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 show that protein VI is engaged in rapid intracellular trafficking that depends on intact microtubules and requires the Nterminal amphipathic helix for microtubule association and the PPxY motif for motion. To our knowledge protein VI is the first Ad capsid protein described that possesses its own microtubuledependent dynamics and future work has to address if other capsid proteins have similar properties. Inactivation of the PPxY in the viral context (Ad5-VI-M1) resulted in a post-entry delay that reduced infectivity and prevented efficient accumulation of the entering virus particles at the MTOC. While we cannot exclude the possibility that the PPxY motif in protein VI also plays a role in adenoviral replication, assembly or egress, our single round infection assays showed that the majority of the titer reduction for the mutant was related to steps prior to the initiation of replication or the delivery and expression of a reporter gene. Moreover, our data show that the efficiency with which a toxin is delivered to the cytosol during Ad infection is similar for mutant and wt virus. This provides strong evidence that the PPSY motif in protein VI has a function during cell entry, but probably only after endosomal membrane lysis has occurred. Current structural data place protein VI inside the assembled capsid, therefore potentially precluding it from functions during egress at least when capsid associated [43] [44] [45] . Our observations show that protein VI is exposed after entry and that capsid disassembly is required for its ubiquitylation, which is consistent with our hypothesis that the PPxY motif is accessible only during Ad entry following partial capsid disassembly. Furthermore, it is currently not clear whether late domains containing PPxY motifs present in other viral systems, which are required for viral egress, have additional functions. Mutational inactivation of PPxY motifs in the VP40 structural protein of Ebola virus and matrix protein of rabies virus both showed an attenuation of the virus and a reduction of infectivity [20, 46] . Interestingly, for Ebola virus, the PPxY mutants showed no budding defect, but virus production was reduced, which could indicate a disruption earlier on in the life cycle than previously thought [46] . For rabies virus, Wirblich et al. describe a Figure 7 . Protein VI interacts with Nedd4 ligases via the PPxY motif. A) Protein VI (VI-wt) was N-terminally fused to mRFP and co-transfected with GFP-Nedd4.1 (first row), GFP-Nedd4.2 (second row), GFP-AIP4/Itch (third row), GFP-WWP1 (fourth row) and GFP-WWP2 (last row). Confocal images of representative cells are shown and the mRFP signal for protein VI (left column), the GFP signal for the ligases (center column) and the merged signals together with DAPI stain of the nucleus (right column) is indicated above each column. Transfected plasmids are indicated. Colocalization of Nedd4 and protein VI results in a yellow signal. The scale bar is 10mm. B) Diverse GFP-tagged Nedd4 ligases were over-expressed in cells and cytosolic extracts were used for pulldown experiments using recombinant protein VI-wt or VI-M1 coupled to beads. 10% of the input material (IP) is shown in the first lane. Bound material for protein VI-wt (lane 2) and VI-M1 (lane 3) was detected with respective antibodies as indicated to the right. Co-eluted protein VI detected with a protein VI specific antibody is shown as loading control in the lower lane. doi:10.1371/journal.ppat.10