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.

Next we asked whether Ad5 endosomal lysis efficiency following internalization is affected by the mutation in the PPxY motif of protein VI. We infected A549 cells with 30, 300 or 3000 particles per cell of either Ad5-VI-wt or Ad5-VI-M1 in the presence of alpha-sarcin, a membrane impermeable toxin that inhibits translation when it enters the cytoplasm. Alpha-sarcin enters the cell by virus-mediated endosomal membrane lysis thus providing a quantifiable marker for endsome membrane lysis ( Figure 2G , [28] ). We measured the incorporation of radiolabeled amino acids over time as a means to determine translational efficiency. As control we used the temperature sensitive Ad mutant Ad2ts1 that is closely related to Ad5. Ad2ts1 mutants poorly lyse the endosomal membrane when the virus is grown at non-permissive temperatures due to an increased capsid stability that lacks intraendosomal disassembly. Ad2ts1 mutants grown at permissive temperatures lyse the endosome with the same efficiency as the wt virus. Incorporation of radiolabeled amino acids was compared to alpha-sarcin treated cells. When we added alpha-Sarcin and Ad5-VI-wt, Ad5-VI-M1 or the Ad2ts1 mutant grown at a permissive temperature translation was diminished in a dosedependent manner over at least two orders of magnitude, showing efficient cytoplasmic delivery of the toxin. Ad2ts1 grown at the non-permissive temperature did not inhibit translation in this assay ( Figure 2G ). We observed no difference between the PPxY-mutant virus and the wt controls. This indicated that an attenuating effect of the PPxY-mutation occurs after endosomal lysis and prior to the onset of replication.