Figure 1

CD63 is required for HPV16, -18 and -31 infections.

(a) HPV16 is internalised into CD63-positive endosomal compartments in primary keratinocytes (NHEK). NHEK cells were infected with HPV16 for 7 hours and stained with CD63 monoclonal antibody (mAb) and L1-polyclonal antibody (pAb) K75. Shown are representative pictures of endogenous CD63 (green) and HPV16 L1 (red). (b) CD63 knockdown correlates with reduced infectivity. Upper panels show efficiency of CD63 knockdown in HeLa, HaCaT and NHEK cells by Western blotting. Bottom panels show HPV16 pseudovirus (PsV) infection assay of HeLa, HaCaT and NHEK cells after CD63 siRNA treatment. Infectivity was measured by luciferase activity and normalized by LDH measurements. Infection rate of control siRNA was set to 100%. (c) HPV16 infectivity can be restored after reexpression in CD63 depleted cells. HeLa cells were treated with CD63 siRNAs, transfected with CD63-expression or control plasmid and infected with HPV16 PsV. Infectivity of HPV16 PsV was analysed as above. Upper panels show the efficiency of knockdown and reexpression. (d) HPV18 and -31 are internalised into CD63-positive endosomal compartments. NHEK cells were infected with HPV18 or HPV31 PsV and analysed as in (a). (e) Quantification of L1 colocalisation with CD63 was performed by analysis of at least 12 images (3–5 cells per image) using Colocalisation Software 4.7 (Zeiss). Shown is the proportion of CD63-colocalising L1 pixels relative to the total amount of L1 pixels. (f) HeLa, HaCaT and NHEK cells were treated as in (b) and HPV18 and HPV31 PsV infectivity after CD63 knockdown was analysed as above. *P < 0.05 significant decrease compared to control, ^$P < 0.05 significant increase compared to cells transfected with CD63 3′UTR siRNA and control plasmid.