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.

This assay dissociates the vertices including fiber, penton, protein VI and peripentonal hexons, but leaves the remainder of the capsid intact ( Figure S5 ; [28] ). Heat and mock-treated samples were subjected to in vitro ubiquitylation reactions, using free ubiquitin, recombinant E1 and E2 enzymes and purified cytosol as source for the E3 ubiquitin ligase(s), and analyzed by western blot (Figure 6 , [28] ). Western blot analysis showed that partial capsid disassembly resulted in the appearance of protein VI reactive signals with discrete size increments suggesting predominant modification with two to three ubiquitin as well as some higher molecular weight bands (lane 2, Figure 6A ). In contrast, the lack of capsid disassembly (lane 1) or cytosol (lane 3) showed no additional protein VI reactive bands. We also tested ubiquitylation of the capsid proteins fiber, protein IIIA and penton base as internal control. We only detected ubiquitylation of the penton base (which also harbors two PPxY domains at its N-terminus), while the fiber and protein IIIA (which lack PPxY motifs) were not modified ( Figure S5 ). The ubiquitylation of protein VI was also confirmed by using GST-ubiquitin in the above assay, followed by GSTpulldown to show covalent modification of protein VI with ubiquitin confirming the predominant modification with two to three ubiquitin-moieties (data not shown).