br G Representative images showing HPV
(G) Representative images showing HPV16 PsV-derived GFP signal in hCerEC-CTRL ML-210 (hCerECs transfected with non-targeting scrambled short interfering RNA [siRNA] as control) and hCerEC-siYAP cells (hCerECs transfected with YAP1-specific siRNA). Cells were incubated in growth media with HPV16 PsV (MOI = 1) for 72 h. Scale bar: 100 mm.
(H) Representative images showing HPV16 PsV-derived GFP signal in Ect1-MX, Ect1-YAP, and Ect1-YAPS127A cells. Ect1 cells is an immortalized cervical epithelial cell line. Scale bar: 100 mm.
(I) Representative images showing HPV16 PsV-derived GFP signal in SiHa-CTRL and SiHa-siYAP cells. SiHa is a cervical cancer cell line. Scale bar: 100 mm. Quantitative data are presented in Figure S5.
Figure 3. Hyperactivation of YAP1 Upregulates Expression of the Putative HPV Receptor Molecules In Vitro and In Vivo
(C) Representative images showing HPV16 PsV-derived GFP signal in hCerEC-MX, hCerEC-YAP, and hCerEC-YAPS127A cells with (siITGA6) or without (siControl) knockdown of ITGA6 protein. Scale bar: 100 mm.
(D) Representative images showing expressions of YAP1 and the well-studied putative HPV receptor molecule ITGA6, in cervical tissues derived from 10-month-old
control (KRT-rtTA mice) and KRT14-YAPS127A transgenic mice. Protein expression was determined using peroxidase-based immunohistochemistry. The nuclei were counterstained with hematoxylin. Note upregulation of ITGA6 protein in the epithelium of KRT14-YAPS127A transgenic mice. Scale bar: 200 mm.
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Following viral entry, the first step in activating an innate im-mune response against viral infection is the detection of viral pathogens, which is mediated by the interaction between pattern recognition receptors (PPRs) of host cells and the path-ogen-associated molecular patterns (PAMPs) of HPV virions (Stanley, 2012). Our RT-PCR results suggested that mRNA expression of the well-studied PRRs for HPV, Toll-like receptors (TLRs) TLR2 (Alphs et al., 2008) and TLR4 (Pannone et al., 2016), was significantly downregulated in YAPS127A-expressing cells (Figure 4B). The signal transduction from TLRs to downstream kinase cascades requires the mediation of several adaptor pro-teins, including myeloid differentiation primary response gene 88 (MYD88) and TIR-domain-containing adaptor-inducing inter-
feron-b (TRIF) (Tartey and Takeuchi, 2017). The expression of MYD88 and TRIF mRNA was also inhibited by YAPS127A in hCer-
ECs (Figure 4C). Western blot analysis further confirmed the downregulation of TLR2 and TRIF in hCerECs overexpressing YAP1 (Figure S8A). These results indicated that hyperactivation of YAP1 can negatively regulate the viral recognition pathway.
The recognition of HPV PAMPs by PRRs may trigger a series of kinase cascades that subsequently leads to activation and nuclear translocation of IRF1, IRF3, IRF7, and NFkB, which are transcription factors regulating expression of type I inter-ferons (IFNa and IFNb) and cytokines, the key molecules for anti-
viral immune response (McNab et al., 2015; Takeuchi and Akira, 2009). Similar to TLRs in hCerEC-YAPS127A cells, expression of
IRF1 and IRF7 genes was also significantly decreased in YAP1- hyperactivated cells (Figure 4D). Consistently, knock-down of YAP1 in these cells significantly increased expression of IRF1 and IRF7 mRNA levels (Figure 4E). Although the level of IRF3 protein was not significantly affected by YAP1 expres-sion, the level of phospho-IRF3 (active form) was decreased in hCerECs expressing YAPS127A (Figure S8A). Immunofluorescent
analysis also showed that ectopic expression of YAP1 or YAPS127A resulted in translocation of IRF3 and NFkB1 from