Figure 2

Infection with HPV-PsVs in vitro. Luciferase reporter gene encapsidating HPV-PsVs composed of HPV18-L1/L2 or HPV16-L1/L2 envelope proteins, respectively, were prepared as described previously [38, 42]. (A) Purity of the preparations was analyzed by SDS-PAGE followed by silver staining. Note that the additional band at approximately 60 kDa in the HPV16-PsVs (luc) preparation is the silver-stained pGL3 plasmid which is not present in the HPV16-PsVs preparation that does not encapsidate any DNA. (B) In order to test functionality of the preparations, HaCaT cells were infected with HPV18-PsVs (luc) or HPV16-PsVs (luc) using a vge of 100 pseudovirion particles per cell. Luciferase activity was measured 48 h post-infection. Neutralization experiments were performed for each pseudovirion preparation with antibodies against HPV16-L1/L2 (H16.V5) and HPV18-L1/L2 (H18.J4) envelope proteins as indicated. The experiments were done in duplicates, repeated three times and are presented as % of luciferase activity to untreated controls. (C) The indicated cell lines were infected with equal amounts of HPV18-PsVs (luc) or HPV16-PsVs (luc), respectively. Luciferase activity was measured 48 h post-infection. The experiments were performed in duplicates, repeated three times and are presented as x-fold increase of luciferase activity to uninfected controls. (D) FACS analysis showing ITGA6 expression on the surface of the indicated cell lines. In each panel, the dotted histogram corresponds to the negative control while the solid line shows the histograms of cells stained with the ITGA6 antibody. (E) Cells were plated on coverslips and infected with equal amounts of HPV18-PsVs or HPV16-PsVs, respectively, which were labeled with Oregon Green®488 carboxylic acid diacetate succinimidyl ester, a fluorchrome that is only activated after cell entry [43]. The cell line pgsD-677 which is defective in HPV uptake compared to the wildtype CHO-K1 counterpart [50] was included as negative control. Pictures were taken on an Olympus IX81S8F fluorescent microscope at 40x magnification.