Oligo sequences.

<p>Oligo sequences.</p

Loss of p53 inhibition by 16E6 restores IRF6 activity.

<p>(A) Table defining the 16E6 mutations that alter p53, E6AP or PDZ binding sites. (B) NIKs were co-transfected with IL-1β promoter luciferase construct along with the HPV16E6 WT and mutated constructs at the indicated concentration. n = 5. (C) Human primary keratinocytes were transfected with cas9/sgRNA for p53 or control and 36h later cells were examined for mRNA levels for p53, IL-1β or IRF6. n = 4. (D) siRNAE6AP (+) or siRNA scramble control (-) was transfected into PLXSN or 16E6 transduced human keratinocytes for 48 h, cells were harvested for protein and RNA. Western blot analysis for p53 and β-actin. Left top, RT-qPCR for IL-1β and, left below IRF6. n = 3, (E) NIKs were co-transfected with the IL-1β promoter and pLXSN, E6, p53 or E6 with p53. Luciferase activity was measured 48 h post infection. n = 4. (F) 16E6 transduced primary keratinocytes were transfected with vector control (-), p53 or IRF6 expression vectors. Twenty-four hours later cells were harvested and pro-IL-1β, p53 or IRF6 levels were examined by immunoblotting. n = 4. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P < 0.0001, based on an one or two way (applicable to > 2 conditions) ANOVA test. For immunoblotting data, 1 out of 4 experiments is shown.</p

IRF6 and not IRF8 is recruited to the IL-1β promoter which is blocked by HPV16E6.

<p>(A) HEK293 cells were co-transfected with IL-1β promoter luciferase construct along with the empty vector pUNO, IRF8 or IRF6 plasmid at the indicated concentration. Post 48 h cells were lysed and luciferase activity measured. n = 4. (B) Oligo pulldown assay for WT or the mutated ISRE site using protein lysates from HEK293 cells transfected with IRF6 or IRF8. Bound proteins were assessed by immunoblotting for IRF8 or IRF6. Input controls (10%). n = 4. (C) Immunoblot analysis of IRF6 protein levels in in pLXSN and 16E6 transduced human primary keratinocytes. n = 4. (D) IRF6 relative levels were measured in pLXSN, 16E6 and 16E7 transduced human primary keratinocytes by RT-qPCR. n = 4. (E) Immunofluorescent staining of IRF6 in human keratinocytes transduced with pLXSN or HPV16E6. Left, semi-quantative analysis of IRF6 was examined by calculating immunofluorescent intensity. The mean and S.E.M of five fields were plotted. n = 4. (F) Immunoblot analysis of IRF6 protein levels in C33A and NIKs. n = 4. (G) IRF6 mRNA levels detected by RT-qPCR in NIKs and CaSki cells. n = 4. (H) NIKs and CaSki cells were co-transfected with IL-1β promoter luciferase construct ± siRNA for 16E6. Post 48 h cells were lysed and luciferase activity measured. (I) C33A cells were treated with control PsV or 16QsV at different v.g.e per cell for 24 h and IRF6 protein levels were examined by immunoblot. n = 3. (J) C33A cells were treated with control PsV or HPV16 at different v.g.e for 24h and IRF6 mRNA levels were examined by RT-qPCR and (below) viral DNA expression of E7 vs β2-microgloubulin. n = 3. (K), ChIP using IgG, IRF6 or IRF8 antibodies was performed for the ISRE site on C33A cells infected with HPV16 or PsV for 24 h. n = 3. Data are representative of n independent experiments performed in triplicate. Panels A, E, J and K are shown as the mean ± SEM with ***, P < 0.0001, * P, < 0,01, based on a two way ANOVA test. Panels D and G are shown as the mean ± SEM with ***, P < 0.0001 based on an unpaired T test. For immunoblotting data, 1 out of 4 experiments is shown. For immunoblotting data, 1 out of 4 experiments is shown.</p

HPV16 induces transient IL-1β secretion by keratinocytes.

<p>(A) Human primary keratinocytes were treated as indicated with 16QsV or PsV (at 200 viral genome equivalents (v.g.e) per cell). IL-1β transcripts were determined by RT-qPCR. n = 5. (B) As in A, E1, E6 and E7 mRNA relative levels were determined by RT-qPCR. n = 4. (C) Human keratinocytes were treated at 4, 8 and 24 h with 16QsV at different v.g.e per cell. Supernatants were harvested and IL-1β or IL-18 production was measured by ELISA. PsV or L1/L2 fractions were added as controls. n = 5. (D) Human keratinocytes were treated with 16QsV or PsV at decreasing v.g.e per cell for 24 h ± recombinant IL-1β (200pg/ml). Cells were harvested and E7 mRNA levels were measured by RT-qPCR. n = 5. (E) Human keratinocytes were treated with 16QsV or PsV (200 v.g.e) for 24 h ± IL-1R inhibitor (Anakinra). Cells were harvested and E1 mRNA levels were measured by RTqPCR. qPCR. n = 5. Data are representative of n independent experiments performed. Shown are the mean ± SEM with ***, P < 0.0001, based on a two way ANOVA test.</p

16E6E7 block IL-1β production in primary human keratinocytes and in cervical cancer derived cells lines.

<p>(A) Analysis of the IL-1β production by ELISA in human keratinocytes transduced with pLXSN or 16E6E7 stimulated with nigericin or poly dA:dT. n = 10. (B) Human keratinocytes transduced with pLXSN or 16E6E7 transfected with a siRNA targeting 16E6E7 (+) or the scramble control (-). Cells were stimulated with the NLRP3 ligand nigericin and IL-1β secretion was measured by ELISA. Middle, western blot of E6 or E7 siRNA efficacy on 16E6E7 or PLXSN transduced cells. n = 4. Left SiHa cell were treated with a siRNA targeting 16E6E7 (+) or the scramble control (-). Cells were stimulated with the NLRP3 ligand nigericin and IL-1β secretion was measured by ELISA. n = 4. (C) IL-8 bioassay: HEK293T cells transiently expressing the IL-8 promoter linked to luciferase gene were treated with increasing concentrations of recombinant IL-1β ± Anakinra. Twenty four h post treatment cells were harvested and luciferase activity was measured. n = 4. (D) IL-8 bioassay using supernatants from human keratinocytes transduced with pLXSN or 16E6E7± AIM2 ligand poly dA:dT. n = 4. (E) Cervical cancer cells (SiHa) were transfected with a siRNA targeting 16E6E7 or the scramble control. The cells were stimulated with the NLRP3 ligand nigericin, AIM2 ligand poly dA:dT or 16QsV (200 v.g.e per cell) and IL-1β was measured by ELISA. n = 4. (F) IL-8 bioassay using supernatants from cervical cancer cell lines ± nigericin. n = 6. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P < 0.0001, based on a two way ANOVA test. For immunoblotting data, 1 out of n = 3 experiments is shown.</p

HPV16 E6 down-regulates the IL-1β promoter in cervical cells via the ISRE site.

<p>(A) NIKs were co-transfected with the IL-1β promoter with increasing concentrations of pLXSN HPV16E6E7 or 16E6 or 16E7 as indicated. After 48 h, cells were harvested and luciferase activity was measured. n = 5. (B) Relative expression of 16E6E7, 16E6 or 16E7 were measured by RT-qPCR. n = 5. (C) Primary human keratinocytes transduced with 16E6 and treated with a scramble or siRNA against 16E6. Protein levels of pro-IL-1β and loading control β-tubulin were evaluated by immunoblotting. n = 4. (D) Schematic representation of IL-1β promoter luciferase deletion mutations. (E) WT and deleted IL-1β promoter constructs were transiently transfected into NIKs expressing pLXSN or 16E6. After 48 h, cells were harvested and luciferase activity was measured. n = 4. (F) Schematic representation of the IL-1β LILRE site. (G) WT and deleted or mutated IL-1β promoter constructs were transiently transfected into NIKs expressing pLXSN or 16E6. After 48 h, cells were harvested and luciferase activity was measured. n = 4. Data are representative of n independent experiments performed in triplicate. Panel A and B shown are the mean ± SEM with ***, P < 0.0001, based on a two way ANOVA test. Panel F, is based on an one way ANOVA test and panel G a paired T test. For immunoblotting data, 1 out of 4 experiments is shown.</p

Four normal and tumor biopsies from (A) were used to perform ChIP analysis for p53 binding onto the IRF6 promoter.

<p>n = 3. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P < 0.0001, **, P < 0.0005 based on a paired Student’s t test (when patient matched) or unpaired when not patient matched.</p

HPV16-positive cervical cancer lesions contain less IRF6 and IL-1β.

<p>(A) Immunofluorescence of normal cervical issue and HPV16+ cervical cancer biopsies. IL-1β (green), p53 (red) with trace indicating the basal layer and nucleus (white). Normal (HPV−) and a neoplastic biopsy (HPV16+) from one representative patient out of six with similar results are shown. Bars represent a scale of 10 μm. For each stained biopsy, six fields were examined IL-1β staining was counted manually and the percentage scored out of 100 cells. n = 4. (B) RNA was extracted from normal (29) and cervical cancer biopsies (29). IL-1β relative and IRF6 mRNA levels were measured by RT-qPCR. n = 4. (C) Table of immunohistochemical scoring IRF6 in patients at different stages of cervical neoplasia. Scoring, *** strong, ** medium, * low and—no staining (4 normal, 8 CINI, 8 CINII, 5 CINIII). n = 2. All tissue staining data were examined by two pathologists. (D), Immunohistochemical staining of IRF6 in cervical tissue in patients with CINI, II or III. n = 2. (E) RT-qPCR of IL-1β and IRF6 mRNA expression levels in normal vs neoplastic cervical tissue. n = 3. <b>A p53 site is required to bind to the IRF6 promoter but is lost in cervical cancer tissues</b>. (F) ChIP analysis was performed on normal and cervical neoplastic tissue for IRF6 binding on ISRE site on the IL-1β promoter. n = 3.</p

HPV16 oncoproteins inhibit pro-IL-1β levels.

<p>(A) Human keratinocytes transduced with pLXSN or 16E6E7 were stimulated with AIM2 and (B) NLPR3 ligands and both pro-IL-1β and IL-1β from cell lysates or supernatants were analysed by immunoblotting. β-actin was used as a loading control. Densitometry analysis was performed n = 3. (C) Immunoblotting of pro-IL-1β in human keratinocytes transduced with pLXSN or 16E6E7. (D) Cervical cancer cell lines were lysed and immunoblotting for pro-IL-1β was performed. n = 4 (E) Human keratinocytes transduced with pLXSN or 16E6E7 were treated for 24 h with N-CBZ-Leu-Leu-Leu-al. Cells were harvested and p53, E6 as well as pro-IL-1β levels were determined by immunoblotting. Right, p53 densitometry levels were normalized to β-actin. Below, immunoblot analysis of the 16E6 protein. n = 3. (F) RNA was extracted from Human keratinocytes transduced with pLXSN or 16E6E7 and IL-1β transcripts relative expression was determined by RT-qPCR. n = 5. (G) RNA was extracted from patient derived cervical cancer cell lines and IL-1β transcripts were determined by RT-qPCR. n = 6. Panels A-E. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P < 0.0001, based on a two way ANOVA test. Panel F P < 0.0001, based on a one way ANOVA test. Panel G Student unpaired T test was performed comparing C33A to CaSki or SiHa. For immunoblotting data, 1 out of 3 experiments is shown.</p

Model representing the induction and inhibition of IL-1β by HPV16.

<p>Infection of the basal keratinocytes with HPV16 induces inflammasome dependent IL-1β production sensed by an unknown innate receptor. p53 transcriptional regulation of IRF6 is increased, which we show drives IL-1β transcription. The pro form of IL-1β is cleaved by caspase 1 (red bar). The active form of IL-1β can block the increase in viral copies. However, when the oncoprotein E6 is expressed this drives p53 degradation by E6AP preventing IRF6 and consequently IL-1β transcription. This mechanism of viral inhibition of innate responses may contribute to HPV16 persistence in the host.</p