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.

Cytoplasmic extracts for pulldowns were prepared from 293T cells (human embryonic kidney cells). All cells except hTERT-RPE were grown in DMEM Glutamax TM (Gibco) supplemented with 10% of fetal calf serum (FCS) (Biowest). hTERT-RPE1 cells were a kind gift from M. Bonhivers (University of Bordeaux 2) and grown in DMEM/HamsF12 media supplemented with 10% FCS according to the suppliers instructions. Prior to infection experiments, cells were serum starved for 24h to induce primary cilia growth [32] . Recombinant Ad5-VI-wt and Ad5-VI-M1 viruses and their GFP expressing counterparts were constructed as described in the supplemental material. Amplification of viruses was done in 293 cells and purified using double CsCl 2 -banding. Virus particle to cell ratios were calculated based on the estimated copy numbers of viral genomes. Copy numbers were calculated according to Mittereder et al. [51] . Briefly, purified particles were diluted 1:10 in virus lysis buffer (0.1% SDS, 10 mM Tris/HCl pH 7.4, 1 mM EDTA) and incubated for 10 min at 56uC to release the viral genomes and the OD 260 was determined. Calculations were based on 1 OD 260 = 1.1610 12 particles/ml [51] . Lentiviral vector production for shRNA encoding vectors was done by the service platform for lentiviral vector production of the indicated (from 25-100%) and transduced with AdGFP. GFP expression levels were determined 24 h later using flow cytometry. GFP expression levels within each condition were normalized to transduction controls of cells treated with shRNA expressing lentiviral vectors against luciferase (arbitrarily set to 1). Values are the mean (+/2 standard deviation) of at least two experiments done in triplicates D) Accumulation of Ad at the MTOC region following Nedd4 depletion. Fluorescently labeled Ad was used to infect cells following control depletions (shLuc) or depletion of Nedd4.1 or Nedd4.2 using (sh4.1 (1) and sh4.2 (1) as in C). Subcellular localization of viral particles was determined at 45 min. post infections. Cells were fixed and stained for the MTOC using a pericentrin antibody. Particles were counted and scored for their relative proximity to the MTOC using two concentric circles with 10 and 20 mm diameter around the MTOC (compare also Figure 3 ). The graph shows percentage of viral particles within 10mm or 10-20 mm proximity to the MTOC as indicated. Note that in Nedd4.1 and Nedd4.2 depleted cells the relation is inverted compared to control depleted cells showing less viral particles accumulating at the MTOC. The error bar represents cell-to-cell variation (n.15, p,0.05). doi:10.1371/journal.ppat.1000808.g008