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.

Nedd4.1 and Nedd4.2 both showed strong interaction with protein VI in pulldown assays. To further investigate the role of each ligase we transduced cells with lentiviral vector expressing shRNAs against either Nedd4.1 or Nedd4.2 or luciferase as a control. We transduced cells in a dose-dependent manner to achieve different levels of knockdown. Transduction efficiency was monitored using a GFP expressing lentiviral vector in control cells. Seven days post-transduction shRNA treated cells were infected with AdGFP virus as described above and the transduction rate was determined by flow cytometry. We observed a dose-dependent decrease in infectivity for two different shRNAs against Nedd4.2, which was similar to what we observed when we used siRNAs ( Figure 8C ). The results for shRNAs against Nedd4.1 were less clear. One shRNA also reduced viral infection at very high transduction rates but to a lesser extent than shRNAs against Nedd4.2 while a second shRNA showed no effect on Ad transduction ( Figure 8C) . A combined treatment of cells with either siRNAs or shRNAs against Nedd4.1 and Nedd4.2 did not further decrease Ad-transduction indicating that the effects of Nedd4-ligase knockdowns on Ad transduction may be complex (data not shown).