Ethics statement

    Not applicable.

  • References
  • 1
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    2
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    3
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    4
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    5
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    Matters14.5/20

    Endosomophagy clears disrupted early endosomes but not virus particles during virus entry into cells

    Affiliation listing not available.
    Abstractlink

    Enveloped viruses fuse with host membranes without affecting cell integrity. Non-enveloped viruses and bacteria penetrate by rupturing endosomal membranes and thus expose complex-type carbohydrates from the endosome lumen to cytosolic proteins. Here we report on the dynamics and initial marker analyses of Galectin-3 (Gal3)-positive membranes triggered by incoming adenovirus species B/C in HeLa cells. Using mCherry-Gal3 reporter constructs, immunolabeling, confocal and electron microscopy, we detected robust signals from Gal3-containing, early endosomal antigen 1-positive membranes 1 h post-infection (pi). Adenoviruses penetrate from non-acidic endosomes with high efficiency, 15 min pi, and largely outnumbered the Gal3-positive membranes, suggesting that Gal3 recruitment to broken membranes is transient, or Gal3-positive membranes are rapidly turned-over. In support of rapid turn-over, Gal3 was found within single-membrane vesicles and degradative autophagosomes. The Gal3 membranes contained ubiquitin and the poly-ubiquitin binding protein p62/sequestosome-1, but only low amounts of virus, or membrane-lytic protein VI exposed from virions. Remarkably, the Gal3-positive membranes were cleared 3 h pi, slower than protein VI, which was cleared 30 min pi. The data show that broken early endosomes, but not virus particles, are rapidly removed by a process involving autophagy, which we term ‘endosomophagy’. We speculate that endosomophagy is pro-viral and attenuates innate immunity.

    Figurelink

    Figure 1. The nature of adenovirus-ruptured Gal3-positive endosomes.

    (A) Infection of HeLa cells with HAdV-B3 or HAdV-C5 for 1 h induces the formation of cytoplasmic Gal3 foci. Results show single optical sections from confocal laser scanning fluorescence microscopy.

    (B) Gal3 foci are low in HAdV-C5 particles. HeLa cells expressing mCherry-Gal3 were infected with HAdV-C5-Alexa488 at 37°C for 1 h, fixed and analyzed for virus co-localization with cytoplasmic mCherry-Gal3 foci using maximal projections of confocal fluorescence microscopy slices from the entire cell. Note that in order to detect good mCherry-Gal3 foci, dozens of virus particles were required per cell. The number and intensity of the Gal3 foci was dependent on the expression levels of mCherry-Gal3 in the cell and was correlated with variable amounts of red fluorescence in the nucleus, presumably from cytosolic mCherry-Gal3. For image quantification, an average size of the Gal3 foci was defined by a computational mask and was used to determine the signal intensity of HAdV-C5-Alexa488 in the Gal3 foci (Gal3, left column of the dot plot). To estimate background co-localization signals, HAdV-C5-Alexa488 fluorescence was determined in randomly placed masks across the cytoplasm (Rand, right column). Each dot represents the signal from one mask. Number of cells and Gal3 foci analyzed are indicated. Mean values with standard deviation (SD) are shown in red. Note that only a small amount of Gal3 foci was positive for virus.

    (C) mCherry-Gal3 foci are low in protein VI from incoming HAdV-C2. Arrows in this representative cell denote co-localization events of mCherry-Gal3 and protein VI observed in single section confocal slices (not shown). Shown here are maximal projections of the signals to give an overall impression.

    (D) Gal3 foci are positive for early endosome antigen 1 (EEA1). Results were analyzed as described in panels A and B from Gal3 foci and cells as indicated. Results show a significant enrichment of EEA1 on mCherry-Gal3 foci. Mean values with SD are shown in red.

    (E) HAdV-C5 entry enhances the vesicular and autophagosomal levels of Gal3. Cells were infected with HAdV-C5 at 37°C for 1 h, fixed and processed for cryo-immunolabeling as described. Ultrathin cryo-sections were stained with rabbit anti-Gal3 antibodies followed by protein A conjugated to 10 nm colloidal gold, and analysis by electron microscopy in a Philips CM100 (100 kV at 46000 magnification). Gold particles were manually counted in vesicles (Ves), autophagosomes (Aph) and the cytosol. Arrows indicate gold in vesicles or autophagosomes; black arrow heads, cytosolic gold; and white arrow heads, endosomal HAdV-C5 particles. Autophagosomes were identified as structures containing electron-dense material and a closely apposed double membrane at least partially visible in the sections, as suggested in the guidelines from the autophagy community[1]. Mean number of gold particles per structure, and p-values (Student’s t-test) were derived from indicated number of vesicles, autophagosomes, and gold particle. Gold in the cytosol was analyzed in the same images as for vesicle and autophagosome analyses. Note the trend to reduced cytosolic Gal3 pools in HAdV-C5 infected cells. Note that 13 from 22 autophagosomes of infected cells contained Gal3, compared to 1 from 10 Gal3 positive autophagosomes of uninfected cells, indicating a strong enhancement of Gal3 positive autophagomes upon HAdV-C5 infection.

    (F) mCherry-Gal3 foci contain epitopes for the pan-ubiquitin antibody FK2. Images were acquired and analyzed as described above. Mean values with SD are shown in red.

    (G) A fraction of the Gal3 foci induced by HAdV-C5 is positive for the poly-ubiquitin-binding protein p62/sequestosome 1 (p62/SQSTM1). Analyses indicate that about 10% of the Gal3 foci contain the poly-ubiquitin-binding protein p62/SQSTM1 (yellow ellipse), while the rest is indistinguishable from the random control, indicated by means and SD values.

    (H) HAdV-C5 induced mCherry-Gal3 foci are rapidly cleared from infected cells. Cells were infected at 37°C for 1 h, washed free of virus, and analyzed directly (1 h time point), or after 2 h incubation at 37°C in fresh medium (3 h time point). The percentage of cells showing mCherry-Gal3 foci was plotted. One dot represents the fraction of positive cells per image. Shown are the mean values with SD, and the total number of cells analyzed per sample with p-values from unpaired t-tests.

    (I) The membrane-lytic protein VI from incoming HAdV-C5 is rapidly cleared from infected cells. HAdV-C5 particles were allowed to bind and internalize into HeLa cells at 37°C for 30 min, after which unbound virus was removed and cells were further incubated at 37°C for the indicated times. Amounts of protein VI and the major capsid protein hexon in cell extracts were determined by SDS-PAGE and Western blotting using β-tubulin as a loading control. Densitometric analyses of protein bands normalized to tubulin are shown as OD values relative to the 0 min time point.

    Raw data and statistical analyses can be found in the supplementary excel sheet.

    Introductionlink

    All viruses and many bacteria that enter eukaryotic cells activate a genetic program for replication and immune evasion[2][3][4]. Invariably, host defense against the pathogens or host cell damage antagonizes the intruders[5][6][7]. Damage to host cells occurs during invasion of bacteria and non-enveloped viruses, including Salmonella, Shigella, Listeria or Adenovirus, which disrupt phagocytic vacuoles and endosomes[8][9]. Disrupted vacuoles recruit beta-galactoside binding lectins, such as galectin-3 (Gal3) and Gal8, both widely expressed in epithelial cells[10][11][12][13]. Cytosolic galectins can also be subject to export by leader peptide-independent mechanisms[14][15][16]. They can bind to enveloped viruses and bacteria, and act as pattern recognition receptors[17]. Intracellular Gal8 coordinates the destruction of disrupted late endosomes and associated bacteria by autophagy, whereas Gal3 is involved in membrane sorting by clustering glycoproteins and glycolipids, and modulation of cell-cell contacts during organogenesis[18][19][20].

    Recently, Gal3 was reported to be recruited to endosomes disrupted by incoming human adenovirus type 5 (HAdV-C5)[13][21]. Adenoviruses are non-enveloped human pathogens and widely used vectors in clinical gene therapy and vaccination[22][23][24][25]. HAdV-C2 and C5 enter epithelial cells by receptor-mediated endocytosis and a stepwise uncoating program initiated at the plasma membrane by the differential movements of two receptors, integrin and coxsackievirus adenovirus receptor (CAR)[26][27][28]. Virus penetration into the cytosol occurs from early endosomes in a pH-independent manner[29][30]. It requires the membrane-lytic viral protein VI[31][32][33], lysosomal secretion, the sphingolipid ceramide[34][35], and gates the pathway for viral DNA genome separation from the capsid and nuclear delivery of the viral genome[36][37]. Here, we describe a novel observation, the recruitment of Gal3, ubiquitin and the poly-ubiquitin binding protein p62/SQSTM1 to ruptured early endosomes, followed by clearance of disrupted membranes without clearance of virus particles from the infected cell. p62/SQSTM1 is an adaptor linking poly-ubiquitination to macroautophagy[38][39]. We speculate that this mode of clearance is pro-viral, in contrast to the recruitment of Gal8 to ruptured phagosomal vacuoles in Salmonella infected cells, which presents a mode of pathogen restriction[12].

    Objectivelink

    Ruptured host membranes are danger signals, and enhance innate immunity against infections. The objective here was to analyze the composition and dynamics of ruptured endosomes induced upon virus entry into cells.

    Results & Discussionlink

    Most viruses engage in receptor-mediated endocytosis and extensive membrane trafficking using cellular mechanisms[40][41]. Enveloped viruses encode membrane proteins, which fuse the viral and endosomal membranes, or, in rare cases, the plasma membrane. Non-enveloped viruses are membrane-free, except for certain picornaviruses which occur in both enveloped and non-enveloped forms[42]. They all encode membrane-interacting proteins, which are typically encased in a capsid, and can be activated or exposed by cues from the host cell during entry[35][43]. Activation of membrane-active proteins leads to pore formation, piercing or rupture of the host membrane[44][45][46]. Rupture of internal membranes by HAdV-C5 exposes sugar epitopes to cytosolic proteins, and recruits cytosolic Gal3 to the broken membranes[13].

    To track broken membranes, we expressed mCherry-Gal3 fusion protein in HeLa cells, followed by continuous infection with HAdV-C5 or HAdV-B3 for 1h. HAdV-C5 enters epithelial cells through CAR and integrin receptors, and HAdV-B3 through CD46 and desmoglein-2 receptors[3][47][48][49]. Both viruses penetrate HeLa cells independent of endosomal acidification with an efficiency higher than 70% (HAdV-C5) or about 40% (HAdV-B3) within 30 min of cold-synchronized infection[30]. Uninfected cells had diffuse cytoplasmic red fluorescence and variable levels of nuclear red fluorescence, in contrast to the prominent cytoplasmic mCherry-Gal3 foci in both types of infected cells, indicating that membrane rupture was independent of the nature of entry receptors (Fig. 1A, and for additional uninfected cells, see panels 1C, F, H). Most of the mCherry-Gal3 foci were devoid of HAdV-C5 particles labeled with Alexa488 fluorophore, as indicated by quantitative image analyses of single confocal slices (Fig. 1B, shown are maximal projections of confocal stacked images). Note that only two of the overlapping puncta co-localized in any of the single confocal slices (indicated by gray arrow heads; single slices not shown). Poor co-localization of HAdV-C2 and mCherry-Gal3 foci was confirmed by a low overlap of mCherry-Gal3 with the incoming membrane-lytic viral protein VI, detected by an affinity-purified polyclonal antibody (Fig. 1C, two arrow heads point to the only co-localization events in these cells by single slice analyses). In contrast, a large fraction of the mCherry-Gal3 foci was robustly positive for the early endosomal antigen 1 (EEA1), indicating that the broken membranes had features of early endosomes (Fig. 1D). These results were in agreement with earlier notions that HAdV escapes quickly from endosomes and is transported by dynein/dynactin mediated processes toward the nucleus[50][51][52]. Protein VI on the other hand likely remains membrane associated, and may be sorted separately for degradation[26][53].

    Remarkably, visual inspection of the images suggested that the number of HAdV-C5 particles, or protein VI puncta, exceeded the number of mCherry-Gal3 foci (Fig. 1B,C). The same observation was made with HAdV-B3-infected cells (not shown). Although the resolution of virus structures and Gal3-positive membranes was diffraction-limited in our confocal micrographs, the number of mCherry-Gal3 foci correlated with virus dose (data not shown). We also noted that Gal3 foci robustly emerged with 1 h continuous infection schemes, but were rarely visible with shorter infection pulses or at 20 min post-warming after cold-synchronized infections (data not shown). Since virus penetration from endosomes is asynchronous, and may involve different types of endosomes (clathrin-derived endosomes and macropinosomes), but not late endosomes and lysosomes[29], we tested if Gal3-positive membranes were turned-over. Ultrastructural analyses of Gal3 by immuno-electron microscopy in cryo-sections of normal HeLa cells (not transfected with mCherry-Gal3 constructs) showed that most Gal3 immuno-gold signal was in the cytosol, as expected, although infected cells tended to have less cytosolic Gal3 than uninfected cells (Fig. 1E). Infected cells, however, had more Gal3 within vesicles and autophagosomes than uninfected cells. We tentatively classified the Gal3-positive autophagosomes as degradative autophagosomes, based on their partly visible double bilayer membranes and on the contents comprising electron-dense organelle-like material[1] (Fig. 1E, structures labeled Aph). The structures described here are different from amphisomes, which are formed when an endosome fuses with an autophagosome[1]. Our Gal3-positive autophagosomes appear to contain internal membrane remnants (Fig. 1E) and are cleared with faster kinetics than calcium-phosphate-damaged endosomes, and silica- or LLOMe-damaged lysosomes, which are cleared by amphisomes[54][55].

    Further immuno-fluorescence microscopy analyses indicated that the HAdV-C5 induced mCherry-Gal3 foci were strongly positive for the pan-ubiquitin antibody FK2 (Fig. 1F), and a fraction of these foci was positive for the poly-ubiquitin-binding protein p62/SQSTM1 (Fig. 1G). Interestingly, the autophagic marker LC3B was not enriched on mCherry-Gal3 or EEA1 positive membranes of infected cells, but remained diffuse in the cytoplasm (data not shown). This suggested that ubiquitin coordinates LC3B-independent steps for recruiting the autophagic machinery to clear the broken early endosomes. In agreement with this notion, we found that the mCherry-Gal3 foci were readily cleared from the infected cells within 3 h of infection (Fig. 1H). The clearance kinetics of the broken early endosomes was considerably slower than the disappearance of protein VI, which was nearly undetectable in western blots 30 min pi (Fig. 1I).

    In sum, the data are reminiscent of a previous observation that ruptured phagocytic vacuoles in Shigella-infected cells are poly-ubiquitinated, p62/SQSTM1 positive and targeted for autophagic destruction, with concomitant reduction of early inflammatory and cytokine responses[10]. Our data support the earlier finding that the levels of Gal3 and p62/SQSTM1 are reduced in HAdV-B3- or C5-infected A549 human epithelial cells compared to uninfected cells[56]. We suggest the term ‘endosomophagy’ for the clearance of broken early endosomes, and speculate that endosomophagy suppresses danger signals and cell death pathways, and enhances virus infection. This is distinct from lysophagy, which can be induced by chemicals, such as the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe), which oligomerizes, forms toxic products in the lysosomal lumen, leads to lysosomal lysis, and thus triggers necrosis[57][58][59].

    Conclusionslink

    The observation here shows that adenovirus-ruptured early endosomal membranes are targeted for clearance. These membranes are Gal3-positive, contain ubiquitin and the polyubiquitin-binding protein p62/SQSTM1, and are associated with degradative autophagosomes. This endosomal clearance is termed here ‘endosomophagy’. It is distinct from lysosomal rupture, which leads to inflammation and necrotic cell death.

    Conjectureslink

    Our observation has implications for innate immunity, inflammation, and infectious disease. Notably, in spite of the robust cross-reacting cellular and humoral immune responses against HAdV infections, latent or persistent adenovirus infections in humans occur, and may last for years[60]. The report here also impinges on gene therapy and vaccination protocols, considering the prominent roles of adenoviruses in clinical gene therapy, and as vaccination adjuvants[61]. Follow-up analyses of this observation can be directed to the identification of host and viral targets for ubiquitin ligases and deubiquitinases, host adaptor proteins, or the role of virus-induced sphingolipids in tuning endosomophagy[34]. Notably, sphingolipids have key roles in the regulation of autophagy at the levels of transcription, translation, and morphogenesis of autophagosomes[62]. Another emerging question is how Gal3 and ubiquitin turnover are orchestrated on the ruptured membranes. Further studies can also be directed toward professional antigen-presenting cells, such as macrophages and dendritic cells, which play a major role in inducing and controlling local and global immune responses[63]. Finally, specific experiments can address the question if the clearance of the membrane-lytic viral protein VI is related to or distinct from the mechanism of endosomophagy.

    Methodslink

    Cells and viruses

    HeLa cervical carcinoma cells, subline Ohio (from L. Kaiser, University Hospital, Geneva, Switzerland) were grown at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 7.5% fetal calf serum (FCS; Life Technologies) and 1% nonessential amino acids (Sigma). HAdV-C2, HAdV-C5 and HAdV-B3 were grown in human bronchial epithelial A549 cells (American Type Culture Collection), isolated, and labeled with Alexa Fluor 488 (Alexa488; Life Technologies) as previously described[30].

    Immunofluorescence analysis of Gal3 foci

    The pmCherry-Gal3 construct was generated by PCR amplification of Gal3 coding sequence from U2OS cDNA flanked by HindIII and EcoRI sites and cloned into pmCherry-C1 (Clontech). HeLa-Ohio cells (0.5×106) were transfected with 8 µg of pmCherry-Gal3 using Neon technology (100 µl tip, 1005 V, 35 ms, 2 pulses; Life Technologies). Cells expressing Gal3 with an N-terminal mCherry tag (mCherry-Gal3) were grown in 24 well glass coverslips for 24 h and then were exposed to continuous infection with 1–5 µg/ml virus for the indicated time points. Cells were subsequently fixed in 4% PFA and stained with an anti-EEA1 antibody (mouse, clone 14; Transduction Laboratories), anti-p62/SQSTM1 antibody (mouse, clone 5F2; MBL) or the anti-ubiquitin antibody FK2 (Life Sensors) that detects K29-, K48-, and K63-linked mono- and poly-ubiquitinated proteins.

    Imaging was performed with a Leica SP5 confocal microscope equipped with a 40x objective (oil immersion, numerical aperture 1.25) and a 63x objective (oil immersion, numerical aperture 1.25). Z-stacks composed of 8×0.5 μm steps were acquired at a frequency of 8000 Hz applying bidirectional scan, line averaging 32x and minimized acquisition time. Maximum projections of Z-stacks were analyzed using a customized Matlab routine (Matlab 2009b, available upon request). Fluorescence intensity of either virus labeling or antibody staining on the position of mCherry-Gal3 foci was determined and mean values per cell are shown. To evaluate background values in uninfected cells without mCherry-Gal3 foci, randomly generated and cytosolic foci of pixel size equivalent to mCherry-Gal3 foci localized were quantified. Further details are described in[30][26]. Statistical analyses were performed using GraphPad Prism software (Version 5, GraphPad Software, Inc. La Jolla). Single cell-based assays are represented as scatter dot plots, where the horizontal bars indicate the mean value and the vertical bars the standard deviation. Two-tailed p-values were calculated by unpaired t-tests with Welch's correction and confidence interval 95%.

    Co-localization of mCherry-Gal3 and protein VI

    pmCherry-Gal3-transfected HeLa-Ohio cells grown on coverslips in 24 well dish were infected with 0.8 µg atto647-labeled HAdV-C2 (kindly provided by I-Hsuan Wang) for 1 h at 37°C. Control cells did not receive any virus. Cells were stained with affinity-purified rabbit anti-protein VI antibodies[26] and secondary AlexaFluor 488-conjugated anti-rabbit antibodies. Samples were imaged with Leica SP5 confocal laser scanning microscope using a 63x objective (oil immersion, numerical aperture 1.4) and zoom factor 4. Stacks were recorded at 0.5 µm intervals using 4x averaging, between frames sequential method and a frequency of 1000 Hz. Shown are maximal projections of Z-stacks, but co-localization of protein VI and mCherry-Gal3 dots were checked also on individual confocal sections.

    Analysis of incoming protein VI

    HeLa cells (1.5×105) were seeded in a 12 well plate the day before the infection. HAdV-C5 (16.8 µg) was added to the cells and allowed to bind and internalize at 37°C for 30 min in RPMI 1640 medium supplemented with 0.2% bovine serum albumin and 20 mM HEPES-NaOH, pH 7.4. Free virus was removed by washing the cells, which were further incubated at 37°C for the indicated times in DMEM medium supplemented with 10% fetal calf serum, and thereafter lysed in lysis buffer (0.2 ml of 200 mM Tris, pH 8.8, 20% glycerol, 5 mM EDTA, 50 mM DTT, 5% SDS, 0.02% bromophenol blue). Lysates were boiled at 95°C for 5 min and centrifuged at 16,000x g for 5 min. Proteins were resolved by SDS-PAGE and hexon and protein VI were detected by western blotting using anti-hexon (Abcam, ab6982) and anti-protein VI antibodies[26], and anti-β-tubulin (Amersham) as a loading control.

    Electron microscopy

    Cryo-ultramicrotomy and immunocytochemistry was performed based on the protocol of Tokuyasu[64][65][66]. The procedure was similar to one described earlier[26]. About 3×106 subconfluent cells were fixed in PBS containing 2% pFA and 0.2% glutaraldehyde for 1 h, scraped off the dish, pelleted at about 500x g for 10 min, washed several times in PBS, embedded in a small volume of 10% gelatine (Sigma, G6650) in PBS at 37°C, pelleted and solidified in a thin-walled Eppendorf tube on ice o/n. The cells embedded in gelatine were removed by slicing the tube wall and then were infiltrated with 2.3 M sucrose in PBS at 4°C for 2 days. The gelatine cell block was mounted onto a metal plate, snap frozen in liquid nitrogen, and placed into a Leica EM Ultra Cut UC6 / FC6 machine. After trimming to about 0.25 mm2 surface area, ultrathin 80 nm thick cryo-sections were obtained with a diamond knife at -120°C. Frozen sections were collected onto a drop of cold sucrose on a wire loop, brought to room temperature, and transferred from the loop onto a Formvar-coated Ni-EM grid (125 µm mesh size). Grids were washed several times in PBS at room temperature, and at 40°C for 10 min to remove the gelatine. Aldehydes were blocked by 3 short incubations in PBS containing 0.15% glycine, pH 7.5, and 2 washes in PBS. Samples were blocked in PBG buffer consisting of PBS, 0.2% gelatine, 0.5% BSA (AppliChem, A6588, 0050) and 0.01% Tween20 (Thermo Scientific, 28320) for 10 min, and then with the primary rabbit immunoglobulin (IgG) anti-Gal3 antibody (PeproTech) at 1:20 in PBG for 1 h, room temperature, washed 4 times for 3 min in PBG, blocked again for 3 min, incubated with the secondary goat anti-rabbit IgG conjugated to 10 nm gold (BBI Solutions) at 1:50 in PBG, and washed several times in PBG, PBS, and H2O. Samples were fixed in 0.5% glutaraldehyde in H2O for 20 min, washed 5 times in H2O, and stained in 1.8% methylcellulose, 0.3% uranyl acetate on ice for 5 min. Excess liquid was blotted off, the sample dried on ice for several minutes, and analyzed in a Philips CM100 (100 kV at 46000 magnification using a digital CCD camera Gatan Orius 1000, 4kx2.6k pixels) or a ZEISS TEM10 (80 kV, 50000 magnification, using digital CCD camera Gatan Erlangshen ES500W, Model 782).

    Funding Statementlink

    The work was supported by a research grant from the Swiss National Science Foundation (310030B_160316), and a Medical Research and Development project from the Swiss initiative for systems biology SystemsX.ch (2014/264 Project VirX, evaluated by the Swiss National Science Foundation) to UFG, and DFG grant Me1626/5-1 to HM.

    Acknowledgementslink

    The authors would like to thank Gery Barmettler, Center for Microscopy and Image Analysis of the University of Zurich, for advice and support in immunocytochemistry experiments.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    Not applicable.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
    1. Daniel J Klionsky, Kotb Abdelmohsen, Akihisa Abe, 2465 More Truncated Authors.
      Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
    2. B. Brett Finlay, Grant McFadden,
      Anti-Immunology: Evasion of the Host Immune System by Bacterial and Viral Pathogens
    3. Nina Wolfrum, Urs F. Greber,
      Adenovirus signalling in entry
      Cellular Microbiology, 15/2012, pages 53-62 DOI: 10.1111/cmi.12053chrome_reader_mode
    4. Pascale Cossart, Ari Helenius
      Endocytosis of Viruses and Bacteria
      Cold Spring Harbor Perspectives in Biology, 6/2014, pages a016972-a016972 DOI: 10.1101/cshperspect.a016972chrome_reader_mode
    5. Christopher M. Robinson, Julie K. Pfeiffer,
      Viruses and the Microbiota
      Annual Review of Virology, 1/2014, pages 55-69 DOI: 10.1146/annurev-virology-031413-085550chrome_reader_mode
    6. Hans-Heinrich Hoffmann, William M. Schneider, Charles M. Rice,
      Interferons and viruses: an evolutionary arms race of molecular interactions
      Trends in Immunology, 36/2015, pages 124-138 DOI: 10.1016/j.it.2015.01.004chrome_reader_mode
    7. Rodinde Hendrickx, Nicole Stichling, Jorien Koelen, Lukasz Kuryk, Agnieszka Lipiec, Urs F. Greber,
      Innate Immunity to Adenovirus
      Human Gene Therapy, 25/2014, pages 265-284 DOI: 10.1089/hum.2014.001chrome_reader_mode
    8. O. Meier, M. Gastaldelli, K. Boucke, S. Hemmi, U. F. Greber,
      Early Steps of Clathrin-Mediated Endocytosis Involved in Phagosomal Escape of Fc  Receptor-Targeted Adenovirus
      Journal of Virology, 79/2005, pages 2604-2613 DOI: 10.1128/jvi.79.4.2604-2613.2005chrome_reader_mode
    9. Ju Huang, John H. Brumell,
      Bacteria–autophagy interplay: a battle for survival
      Nature Reviews Microbiology, 12/2014, pages 101-114 DOI: 10.1038/nrmicro3160chrome_reader_mode
    10. Nicolas Dupont, Sandra Lacas-Gervais, Julie Bertout, Irit Paz, Barbara Freche, Guy Tran van Nhieu, F. Gisou van der Goot, Philippe J. Sansonetti, Frank Lafont,
      Shigella Phagocytic Vacuolar Membrane Remnants Participate in the Cellular Response to Pathogen Invasion and Are Regulated by Autophagy
      Cell Host & Microbe, 6/2009, pages 137-149 DOI: 10.1016/j.chom.2009.07.005chrome_reader_mode
    11. Irit Paz, Martin Sachse, Nicolas Dupont, Joelle Mounier, Cecilia Cederfur, Jost Enninga, Hakon Leffler, Francoise Poirier, Marie-Christine Prevost, Frank Lafont, Philippe Sansonetti,
      Galectin-3, a marker for vacuole lysis by invasive pathogens
      Cellular Microbiology, 12/2010, pages 530-544 DOI: 10.1111/j.1462-5822.2009.01415.xchrome_reader_mode
    12. Teresa L. M. Thurston, Michal P. Wandel, Natalia von Muhlinen, Ágnes Foeglein, Felix Randow,
      Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion
      Nature, 482/2012, pages 414-418 DOI: 10.1038/nature10744chrome_reader_mode
    13. O. Maier, S. A. Marvin, H. Wodrich, E. M. Campbell, C. M. Wiethoff,
      Spatiotemporal Dynamics of Adenovirus Membrane Rupture and Endosomal Escape
      Journal of Virology, 86/2012, pages 10821-10828 DOI: 10.1128/jvi.01428-12chrome_reader_mode
    14. Delphine Delacour, Valérie Gouyer, Jean-Pierre Zanetta, Hervé Drobecq, Emmanuelle Leteurtre, Georges Grard, Odile Moreau-Hannedouche, Emmanuel Maes, Alexandre Pons, Sabine André, André Le Bivic, Hans Joachim Gabius, Aki Manninen, Kai Simons, Guillemette Huet,
      Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells
      The Journal of Cell Biology, 169/2005, pages 491-501 DOI: 10.1083/jcb.200407073chrome_reader_mode
    15. Gabriel A. Rabinovich, Marta A. Toscano,
      Turning 'sweet' on immunity: galectin–glycan interactions in immune tolerance and inflammation
      Nature Reviews Immunology, 9/2009, pages 338-352 DOI: 10.1038/nri2536chrome_reader_mode
    16. Walter Nickel, Catherine Rabouille,
      Mechanisms of regulated unconventional protein secretion
      Nature Reviews Molecular Cell Biology, 10/2008, pages 148-155 DOI: 10.1038/nrm2617chrome_reader_mode
    17. Gerardo R. Vasta,
      Roles of galectins in infection
      Nature Reviews Microbiology, 7/2009, pages 424-438 DOI: 10.1038/nrmicro2146chrome_reader_mode
    18. Delphine Delacour, Annett Koch, Ralf Jacob,
      The Role of Galectins in Protein Trafficking
    19. I. R. Nabi, J. Shankar, J. W. Dennis,
      The galectin lattice at a glance
      Journal of Cell Science, 128/2015, pages 2213-2219 DOI: 10.1242/jcs.151159chrome_reader_mode
    20. Ludger Johannes, Robert G. Parton, Patricia Bassereau, Satyajit Mayor,
      Building endocytic pits without clathrin
      Nature Reviews Molecular Cell Biology, 16/2015, pages 311-321 DOI: 10.1038/nrm3968chrome_reader_mode
    21. Ruben Martinez, Andrew M. Burrage, Christopher M. Wiethoff, Harald Wodrich,
      High Temporal Resolution Imaging Reveals Endosomal Membrane Penetration and Escape of Adenoviruses in Real Time
      Virus-Host Interactions, Methods in Molecular Biology (Methods and Protocols), 1064/2013, pages 211-226 DOI: 10.1007/978-1-62703-601-6_15chrome_reader_mode
    22. V Ann Chailertvanitkul, Colin W Pouton,
      Adenovirus: a blueprint for non-viral gene delivery
      Current Opinion in Biotechnology, 21/2010, pages 627-632 DOI: 10.1016/j.copbio.2010.06.011chrome_reader_mode
    23. Dragomira Majhen, Hugo Calderon, Naresh Chandra, Carlos Alberto Fajardo, Anandi Rajan, Ramon Alemany, Jerome Custers,
      Adenovirus-Based Vaccines for Fighting Infectious Diseases and Cancer: Progress in the Field
      Human Gene Therapy, 25/2014, pages 301-317 DOI: 10.1089/hum.2013.235chrome_reader_mode
    24. Walter Mangel, Carmen San Martín,
      Structure, Function and Dynamics in Adenovirus Maturation
      Viruses, 6/2014, pages 4536-4570 DOI: 10.3390/v6114536chrome_reader_mode
    25. Ronald G. Crystal,
      Adenovirus: The First Effective In Vivo Gene Delivery Vector
      Human Gene Therapy, 25/2014, pages 3-11 DOI: 10.1089/hum.2013.2527chrome_reader_mode
    26. Christoph J. Burckhardt, Maarit Suomalainen, Philipp Schoenenberger, Karin Boucke, Silvio Hemmi, Urs F. Greber,
      Drifting Motions of the Adenovirus Receptor CAR and Immobile Integrins Initiate Virus Uncoating and Membrane Lytic Protein Exposure
      Cell Host & Microbe, 10/2011, pages 105-117 DOI: 10.1016/j.chom.2011.07.006chrome_reader_mode
    27. Christopher M. Wiethoff, Glen R. Nemerow,
      Adenovirus membrane penetration: Tickling the tail of a sleeping dragon
      Virology, 479-480/2015, pages 591-599 DOI: 10.1016/j.virol.2015.03.006chrome_reader_mode
    28. Urs F. Greber,
      Virus and Host Mechanics Support Membrane Penetration and Cell Entry
      Journal of Virology, 90/2016, pages 3802-3805 DOI: 10.1128/jvi.02568-15chrome_reader_mode
    29. Michele Gastaldelli, Nicola Imelli, Karin Boucke, Beat Amstutz, Oliver Meier, Urs F. Greber,
      Infectious Adenovirus Type 2 Transport Through Early but not Late Endosomes
    30. M. Suomalainen, S. Luisoni, K. Boucke, S. Bianchi, D. A. Engel, U. F. Greber,
      A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells
      Journal of Virology, 87/2013, pages 12367-12379 DOI: 10.1128/jvi.01833-13chrome_reader_mode
    31. C. M. Wiethoff, H. Wodrich, L. Gerace, G. R. Nemerow,
      Adenovirus Protein VI Mediates Membrane Disruption following Capsid Disassembly
      Journal of Virology, 79/2005, pages 1992-2000 DOI: 10.1128/jvi.79.4.1992-2000.2005chrome_reader_mode
    32. C. L. Moyer, C. M. Wiethoff, O. Maier, J. G. Smith, G. R. Nemerow,
      Functional Genetic and Biophysical Analyses of Membrane Disruption by Human Adenovirus
      Journal of Virology, 85/2011, pages 2631-2641 DOI: 10.1128/jvi.02321-10chrome_reader_mode
    33. Crystal L. Moyer, Glen R. Nemerow,
      Disulfide-bond formation by a single cysteine mutation in adenovirus protein VI impairs capsid release and membrane lysis
    34. Stefania Luisoni, Maarit Suomalainen, Karin Boucke, Lukas B. Tanner, Markus R. Wenk, Xue Li Guan, Michał Grzybek, Ünal Coskun, Urs F. Greber,
      Co-option of Membrane Wounding Enables Virus Penetration into Cells
      Cell Host & Microbe, 18/2015, pages 75-85 DOI: 10.1016/j.chom.2015.06.006chrome_reader_mode
    35. Yohei Yamauchi, Urs F. Greber,
      Principles of Virus Uncoating: Cues and the Snooker Ball
      Traffic, 17/2016, pages 569-592 DOI: 10.1111/tra.12387chrome_reader_mode
    36. I-Hsuan Wang, Maarit Suomalainen, Vardan Andriasyan, Samuel Kilcher, Jason Mercer, Anne Neef, Nathan W. Luedtke, Urs F. Greber,
      Tracking Viral Genomes in Host Cells at Single-Molecule Resolution
      Cell Host & Microbe, 14/2013, pages 468-480 DOI: 10.1016/j.chom.2013.09.004chrome_reader_mode
    37. Yohei Yamauchi, Ari Helenius
      Virus entry at a glance
      Journal of Cell Science, 126/2013, pages 1289-1295 DOI: 10.1242/jcs.119685chrome_reader_mode
    38. M. L. Seibenhener, J. R. Babu, T. Geetha, H. C. Wong, N. R. Krishna, M. W. Wooten,
      Sequestosome 1/p62 Is a Polyubiquitin Chain Binding Protein Involved in Ubiquitin Proteasome Degradation
      Molecular and Cellular Biology, 24/2004, pages 8055-8068 DOI: 10.1128/mcb.24.18.8055-8068.2004chrome_reader_mode
    39. Junyan Shi, Jerry Wong, Paulina Piesik, Gabriel Fung, Jingchun Zhang, Julienne Jagdeo, Xiaotao Li, Eric Jan, Honglin Luo,
      Cleavage of sequestosome 1/p62 by an enteroviral protease results in disrupted selective autophagy and impaired NFKB signaling
      Autophagy, 9/2013, pages 1591-1603 DOI: 10.4161/auto.26059chrome_reader_mode
    40. Jason Mercer, Mario Schelhaas, Ari Helenius,
      Virus Entry by Endocytosis
      Annual Review of Biochemistry, 79/2010, pages 803-833 DOI: 10.1146/annurev-biochem-060208-104626chrome_reader_mode
    41. S. Sigismund, S. Confalonieri, A. Ciliberto, S. Polo, G. Scita, P. P. Di Fiore,
      Endocytosis and Signaling: Cell Logistics Shape the Eukaryotic Cell Plan
      Physiological Reviews, 92/2012, pages 273-366 DOI: 10.1152/physrev.00005.2011chrome_reader_mode
    42. Zongdi Feng, Lucinda Hensley, Kevin L. McKnight, Fengyu Hu, Victoria Madden, Lifang Ping, Sook-Hyang Jeong, Christopher Walker, Robert E. Lanford, Stanley M. Lemon,
      A pathogenic picornavirus acquires an envelope by hijacking cellular membranes
      Nature, 496/2013, pages 367-371 DOI: 10.1038/nature12029chrome_reader_mode
    43. Maarit Suomalainen, Urs F Greber,
      Uncoating of non-enveloped viruses
      Current Opinion in Virology, 3/2013, pages 27-33 DOI: 10.1016/j.coviro.2012.12.004chrome_reader_mode
    44. Christopher Browning, Mikhail M. Shneider, Valorie D. Bowman, David Schwarzer, Petr G. Leiman,
      Phage Pierces the Host Cell Membrane with the Iron-Loaded Spike
      Structure, 20/2012, pages 326-339 DOI: 10.1016/j.str.2011.12.009chrome_reader_mode
    45. Aliaa H. Abdelhakim, Eric N. Salgado, Xiaofeng Fu, Mithun Pasham, Daniela Nicastro, Tomas Kirchhausen, Stephen C. Harrison,
      Structural Correlates of Rotavirus Cell Entry
      PLOS Pathogens, 10/2014, page e1004355 DOI: 10.1371/journal.ppat.1004355chrome_reader_mode
    46. Dieter Blaas, Renate Fuchs,
      Mechanism of human rhinovirus infections
      Molecular and Cellular Pediatrics, 3/2016, page 21 DOI: 10.1186/s40348-016-0049-3chrome_reader_mode
    47. G.R. Nemerow, L. Pache, V. Reddy, P.L. Stewart,
      Insights into adenovirus host cell interactions from structural studies
      Virology, 384/2009, pages 380-388 DOI: 10.1016/j.virol.2008.10.016chrome_reader_mode
    48. H. V. Trinh, G. Lesage, V. Chennamparampil, B. Vollenweider, C. J. Burckhardt, S. Schauer, M. Havenga, U. F. Greber, S. Hemmi,
      Avidity Binding of Human Adenovirus Serotypes 3 and 7 to the Membrane Cofactor CD46 Triggers Infection
      Journal of Virology, 86/2011, pages 1623-1637 DOI: 10.1128/jvi.06181-11chrome_reader_mode
    49. Hongjie Wang, Zong-Yi Li, Ying Liu, Jonas Persson, Ines Beyer, Thomas Möller, Dilara Koyuncu, Max R Drescher, Robert Strauss, Xiao-Bing Zhang, James K Wahl, Nicole Urban, Charles Drescher, Akseli Hemminki, Pascal Fender, André Lieber,
      Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14
      Nature Medicine, 17/2010, pages 96-104 DOI: 10.1038/nm.2270chrome_reader_mode
    50. S. A. Kelkar, K. K. Pfister, R. G. Crystal, P. L. Leopold,
      Cytoplasmic Dynein Mediates Adenovirus Binding to Microtubules
      Journal of Virology, 78/2004, pages 10122-10132 DOI: 10.1128/jvi.78.18.10122-10132.2004chrome_reader_mode
    51. K. Helen Bremner, Julian Scherer, Julie Yi, Michael Vershinin, Steven P. Gross, Richard B. Vallee,
      Adenovirus Transport via Direct Interaction of Cytoplasmic Dynein with the Viral Capsid Hexon Subunit
      Cell Host & Microbe, 6/2009, pages 523-535 DOI: 10.1016/j.chom.2009.11.006chrome_reader_mode
    52. Martin F. Engelke, Christoph J. Burckhardt, Matthias K. Morf, Urs F. Greber
      The Dynactin Complex Enhances the Speed of Microtubule-Dependent Motions of Adenovirus Both Towards and Away from the Nucleus
      Viruses, 3/2011, pages 233-253 DOI: 10.3390/v3030233chrome_reader_mode
    53. Harald Wodrich, Daniel Henaff, Baptist Jammart, Carolina Segura-Morales, Sigrid Seelmeir, Olivier Coux, Zsolt Ruzsics, Christopher M. Wiethoff, Eric J. Kremer,
      A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry
      PLOS Pathogens, 6/2010, page e1000808 DOI: 10.1371/journal.ppat.1000808chrome_reader_mode
    54. S. Lepine, J. C. Allegood, Y. Edmonds, S. Milstien, S. Spiegel
      Autophagy Induced by Deficiency of Sphingosine-1-phosphate Phosphohydrolase 1 Is Switched to Apoptosis by Calpain-mediated Autophagy-related Gene 5 (Atg5) Cleavage
      Journal of Biological Chemistry, 286/2011, pages 44380-44390 DOI: 10.1074/jbc.m111.257519chrome_reader_mode
    55. Ikuko Maejima, Atsushi Takahashi, Hiroko Omori, Tomonori Kimura, Yoshitsugu Takabatake, Tatsuya Saitoh, Akitsugu Yamamoto, Maho Hamasaki, Takeshi Noda, Yoshitaka Isaka, Tamotsu Yoshimori
      Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury
      The EMBO Journal, 32/2013, pages 2336-2347 DOI: 10.1038/emboj.2013.171chrome_reader_mode
    56. Hung V. Trinh, Jonas Grossmann, Peter Gehrig, Bernd Roschitzki, Ralph Schlapbach, Urs F. Greber, Silvio Hemmi,
      iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections
      International Journal of Proteomics, 2013/2013, pages 1-16 DOI: 10.1155/2013/581862chrome_reader_mode
    57. Ikuko Maejima, Atsushi Takahashi, Hiroko Omori, Tomonori Kimura, Yoshitsugu Takabatake, Tatsuya Saitoh, Akitsugu Yamamoto, Maho Hamasaki, Takeshi Noda, Yoshitaka Isaka, Tamotsu Yoshimori,
      Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury
      The EMBO Journal, 32/2013, pages 2336-2347 DOI: 10.1038/emboj.2013.171chrome_reader_mode
    58. Jürgen Brojatsch, Heriberto Lima, Alak K. Kar, Lee S. Jacobson, Stefan M. Muehlbauer, Kartik Chandran, Felipe Diaz-Griffero,
      A Proteolytic Cascade Controls Lysosome Rupture and Necrotic Cell Death Mediated by Lysosome-Destabilizing Adjuvants
    59. Heriberto Lima Jr., Lee Jacobson, Michael Goldberg, Kartik Chandran, Felipe Diaz-Griffero, Michael P. Lisanti, Jürgen Brojatsch,
      Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death
      Cell Cycle, 12/2013, pages 1868-1878 DOI: 10.4161/cc.24903chrome_reader_mode
    60. Thomas Lion
      Adenovirus Infections in Immunocompetent and Immunocompromised Patients
      Clinical Microbiology Reviews, 27/2014, pages 441-462 DOI: 10.1128/cmr.00116-13chrome_reader_mode
    61. Katie J. Ewer, Kailan Sierra-Davidson, Ahmed M. Salman, Joseph J. Illingworth, Simon J. Draper, Sumi Biswas, Adrian V.S. Hill,
      Progress with viral vectored malaria vaccines: A multi-stage approach involving “unnatural immunity”
    62. Wenhui Jiang, Besim Ogretmen,
      Autophagy paradox and ceramide
      Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1841/2014, pages 783-792 DOI: 10.1016/j.bbalip.2013.09.005chrome_reader_mode
    63. F. Geissmann, M. G. Manz, S. Jung, M. H. Sieweke, M. Merad, K. Ley,
      Development of Monocytes, Macrophages, and Dendritic Cells
      Science, 327/2010, pages 656-661 DOI: 10.1126/science.1178331chrome_reader_mode
    64. Griffiths, Gareth
      Fine structure immunocytochemistry
      Springer Verlag, Heidelberg, Germany, 1993, ISBN-13: 978-3642770975, 1st edn/1993, pages 1-484 chrome_reader_mode
    65. K. T. Tokuyasu
      Immuno-cytochemistry on ultrathin cryosections
      Cells: A Laboratory Manual: Subcellular Localization of Genes and Their Products , 3/1997, pages 131.1 - 131.27 chrome_reader_mode
    66. K. T. Tokuyasu
      A technique for ultracryotomy of cell suspensions and tissues
      The Journal of Cell Biology, 57/1973, pages 551-565 chrome_reader_mode
    Commentslink

    Create a Matters account to leave a comment.