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.

To address the role of the PPxY motif in protein VI ubiquitylation, we repeated the in vitro ubiquitylation assay using wt or M1 mutant protein VI purified from E. coli followed by western blot analysis. We detected protein VI-reactive bands, consistent with protein VI modified with two to three ubiquitin ( Figure 6B , lane 2). In contrast, no modification was observed when the PPxY motif was mutated ( Figure 6B , lane 1) or in the absence of ATP ( Figure 6B, lane 3 and 4) . Using viral particles in in vitro ubiquitylation reactions (following partial capsid disassembly) protein VI of Ad5-VI-M1 was not ubiquitylated ( Figure 6C , Figure 5 . Subcellular dynamics of protein VI. A) U2OS cells were transfected with VI-wt (top row) or VI-M1 (bottom row) fused to mRFP and analyzed by live-cell imaging. Frames were taken at 1 sec intervals using a Cascade 512B 2 camera. The left image in all rows shows maximum image projections of the full frame depicting the cell with scale bar of 10 mm. The four inverted images to the right show magnifications from the boxed inset of four consecutive frames (in the case of VI-wt) and every fifths frame (in the case of VI-M1). In the top panel a moving particle is depicted by a grey arrow within each frame using the departure point depicted by a white arrow as reference. The lower panel shows VI-M1 not moving. B) Analysis of trajectory length and relative particle speed of VI-wt or VI-M1 (left side of both panels) or trajectory length and relative particle speed of VI-wt using drugs as indicated (right side of both panels). Movies were processed and all recorded trajectories were analyzed for length and relative speed using the Imaris TM software package. Individual particles were plotted for length of trajectories (in mm, left panel) and the net movement (in mm/ 20sec, right panel) under the conditions tested (indicated below each chart). Significant differences are indicated by bars. doi:10.1371/journal.ppat.1000808.g005 lane 3 and 4), while protein VI from Ad5-VI-wt was ( Figure 6C , lane 1 and 2). Thus, the PPxY motif in protein VI is inaccessible in intact capsids but can recruit ubiquitin ligase activity from cytosol when protein VI is released from the capsid interior. Together our results show that protein VI ubiquitylation depends on i) virus disassembly, ii) an intact PPxY domain and iii) the presence of a cytosolic ubiquitylation activity.