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.
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).