Author: Wodrich, Harald; Henaff, Daniel; Jammart, Baptist; Segura-Morales, Carolina; Seelmeir, Sigrid; Coux, Olivier; Ruzsics, Zsolt; Wiethoff, Christopher M.; Kremer, Eric J.
Title: A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry Document date: 2010_3_19
ID: 1mjmttec_34
Snippet: Because Nedd4.2 showed the strongest effect on Ad transduction we determined whether bacterially expressed and purified Nedd4.2 could ubiquitylate purified protein VI in vitro. A minimal system where cytosol was replaced by recombinant Nedd4.2 was sufficient for protein VI ubiquitylation ( Figure 8B, lane 1) . The ubiquitylation pattern was similar to that obtained with cytosol anti-protein VI antibodies is shown with ubiquitylated proteins marke.....
Document: Because Nedd4.2 showed the strongest effect on Ad transduction we determined whether bacterially expressed and purified Nedd4.2 could ubiquitylate purified protein VI in vitro. A minimal system where cytosol was replaced by recombinant Nedd4.2 was sufficient for protein VI ubiquitylation ( Figure 8B, lane 1) . The ubiquitylation pattern was similar to that obtained with cytosol anti-protein VI antibodies is shown with ubiquitylated proteins marked with grey arrows (VI-Ubi) and unmodified protein VI (VI) is marked with a black arrow. C) Reactions as in A) followed by detection of protein VI by western blot using Ad5-VI-wt (lane 1 and 2) and Ad5-VI-M1 capsids (lane 3 and 4). Disassembly of capsids is indicated above each lane and protein VI reactive bands are marked as in B to the right. doi:10.1371/journal.ppat.1000808.g006 ( Figure 6 ) and required an intact PPxY and a catalytically active Nedd4.2 ( Figure 8B, lane 2 and 3) .
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