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. 2020 Mar 2;117(11):6121–6128. doi: 10.1073/pnas.1917748117

Fig. 1.

Fig. 1.

Identification and analysis of peptides that inhibit HPV infection. (A, Top) Sequences of L2 peptides with wild-type CPP segments (red) and RBS (green) highlighted. Amino acid substitutions in the CPP or RBS mutant peptides are shown in blue. (A, Bottom) RBS and flanking sequences of indicated HPV L2 proteins or DMT1-II. (B) Inhibitory dose–response curve for the wild-type P16/16 peptide. HeLa cells were pretreated with various concentrations of P16/16 for 1 h prior to infection with HPV16 PsV at an MOI of 1. The peptide and PsV were left in the medium for the duration of the experiment. At 48 h.p.i., flow cytometry was used to determine the fraction of cells expressing reporter protein HcRed. The graph shows mean results of three experiments, +/− SD. (C) HeLa cells were pretreated for 1 h with 14 µM (orange line) or 28 µM (green line) P16/16 or 14 µM PDM/16 (gray line), or left untreated (blue line). Cells were then infected with HPV16 PsV at the indicated MOI, and at 48 h.p.i. infectivity was measured by flow cytometry for reporter gene expression as in B. (D) Inhibition of authentic HPV16. HeLa cells were infected with HPV16 harvested from organotypic cultures of human keratinocytes or with HPV16 PsV in the presence (gray bars) or absence (black bars) of 14 μM P16/16. Infection by HPV16 and HPV16 PsV was assessed by qRT-PCR for expression of HPV E7 and HcRed mRNA, respectively, and normalized to infection by the cognate virus in the absence of the peptide. The graph shows average results of three independent experiments, +/− SD, where infection of untreated cells is set at 100%. The background signal determined with noncognate primers was <0.01%. (E) Inhibition of HPV infection of HaCaT cells. HaCaT keratinocytes were infected at an MOI of 1 with HPV16 PsV in the presence (gray bars) or absence (black bars) of 14 μM P16/16, and infectivity was assessed at 48 h.p.i. by flow cytometry for HcRed fluorescence and displayed as in B. (F) HeLa cells were treated for 24 h with no peptide (black bars) or 14 µM P16/16 (gray bars). The peptide was then removed, and the cells were incubated for the indicated period of time prior to infection with HPV16 PsV at an MOI of 1. At 48 h.p.i, infectivity was measured by flow cytometry as in Fig. 1B. The graph shows results of three experiments +/− SD, normalized to no peptide control at time = 0. Numbers indicate the P value for each pairwise comparison.