Figure 1.

S100A10 knockout (KO) and AnxA2 KO (resulting in loss of full A2t) does not affect cell proliferation and inhibits multiple high-risk HPV infections in HeLa cells. (a) *Because S100A10 is rapidly degraded in the absence of AnxA2, targeting AnxA2 results in full A2t KO. Here, either dimeric S100A10 or heterotetrameric A2t (S100A10 + AnxA2) was knocked out in wild type (WT) HeLa cells via CRISPR/Cas9 and confirmed by western blot. Western blot images were cropped. See full-length blots with molecular weight standards in Supplementary Fig. 1a. (b,c) WT, S100A10 KO, and A2t KO HeLa cells were seeded with equal cell number, grown for 48 h, and then analyzed for differences in cell proliferation via (b) analyzing cell number and viability via trypan blue exclusion test, and (c) quantification of nucleic acid content via CyQUANT Cell Proliferation Assay Kit (Thermo Fisher) and then analyzed relative to WT HeLa cells. Results (b,c) represent N = 8 ± s.d. and are representative of at least 3 independent experiments. Statistics: 1-way ANOVA with Dunnett’s multiple comparison test. ns = not significant. (d) WT, S100A10 KO, and A2t KO HeLa cells were treated with HPV PsVs (TCID₅₀) carrying a GFP reporter plasmid. Infection (GFP gene transduction) was measured 48 h p.i. via flow cytometry. Neutralizing anti-HPV L1 antibody H16.V5 (neut. Ab) was used as a positive control for infection inhibition, and background from mock infected cells was subtracted. At least 3 independent S100A10 KO and 3 independent A2t KO clones were screened for consistent inhibition of HPV16 infection. Results are representative of at least 3 independent experiments and show the mean %GFP-positive cells ± s.d. (n = 3, normalized to WT). Statistics: 1-way ANOVA with Dunnett’s multiple comparisons test – ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.