A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry

Wodrich, Harald; Henaff, Daniel; Jammart, Baptist; Segura-Morales, Carolina; Seelmeir, Sigrid; Coux, Olivier; Ruzsics, Zsolt; Wiethoff, Christopher M.; Kremer, Eric J.

Confocal pictures were taken on a Zeiss LSM 510 Meta confocal microscope or a Leica SP5 confocal microscope and epifluorescence pictures were taken on a Zeiss Axiolmager Z1 microscope with CoolSnap HQ Photometrics camera both equipped with Metamorph TM software. Confocal stacks where taken every 0.5 mm with a pinhole setting of 1 for all channels to achieve high local resolution. Images were processed using ImageJ and Adobe Photoshop TM . Counting of viral particles was performed using the semi-automated cell counting tool from ImageJ. Colocalization analysis: Stacks from confocal images where combined as Z-projection using maximum intensity, converted into 8-bit images for each channel. Colocalization between protein VI and Ad was then determined using the colocalization finder plugin from ImageJ. Live-Cell Imaging: U2OS cells were seeded in 3.5cm glass-bottom dishes and transfected with 1.5 mg of protein VI-expressing vectors. Twenty-four h later the medium was replaced by pre-warmed OPTI-MEM (Gibco). Movies were acquired on a Nikon TE 2000 microscope with Cascade 512B 2 camera using Metamorph TM software for data acquisition (120 frames, 1 frame/sec). In some cases, 2 h before the acquisition, cells were incubated with either Nocodazole (Sigma) or Cytochalasin B (Sigma) diluted respectively at 0.4 mg/ml and 5 mg/ml in OPTI-MEM. Drugs were not removed during acquisition. Acquired movies were further processed using ImageJ to enhance protein VI particle detection by background subtraction and bleach correction. Particles were tracked using the spot-tracking tool of Imaris TM software to determine the length of their trajectories and the speed of their movement. Particle detection size was scaled to 0.75 mm and tracks were built with a maximum displacement of 1.5 mm between consecutive frames, a maximum gap size of 3 frames and a minimal track length of 20 s. At least 5 cells were analyzed for each condition that equals a minimum of 300 analyzed tracks per condition.