Figure 2
From: The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses
CD63 associates with L1 and is involved in HPV16 post-uncoating processing but not in cell surface binding or virus endocytosis.
(a) CD63 is coprecipitated by L1 pAb K75 immunoprecipitation (IP) from lysates of HPV16 PsV infected HeLa cells. Expressed (left) and precipitated proteins (right) were detected by Western blotting using L1 mAb 321-F (upper panels) and mAb CD63 (lower panels). (b–d) CD63 knockdown does not affect HPV16 cell binding. (b) HeLa cells were pretreated with CD63 or control siRNA and incubated with HPV16 PsV. Cell-bound PsV were detected in cell lysates by Western blotting (WB) using L1 mAb 312F. Polyethylenimine (PEI) treated cells were used as control for binding deficiency75. (c) Quantification of WB bands shows HPV16 cell surface binding independent of CD63. (d) PsV binding and endocytosis is unaffected by CD63 knockdown. HeLa cells were pretreated as in (b). Cells were incubated with HPV16 PsV as indicated. The amount of surface associated PsV was assessed by flow cytometry using L1 pAb K75. The difference between the amount of surface bound PsV at 1 h and 24 h serves as a measure for endocytosis. Mean fluorescence intensity of cells incubated with PsV for 1 h was adjusted to 100%. (e,f) L1-7 reactivity is reduced after CD63 knockdown. Representative pictures of immunofluorescence with HPV16 L1 pAb K75 (blue), mAb 33L1-7 (green) and CD63 mAb (red) are shown in (e). Hela cells were treated with siRNAs as in (b) and infected for 7 hours. K75 Ab recognises bound and internalized virus particles. 33L1-7 Ab recognises HPV16 epitope only accessible after capsid disassembly. (f) Shows quantification of disassembled viral capsids performed by analysis of L1-7 positive pixels of at least 15 images using ImageJ-script. Shown are the results of three independent experiments using CD63 siRNAs normalized to control siRNA treated cells. *P < 0.05 compared to control.
