Apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3) proteins are interferon (IFN)-inducible antiviral factors that counteract various viruses such as hepatitis B virus (HBV) and human immunodeficiency virus type 1 (HIV-1) by inducing cytidine (C)-to-uracil (U) mutations in viral DNA and inhibiting reverse transcription. However, whether APOBEC3 proteins (A3s) can hypermutate human papillomavirus (HPV) viral DNA and exhibit antiviral activity in human keratinocyte remains unknown. Here we examined the involvement of A3s in the HPV life cycle using cervical keratinocyte W12 cells, which are derived from low-grade lesions and retain episomal HPV16 genomes in their nuclei. We focused on the viral E2 gene as a potential target for A3-mediated hypermutation because this gene is frequently found as a boundary sequence in integrated viral DNA. Treatment of W12 cells with beta interferon (IFN-β) increased expression levels of A3s such as A3A, A3F, and A3G and induced C-to-U conversions in the E2 gene in a manner depending on inhibition of uracil DNA glycosylase. Exogenous expression of A3A and A3G also induced E2 hypermutation in W12 cells. IFN-β-induced hypermutation was blocked by transfection of small interfering RNAs against A3G (and modestly by those against A3A). However, the HPV16 episome level was not affected by overexpression of A3A and A3G in W12 cells. This study demonstrates that endogenous A3s upregulated by IFN-β induce E2 hypermutation of HPV16 in cervical keratinocytes, and a pathogenic consequence of E2 hypermutation is discussed.

Human papillomavirus vaccines are being introduced worldwide and are expected to reduce the incidence of cervical cancer. Here we report a cross-sectional study using a validated human papillomavirus genotyping method to reveal the human papillomavirus prevalence and genotype distribution in Japanese women with cervical intraepithelial neoplasia Grade 2/3 and invasive cervical cancer. Cervical exfoliated cells were collected from 647 patients with abnormal cervical histology (cervical intraepithelial neoplasia Grade 2, n = 164; cervical intraepithelial neoplasia Grade 3, n = 334; and invasive cervical cancer, n = 149), and subjected to the PGMY-PCR-based genotyping assay. The association between human papillomavirus infection and lesion severity was calculated using a prevalence ratio. Overall, the prevalence of human papillomavirus deoxyribonucleic acid was 96.3% in cervical intraepithelial neoplasia Grade 2, 98.8% in cervical intraepithelial neoplasia Grade 3 and 88.0% in invasive cervical cancer (97.8% in squamous cell carcinoma and 71.4% in adenocarcinoma). The three most prevalent types were as follows: human papillomavirus 16 (29.3%), human papillomavirus 52 (27.4%) and human papillomavirus 58 (22.0%) in cervical intraepithelial neoplasia Grade 2; human papillomavirus 16 (44.9%), human papillomavirus 52 (26.0%) and human papillomavirus 58 (17.4%) in cervical intraepithelial neoplasia Grade 3; and human papillomavirus 16 (47.7%), human papillomavirus 18 (23.5%) and human papillomavirus 52 (8.7%) in invasive cervical cancer. The prevalence ratio of human papillomavirus 16 was significantly higher in cervical intraepithelial neoplasia Grade 3 compared with cervical intraepithelial neoplasia Grade 2 (prevalence ratio, 1.62; 95% confidence interval, 1.26–2.13) and in squamous cell carcinoma compared with cervical intraepithelial neoplasia Grade 3 (prevalence ratio, 1.55; 95% confidence interval, 1.25–1.87). Multiple infections decreased from cervical intraepithelial neoplasia Grade 2/3 (38.4/29.6%) to invasive cervical cancer (14.1%), whereas co-infections with human papillomavirus 16/52/58 were found in cervical intraepithelial neoplasia Grade 2/3. The results of this study provide pre-vaccination era baseline data on human papillomavirus type distribution in Japanese women and serve as a reliable basis for monitoring the future impact of human papillomavirus vaccination in Japan.

The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis. IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1/4 protein, thereby leading to A3B upregulation. Since increased levels of TEAD4 are frequently observed in many cancers, an understanding of the direct link between TEAD and A3B upregulation is of broad oncological interest.