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.

Flp expression by a temperature shift to 43uC. This treatment induced a recombination between the two FRT sites (one in the pAd5-FRT derivative and one on the pO6-A5-gfp) and induced unification of the BAC and the pO6 construct. Only the recombined construct survived the double selection because pO6 constructs are not maintained in DH10B. This approach essentially replaced the old left end of the Ad5 BAC by a CMV promoter driven GFP-expression cassette containing fragment, which also possessed all the cis elements needed for virus reconstitution as described above. Found at: doi:10.1371/journal.ppat.1000808.s003 (0.45 MB TIF) Figure S4 The PPxY-mutant Ad5GFP-VI-M1 forms smaller and fewer plaques. A) Shown is a comparison of the growth of individual plaques starting from a single infected cell. E1 complementing 911 cells were infected at low multiplicity of infection with Ad5GFP-VI-wt and the PPxY mutant virus Ad5GFP-VI-M1 for 24 h and then washed and overlayed with agarose. Virus growth was monitored by the appearance of GFPpositive cells and images of representative cells/plaques were taken on days 1, 3, 9 and 12. The images in the left row show the plaque formation of the wild type virus 1, 3, 9 and 12 days after the initial infection (top-to-bottom). At day 9 and 12 significant large plaques with lesions of the cell monolayer can be observed. In contrast the mutant virus to the right shows a slow expansion of GFP-positive plaques with less damage to the cell monolayer. Images are superimpositions of the GFP signal and the phase contrast image of the monolayer. B) The image shows the damage in the cell monolayer caused by plaque formation on day 12. The arrow indicates the average size of the plaque. The scale bar is 50 mm. Found at: doi:10.1371/journal.ppat.1000808.s004 (2.12 MB TIF) Figure S5 Penton is a target for ubiquitylation following partial disassembly of the virus. A) Localization and sequences of conserved PPxY-motifs in protein VI and penton for Ad5 (black box). Note that processed protein VI is shown as it is present in the capsid during viral entry. B) Schematic representation of the in vitro ubiquitylation assay. Virus disassembly was induced by mild heattreatment, in vitro ubiquitylated using ubiquitin or recombinant GST-ubiquitin, recombinant E1 and E2, an energy regenerating system and purified cytosol as source for the E3-ligase and analyzed by western blot. Controls lack the mild heat-treatment. C) Western blot analysis of viral capsid proteins penton, fiber and protein IIIA following capsid disassembly and in vitro ubiquitylation. Heat-treatment is indicated above each lane. Antibodies are indicated to the right of each blot. Specific bands are labeled accordingly. Grey arrows indicate band shifts due to ubiquitylation, black arrows indicates the unmodified protein. D) Western blot analysis of in vitro ubiquitylation reactions of heat-treated viral particles. Heat treatment is indicated above each lane. For individual reactions the cytosol was depleted with recombinant fiber beads (control), recombinant VI-wt beads or recombinant VI-M1 beads as indicated above each lane. Reactions were blotted with anti-penton. The assay shows that the ubiquitylation activity can be depleted from cytosol with recombinant wt protein VI but not when the PPSY motif is mutated. The same assay also abolishes protein VI ubiquitylation showing that similar ubiquitylation activities are responsible (data not shown). E) Protein VI deplete