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.

We next characterized the release of protein VI from fluorescently labeled Ad-VI-M1 and Ad5-VI-wt following synchronous infections of U2OS cells using the infection assay as described for Figure 1 . Ad5-VI-wt released protein VI within 5 min ( Figure 3A , left panel). In contrast, we observed a delayed release of protein VI from Ad5-VI-M1 (15% of M1 after 5 min compared to 38% of wt, Figure 3A ) and an increase in colocalization of viral particles with protein VI at the nucleus ( Figure 3A ). This observation suggests a delayed accessibility of protein VI within the virus or a defect in protein VI dissociation from the virion ( Figure 3A , images to the right). In addition to the increase of protein VI capsid association, Ad5-VI-M1 appeared to be more evenly distributed throughout the cell and did not efficiently accumulate at the MTOC region ( Figure 3B , images to the left). Quantification revealed that 45 min post-infection approximately 60% of the wt viral particles in proximity of the MTOC could be found within a 10 mm radius around the MTOC and 40% within 10-20 mm. In contrast, the localization for the M1 virus was 50% for each region showing a decreased targeting towards the MTOC ( Figure 3B , right panel). In summary these data suggest that the PPxY motif in protein VI is required for proper uncoating and normal nuclear targeting.