FIG 4.

KLK8 cleavage is not rate limiting for HPV16 internalization. (A) Kinetics of L1 cleavage by KLK8 was analyzed by incubation of HPV16 PsVs with 10 mg/ml heparin, binding to ECM, and incubation with conditioned medium or, as a control, DMEM. Samples were analyzed at the indicated times following conditioned medium incubation by SDS-PAGE and Western blotting using L1-specific CAMVIR-1 antibody. L1 cleavage bands (L1 A) were quantified by densitometry using Fiji. (B) Depicted is the increase of the cleaved L1 band (L1 A) normalized to the average maximal cleavage reached after 16 h of incubation with the batches of conditioned medium used in the experiments in percentages ± SD (n = 2). (C) Infection levels of HPV16 or FPC-HPV16 after 6 or 16 h of preincubation with conditioned medium (CM) relative to levels after preincubation with DMEM in percentages ± SD. (D) Schematic depiction of the experimental setup for KLK8 pretreatment. Virus particles are pretreated with heparin for structural activation (1) and then bound to HaCaT ECM and treated with conditioned medium or DMEM for 16 h (2). Seed-over infectious internalization (3) was performed as described before. Infection of HPV16 (left) or FPC-HPV16 (right) preincubated with growth medium (black) or conditioned medium (gray) for 16 h as described for panel A in a seed-over setup (compare with Fig. 3B). Depicted are infectious internalization values normalized to the 48-h samples in percentages ± SD.