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 delineate the fate of protein VI during Ad entry we performed infection assays and followed the intracellular distribution of the viral capsid and protein VI as a function of time. To this end, fluorescently labeled Ad particles were adsorbed to either human retina epithelia pigment cells (hTERT-RPE1, Figure 1 ) or human osteosarcoma cells (U2OS, Figure S1 ) at 4uC and then transferred to 37uC to synchronize internalization. Cells were fixed at various times and analyzed by confocal microscopy (Figure 1 ). Protein VI was detected using an affinity purified polyclonalantiserum and the location of the MTOC was marked by detecting the primary cilia, which originates at the MTOC using antibodies against acetylated tubulin [32] . At 4uC viral particles accumulated at the cell periphery showing sporadic positive staining with the protein VI antiserum (,1%) possibly due to the recognition of protein VI from damaged particles (Figure 1 first row). In contrast, 5 min after the temperature shift, Ad particles were still localized close to the cell periphery but approximately 40% of them gave a signal with the protein VI antibody indicating that more protein VI was accessible ( Figure 1 , second row). After 15 min, particles had entered the cell with some localized at the MTOC region ( Figure 1 , third row) and some at the nuclear rim as described previously [6] . About 10% of the particles remained protein VI positive, including particles at the nuclear rim. After 45 min the majority of the particles were concentrated at the MTOC region ( Figure 1 , bottom row) as previously reported [3] . Protein VI staining was also concentrated at the MTOC region but most of the signal was not particle associated. Similar results were obtained in U2OS cells ( Figure S1 ). Together these data suggested that structural rearrangements leading to protein VI exposure take place at or close to the cell surface during Ad entry. In addition, the data showed that protein VI trafficked to the MTOC region and partially remained associated with the capsid.