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 identify the ligase responsible for protein VI ubiquitylation, we focused on the Nedd4-family members Nedd4.1, Nedd4.2, AIP4/Itch, WWP1 and WWP2 because they can interact with viral late domains that harbor PPxY motifs [38] . We first coexpressed the VI-wt or VI-M1 mRFP fusion protein together with each of the E3 ligases fused to GFP in U2OS cells. When expressed alone, most ligases localized primarily to the cytoplasm (data not shown, WWP1 localized to the plasma membrane and WWP2 accumulated in an uncharacterized intracellular membrane compartment). In contrast, when VI-wt is coexpressed with Nedd4.1, Nedd4.2 or AIP4/Itch, the ligases are recruited to the same membrane compartment as protein VI ( Figure 7A, row 1-3 ). WWP1 appears to sequester protein VI at the plasma membrane ( Figure 7A, row 4) . WWP2 does not colocalize with VI-wt ( Figure 7A , row 5). We did not detect significant colocalization between VI-M1 and the E3 ligases, consistent with a PPxYdependent interaction ( Figure S6 ).