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.

Data are presented as mean, error bars as STD. Statistical analysis if not indicated otherwise was done using unpaired students t-test (*:P,0.05; **:P,0.01; ***:P,0.005). Figure S1 Protein VI release in U2OS cells during Ad entry. Ad5-VI-wt-488 was pre-bound to cells at 4uC (top row) and shifted to 37uC for 5min (second row), 15min (third row) and 45min (bottom row). Protein VI was detected using affinity purified antiprotein VI antibodies (left column) and Ad by detecting the Alexa-488 fluorescent signal (middle column). A composite of both signals including the nucleus (in greyscale) is shown in the left column. The inset shows a magnification of representative virus and protein VI signals in the small white box. Protein VI signals are shown in red, Ad is shown in green and colocalization of protein VI and Ad is shown in yellow. The scale bar is 10 mm. The rabbit polyclonal serum against protein VI was generated against recombinant purified His-tagged protein VI. Rabbit serum that reacted positive and specific against protein VI in western blots of purified viruses was used for further affinity purification for use in immunofluorescence applications. Affinity purification was done using recombinant purified protein VI coupled to CnBr + -activated sepharose beads. Bound antibodies were eluted with 0.1 M glycin ph2, neutralized with 2M Tris pH 8.8 and dialyzed against PBS. Affinity purified antibodies were used at 1:250 dilutions in immunofluorescence. Found at: doi:10.1371/journal.ppat.1000808.s001 (2.69 MB TIF) Figure S2 Alignment of the PPxY motif in the sequence of protein VI from different human and non-human adenovirus serotypes. A partial alignment of protein VI sequences from different human adenoviral serotypes as well as non-human adenoviruses from the genus Mastadenovirus is shown. The conserved ubiquitin ligase-recruiting motif is boxed. Sequences were retrieved from public databases with the following accession numbers; canine (CAV-2, AP_000621), bovine (boAd3, AP_000031), huAd3 (serotype B, ABB17802), huAd35 (serotype B, AP_000584), huAd4 (serotype E, YP_068031), huAd17 (serotype D, AP_000149), huAd2 (serotype C, AP_000174), huAd5 (serotype C, AP_000210), huAd12 (serotype A, AP 000120), huAd40 (serotype F, NP_040861), murine (muAd1, AP_000350). Found at: doi:10.1371/journal.ppat.1000808.s002 (0.45 MB TIF) Figure S3 Construction of mutant Ad5 with altered PPxY motif in protein VI using BAC technology. A) To construct a bacterial artificial chromosome (BAC) carrying an infectious Ad5 genome, we cloned an AdEasy system (Stratagene) based virus genome into pKSB2 vector as described previously (Warming et al. [57] ; Ruzsics et al. [34] ). This recombinant Ad5 lacked the E1 and E3 regions and carried an FRT site in the place of its E1 region, which was introduced through an FRT containing pShuttle (Stratagene) clone. The resulting BAC, was termed pAd5-FRT and can be reconstituted to fully infectious recombinant Ad5 viruses after transfection of E1 complementing cell lines such as 293 cells. To construct protein VI-modified viruses, pAd5-FRT was transformed into the E. coli strain SW102, which encodes the l-red recombination system from the bacteriophage under a heatinducible promoter [57] . We next amplified a Kanamycin resistance cassette using primers with 50 nt 59 extensions homologous to protein VI coding regions. The forward primer was flanked with a homology located upstream to the PPSY motif and introduced a ClaI site int