Figure 9

ALIX interacts with syntenin-1 and is required for papillomavirus infection.

(a) HPV16 L1 colocalises with syntenin-1 interacting protein ALIX. Representative pictures of ALIX-HA and syntenin-GFP overexpression show colocalisation of syntenin-1 (green), ALIX (red) and L1 (blue) marked by arrows. (b) ALIX knockdown correlates with reduced infectivity. Upper panels show efficiency of ALIX knockdown in HeLa and HaCaT cells. Bottom panels show HPV16 infection assay of HeLa and HaCaT cells after ALIX siRNA treatment. Infectivity was measured as in Fig. 2b. (c,d) Affinity of ALIX V-domain-syntenin1 interaction by SPR equilibrium binding analysis. (c) Double referenced sensorgrams are shown as a plot of relative response of ALIX V-domain/syntenin-1 N-terminal wild-type peptide (654 RU) immobilized surfaces over time. Streptavidin coated non-peptide immobilized surface served as reference. RU, Response units. (d) Data points of ALIX V-domain binding to syntenin-1 wild-type peptide (filled circle), Y4F peptide (filled triangle) or S6A peptide (filled square) fitted to a one-site binding model. Syntenin-1 wild-type peptide showed a KD of 29 μM. (e,f) Syntenin-1-depleted HeLa cells were transfected with control, syntenin-1 wild-type or syntenin-1 mutant expression plasmid. L1-7 reactivity assay was performed as in Fig. 2e,f. (e) Shows representative pictures. (f) Shows quantification of L1-7 reactivity. *P < 0.05 compared to control, ^$P < 0.05 compared to cells transfected with CD63 siRNA and control plasmid.