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.

d cytosol renders Nedd4.2 active for penton ubiquitylation. In vitro ubiquitylation reactions using catalytically active or inactive Nedd4.2 substituted with cytosol depleted by protein VI-wt (as indicated above each lane) and analyzed by western blot with antipenton antiserum. Black arrows indicate ubiquitylated proteins, grey arrows the unmodified protein. This assay shows that additional cytosolic factors are required for full Nedd4.2 activity for penton ubiquitylation. Found at: doi:10.1371/journal.ppat.1000808.s005 (0.73 MB TIF) Figure S6 Protein VI with altered PPxY motif does not colocalize with Nedd4 ligases. Protein VI with PPSY motif mutated to PGAA was N-terminally fused to mRFP and cotransfected with GFP-Nedd4.1 (top row), GFP-Nedd4.2 (second row), GFP-AIP4/Itch (third row), GFP-WWP1 (forth row) and GFP-WWP2 (bottom row). Confocal images of representative cells are shown and the mRFP signal for VI (left column), the GFP signal for the ligases (centre column) and the merged signals together with DAPI stain of the nucleus (right column) is indicated above each column. Transfected plasmids are indicated left of each row. Note that the cytoplasmic localization of each ligase is similar to that in cells without cotransfection of protein VI (data not shown). Found at: doi:10.1371/journal.ppat.1000808.s006 (3.90 MB TIF)