Differential requirement of the transcription factor HOXC13 for the stable maintenance of human papillomavirus genome among high-risk genotypes.

The viral genome of the high-risk human papillomavirus (HPV), the causative agent of cervical cancer, is stably maintained as extrachromosomal episomes that establish persistent infection. We previously identified homeobox-transcription factor HOXC13 as an important host protein mediating the short-term retention of the HPV16 and HPV18 genomes in normal human immortalized keratinocytes (NIKS). Here, we used CRISPR-Cas9 technology to construct HOXC13 knockout (KO) NIKS cells to determine whether HOXC13 is required for the long-term maintenance of high-risk HPV genomes. HPV16, HPV18, HPV52, and HPV58 whole genomes were transfected into HOXC13 KO cells, and the copy number of viral genomes per cell was monitored over cell passages. Copy numbers of HPV16, HPV52, and HPV58 genomes decreased continuously in HOXC13 KO cells, whereas HPV18 genomes remained stable throughout passages. Thus, HOXC13 is critical for the stable maintenance of the viral genomes of HPV16, HPV52, and HPV58, but not HPV18.

Allelic loss of HLA class I facilitates evasion from immune surveillance in cervical intraepithelial neoplasia.

Loss of heterozygosity (LOH) has been reported to occur in HLA regions in cervical intraepithelial neoplasia (CIN) and cervical cancer. However, the details of how this is related to the progression of CIN have been unclear. In this study, we examined the human papillomavirus (HPV) antigen-presenting capacity of people with CIN and the significance of LOH of HLA class I in the progression of CIN. It was shown that differences in antigen-presenting capacity among each case depended on HLA types, not HPV genotypes. Focusing on the HLA type, there was a positive correlation between antigen-presenting capacity against HPV and the frequency of allelic loss. Furthermore, the lost HLA-B alleles had a higher HPV antigen-presenting capacity than intact alleles. In addition, frequency of LOH of HLA class I was significantly higher in advanced CIN (CIN2-3) than in cervicitis or early-stage CIN (CIN1): around half of CIN2-3 had LOH of any HLA class I. Moreover, the antigen-presenting capacity against E5, which is the HPV proteins that facilitate viral escape from this immune surveillance by suppressing HLA class I expression, had the most significant impact on the LOH in HLA-B. This study suggests that HPV evades immune surveillance mechanisms when host cells lose the capacity for antigen presentation by HLA class I molecules, resulting in long-term infection and progression to advanced lesions.

Nuclear proinflammatory cytokine S100A9 enhances expression of human papillomavirus oncogenes via transcription factor TEAD1.

Transcription of the human papillomavirus (HPV) oncogenes, E6 and E7, is regulated by the long control region (LCR) of the viral genome. Although various transcription factors have been reported to bind to the LCR, little is known about the transcriptional cofactors that modulate HPV oncogene expression in association with these transcription factors. Here, we performed in vitro DNA-pulldown purification of nuclear proteins in cervical cancer cells, followed by proteomic analyses to identify transcriptional cofactors that bind to the HPV16 LCR via the transcription factor TEAD1. We detected the proinflammatory cytokine S100A9 that localized to the nucleus of cervical cancer cells and associated with the LCR via direct interaction with TEAD1. Nuclear S100A9 levels and its association with the LCR were increased in cervical cancer cells by treatment with a proinflammatory phorbol ester. Knockdown of S100A9 decreased HPV oncogene expression and reduced the growth of cervical cancer cells and their susceptibility to cisplatin, whereas forced nuclear expression of S100A9 using nuclear localization signals exerted opposite effects. Thus, we conclude that nuclear S100A9 binds to the HPV LCR via TEAD1 and enhances viral oncogene expression by acting as a transcriptional coactivator. IMPORTANCE Human papillomavirus (HPV) infection is the primary cause of cervical cancer, and the viral oncogenes E6 and E7 play crucial roles in carcinogenesis. Although cervical inflammation contributes to the development of cervical cancer, the molecular mechanisms underlying the role of these inflammatory responses in HPV carcinogenesis are not fully understood. Our study shows that S100A9, a proinflammatory cytokine, is induced in the nucleus of cervical cancer cells by inflammatory stimuli, and it enhances HPV oncogene expression by acting as a transcriptional coactivator of TEAD1. These findings provide new molecular insights into the relationship between inflammation and viral carcinogenesis.

Folliculin Prevents Lysosomal Degradation of Human Papillomavirus To Support Infectious Cell Entry.

Human papillomavirus (HPV) infects epithelial basal cells in the mucosa and either proliferates with the differentiation of the basal cells or persists in them. Multiple host factors are required to support the HPV life cycle; however, the molecular mechanisms involved in cell entry are not yet fully understood. In this study, we performed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout (KO) screen in HeLa cells and identified folliculin (FLCN), a GTPase-activating protein for Rag GTPases, as an important host factor for HPV infection. The introduction of single guide RNAs for the FLCN gene into HeLa, HaCaT, and ectocervical Ect1 cells reduced infection by HPV18 pseudovirions (18PsVs) and 16PsVs. FLCN KO HeLa cells also exhibited strong resistance to infection with 18PsVs and 16PsVs; nevertheless, they remained highly susceptible to infections with vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus. Immunofluorescence microscopy revealed that the numbers of virions binding to the cell surface were slightly increased in FLCN KO cells. However, virion internalization analysis showed that the internalized virions were rapidly degraded in FLCN KO cells. This degradation was blocked by treatment with the lysosome inhibitor bafilomycin A1. Furthermore, the virion degradation phenotype was also observed in Ras-related GTP-binding protein C (RagC) KO cells. These results suggest that FLCN prevents the lysosomal degradation of incoming HPV virions by enhancing lysosomal RagC activity. IMPORTANCE Cell entry by human papillomavirus (HPV) involves a cellular retrograde transport pathway from the endosome to the trans-Golgi network/Golgi apparatus. However, the mechanism by which this viral trafficking is safeguarded is poorly understood. This is the first study showing that the GTPase-activating protein folliculin (FLCN) protects incoming HPV virions from lysosomal degradation and supports infectious cell entry by activating the Rag GTPases, presumably through the suppression of excessive lysosomal biosynthesis. These findings provide new insights into the effects of small GTPase activity regulation on HPV cell entry and enhance our understanding of the HPV degradation pathway.

Whole-genome analysis of human papillomavirus 67 isolated from Japanese women with cervical lesions.

BACKGROUND: Human papillomavirus (HPV) type 67 is phylogenetically classified into Alphapapillomavirus species 9 (alpha-9) together with other carcinogenic types (HPV16, 31, 33, 35, 52 and 58), but is the only alpha-9 type defined as possibly carcinogenic. This study aimed to determine whole-genome sequences of HPV67 isolated from Japanese women with cervical cancer or cervical intraepithelial neoplasia (CIN) to better understand the genetic basis of the oncogenic potential of HPV67. METHODS: Total cellular DNA isolated from cervical exfoliated cells that were single positive for HPV67 (invasive cervical cancer, n = 2; CIN3, n = 6; CIN 2, n = 1; CIN1, n = 2; the normal cervix, n = 1) was subjected to PCR to amplify HPV67 DNA, followed by next generation sequencing to determine the complete viral genome sequences. Variant lineages/sublineages were assigned through viral whole-genome phylogenetic analysis. The transcriptional activity of the virus early promoter was assessed by luciferase reporter assays in cervical cancer cell lines. RESULTS: Phylogenetic analyses of HPV67 genomes from Japan (n = 12) revealed that 11 belonged to lineage A (sublineage A1, n = 9; sublineage A2, n = 2) and one belonged to lineage B. All cancer cases contained sublineage A1 variants, and one of these contained an in-frame deletion in the E2 gene. The long control region of the HPV67 genome did not induce transcription from the virus early promoter in HeLa cells, but showed weak transcriptional activity in CaSki cells. CONCLUSIONS: The single detection of HPV67 in cervical cancer and precancer specimens strongly suggests the carcinogenic potential of this rare genotype. The phylogenetic analysis indicates a predominance of lineage A variants among HPV67 infections in Japan. Since only 23 complete genome sequences of HPV67 had been obtained until now, the newly determined genome sequences in this study are expected to contribute to further functional and evolutionary studies on the genetic diversity of HPV67.

Transcription Factor Homeobox D9 Drives the Malignant Phenotype of HPV18-Positive Cervical Cancer Cells via Binding to the Viral Early Promoter.

Persistent infections with two types of human papillomaviruses (HPV), HPV16 and HPV18, are the most common cause of cervical cancer (CC). Two viral early genes, E6 and E7, are associated with tumor development, and expressions of E6 and E7 are primarily regulated by a single viral promoter: P97 in HPV16 and P105 in HPV18. We previously demonstrated that the homeobox D9 (HOXD9) transcription factor is responsible for the malignancy of HPV16-positive CC cell lines via binding to the P97 promoter. Here, we investigated whether HOXD9 is also involved in the regulation of the P105 promoter using two HPV18-positive CC cell lines, SKG-I and HeLa. Following the HOXD9 knockdown, cell viability was significantly reduced, and E6 expression was suppressed and was accompanied by increased protein levels of P53, while mRNA levels of TP53 did not change. E7 expression was also downregulated and, while mRNA levels of RB1 and E2F were unchanged, mRNA levels of E2F-target genes, MCM2 and PCNA, were decreased, which indicates that the HOXD9 knockdown downregulates E7 expression, thus leading to an inactivation of E2F and the cell-cycle arrest. Chromatin immunoprecipitation and promoter reporter assays confirmed that HOXD9 is directly associated with the P105 promoter. Collectively, our results reveal that HOXD9 drives the HPV18 early promoter activity to promote proliferation and immortalization of the CC cells.

The Transcriptional Cofactor VGLL1 Drives Transcription of Human Papillomavirus Early Genes via TEAD1.

The TEAD family of transcription factors requires associating cofactors to induce gene expression. TEAD1 is known to activate the early promoter of human papillomavirus (HPV), but the precise mechanisms of TEAD1-mediated transactivation of the HPV promoter, including its relevant cofactors, remain unexplored. Here, we reveal that VGLL1, a TEAD-interacting cofactor, contributes to HPV early gene expression. Knockdown of VGLL1 and/or TEAD1 led to a decrease in viral early gene expression in human cervical keratinocytes and cervical cancer cell lines. We identified 11 TEAD1 target sites in the HPV16 long control region (LCR) by in vitro DNA pulldown assays; 8 of these sites contributed to the transcriptional activation of the early promoter in luciferase reporter assays. VGLL1 bound to the HPV16 LCR via its interaction with TEAD1 both in vitro and in vivo Furthermore, introducing HPV16 and HPV18 whole genomes into primary human keratinocytes led to increased levels of VGLL1, due in part to the upregulation of TEADs. These results suggest that multiple VGLL1/TEAD1 complexes are recruited to the LCR to support the efficient transcription of HPV early genes.IMPORTANCE Although a number of transcription factors have been reported to be involved in HPV gene expression, little is known about the cofactors that support HPV transcription. In this study, we demonstrate that the transcriptional cofactor VGLL1 plays a prominent role in HPV early gene expression, dependent on its association with the transcription factor TEAD1. Whereas TEAD1 is ubiquitously expressed in a variety of tissues, VGLL1 displays tissue-specific expression and is implicated in the development and differentiation of epithelial lineage tissues, where HPV gene expression occurs. Our results suggest that VGLL1 may contribute to the epithelial specificity of HPV gene expression, providing new insights into the mechanisms that regulate HPV infection. Further, VGLL1 is also critical for the growth of cervical cancer cells and may represent a novel therapeutic target for HPV-associated cancers.

Differential expression of human papillomavirus 16-, 18-, 52-, and 58-derived transcripts in cervical intraepithelial neoplasia.

BACKGROUND: Human papillomavirus (HPV) infection is a primary cause of cervical cancer. Although epidemiologic study revealed that carcinogenic risk differs according to HPV genotypes, the expression patterns of HPV-derived transcripts and their dependence on HPV genotypes have not yet been fully elucidated. METHODS: In this study, 382 patients with abnormal cervical cytology were enrolled to assess the associations between HPV-derived transcripts and cervical intraepithelial neoplasia (CIN) grades and/or HPV genotypes. Specifically, four HPV-derived transcripts, namely, oncogenes E6 and E6*, E1^E4, and viral capsid protein L1 in four major HPV genotypes-HPV 16, 18, 52, and 58-were investigated. RESULTS: The detection rate of E6/E6* increased with CIN progression, whereas there was no significant change in the detection rate of E1^E4 or L1 among CIN grades. In addition, we found that L1 gene expression was HPV type-dependent. Almost all HPV 52-positive specimens, approximately 50% of HPV 58-positive specimens, around 33% of HPV 16-positive specimens, and only one HPV18-positive specimen expressed L1. CONCLUSIONS: We demonstrated that HPV-derived transcripts are HPV genotype-dependent. Especially, expression patterns of L1 gene expression might reflect HPV genotype-dependent patterns of carcinogenesis.

Whole-Genome Analysis of Human Papillomavirus Type 16 Prevalent in Japanese Women with or without Cervical Lesions.

Recent large-scale genomics studies of human papillomaviruses (HPVs) have shown a high level of genomic variability of HPV16, the most prevalent genotype in HPV-associated malignancies, and provided new insights into the biological and clinical relevance of its genetic variations in cervical cancer development. Here, we performed deep sequencing analyses of the viral genome to explore genetic variations of HPV16 that are prevalent in Japan. A total of 100 complete genome sequences of HPV16 were determined from cervical specimens collected from Japanese women with cervical intraepithelial neoplasia and invasive cervical cancer, or without cervical malignancies. Phylogenetic analyses revealed the variant distribution in the Japanese HPV16 isolates; overall, lineage A was the most prevalent (94.0%), in which sublineage A4 was dominant (52.0%), followed by sublineage A1 (21.0%). The relative risk of sublineage A4 for cervical cancer development was significantly higher compared to sublineages A1/A2/A3 (odds ratio = 6.72, 95% confidence interval = 1.78-28.9). Interestingly, a novel cluster of variants that branched from A1/A2/A3 was observed for the Japanese HPV16 isolates, indicating that unique HPV16 variants are prevalent among Japanese women.

Genome-wide association study of cervical cancer suggests a role for ARRDC3 gene in human papillomavirus infection.

The development of cervical cancer is initiated by human papillomavirus (HPV) infection and involves both viral and host genetic factors. Genome-wide association studies (GWAS) of cervical cancer have identified associations in the HLA locus and two loci outside HLA, but the principal genes that control infection and pathogenesis have not been identified. In the present study, we performed GWAS of cervical cancer in East Asian populations, involving 2609 cases and 4712 controls in the discovery stage and 1461 cases and 3295 controls in the follow-up stage. We identified novel-significant associations at 5q14 with the lead single nucleotide polymorphism (SNP) rs59661306 (P = 2.4 × 10-11) and at 7p11 with the lead SNP rs7457728 (P = 1.2 × 10-8). In 5q14, the chromatin region of the GWAS-significant SNPs was found to be in contact with the promoter of the ARRDC3 (arrestin domain-containing 3) gene. In our functional studies, ARRDC3 knockdown in HeLa cells caused significant reductions in both cell growth and susceptibility to HPV16 pseudovirion infection, suggesting that ARRDC3 is involved in the infectious entry of HPV into the cell. Our study advances the understanding of host genes that are responsible for cervical cancer susceptibility and guides future research on HPV infection and cancer development.

Within-Host Variations of Human Papillomavirus Reveal APOBEC Signature Mutagenesis in the Viral Genome.

Persistent infection with oncogenic human papillomaviruses (HPVs) causes cervical cancer, accompanied by the accumulation of somatic mutations into the host genome. There are concomitant genetic changes in the HPV genome during viral infection; however, their relevance to cervical carcinogenesis is poorly understood. Here, we explored within-host genetic diversity of HPV by performing deep-sequencing analyses of viral whole-genome sequences in clinical specimens. The whole genomes of HPV types 16, 52, and 58 were amplified by type-specific PCR from total cellular DNA of cervical exfoliated cells collected from patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) and were deep sequenced. After constructing a reference viral genome sequence for each specimen, nucleotide positions showing changes with >0.5% frequencies compared to the reference sequence were determined for individual samples. In total, 1,052 positions of nucleotide variations were detected in HPV genomes from 151 samples (CIN1, n = 56; CIN2/3, n = 68; ICC, n = 27), with various numbers per sample. Overall, C-to-T and C-to-A substitutions were the dominant changes observed across all histological grades. While C-to-T transitions were predominantly detected in CIN1, their prevalence was decreased in CIN2/3 and fell below that of C-to-A transversions in ICC. Analysis of the trinucleotide context encompassing substituted bases revealed that TpCpN, a preferred target sequence for cellular APOBEC cytosine deaminases, was a primary site for C-to-T substitutions in the HPV genome. These results strongly imply that the APOBEC proteins are drivers of HPV genome mutation, particularly in CIN1 lesions.IMPORTANCE HPVs exhibit surprisingly high levels of genetic diversity, including a large repertoire of minor genomic variants in each viral genotype. Here, by conducting deep-sequencing analyses, we show for the first time a comprehensive snapshot of the within-host genetic diversity of high-risk HPVs during cervical carcinogenesis. Quasispecies harboring minor nucleotide variations in viral whole-genome sequences were extensively observed across different grades of CIN and cervical cancer. Among the within-host variations, C-to-T transitions, a characteristic change mediated by cellular APOBEC cytosine deaminases, were predominantly detected throughout the whole viral genome, most strikingly in low-grade CIN lesions. The results strongly suggest that within-host variations of the HPV genome are primarily generated through the interaction with host cell DNA-editing enzymes and that such within-host variability is an evolutionary source of the genetic diversity of HPVs.

Whole-genome analysis of human papillomavirus genotypes 52 and 58 isolated from Japanese women with cervical intraepithelial neoplasia and invasive cervical cancer.

BACKGROUND: Human papillomavirus genotypes 52 and 58 (HPV52/58) are frequently detected in patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) in East Asian countries including Japan. As with other HPV genotypes, HPV52/58 consist of multiple lineages of genetic variants harboring less than 10% differences between complete genome sequences of the same HPV genotype. However, site variations of nucleotide and amino acid sequences across the viral whole-genome have not been fully examined for HPV52/58. The aim of this study was to investigate genetic variations of HPV52/58 prevalent among Japanese women by analyzing the viral whole-genome sequences. METHODS: The entire genomic region of HPV52/58 was amplified by long-range PCR with total cellular DNA extracted from cervical exfoliated cells isolated from Japanese patients with CIN or ICC. The amplified DNA was subjected to next generation sequencing to determine the complete viral genome sequences. Phylogenetic analyses were performed with the whole-genome sequences to assign variant lineages/sublineages to the HPV52/58 isolates. The variability in amino acid sequences of viral proteins was assessed by calculating the Shannon entropy scores at individual amino acid positions of HPV proteins. RESULTS: Among 52 isolates of HPV52 (CIN1, n = 20; CIN2/3, n = 21; ICC, n = 11), 50 isolates belonged to lineage B (sublineage B2) and two isolates belonged to lineage A (sublineage A1). Among 48 isolates of HPV58 (CIN1, n = 21; CIN2/3, n = 19; ICC, n = 8), 47 isolates belonged to lineage A (sublineages A1/A2/A3) and one isolate belonged to lineage C. Single nucleotide polymorphisms specific for individual variant lineages were determined throughout the viral genome based on multiple sequence alignments of the Japanese HPV52/58 isolates and reference HPV52/58 genomes. Entropy analyses revealed that the E1 protein was relatively variable among the HPV52 isolates, whereas the E7, E4, and L2 proteins showed some variations among the HPV58 isolates. CONCLUSIONS: Among the HPV52/58-positive specimens from Japanese women with CIN/ICC, the variant distributions were strongly biased toward lineage B for HPV52 and lineage A for HPV58 across histological categories. Different patterns of amino acid variations were observed in HPV52 and HPV58 across the viral whole-genome.

Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor.

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.

Cryopreserved cultured epithelial allografts for pediatric deep partial dermal burns: Early wound closure and suppression of scarring.

BACKGROUND: In deep partial thickness dermal burns (DDB) where greater than 50% of the dermis is lost, severe pain, scarring and contractures occur. Therefore, skin grafting may be required. In children, scar contracture occurs because scarred skin does not stretch with growth creating the need for additional scar-releasing or skin-grafting surgeries. In order to resolve this problem, we used cryopreserved cultured epithelial allograft (cryopreserved allo-CEG), which can be grafted shortly after sustaining a wound. We reevaluated the promotion of early wound closure of burns and suppression of scarring by this treatment. METHODS: Cryopreserved allo-CEGs were used to treat 50 cases of pediatric DDB from 1992 to 2000. These cases were reviewed with regard to the time until epithelialization, take percentage, and pain level. Also, in order to examine why cryopreserved allo-CEG promotes healing of burns and suppresses scarring, growth factors and cytokines in the cryopreserved allo-CEG were measured. Cryopreserved allo-CEG sheets were solubilized and concentrations of TGF-α, TGF-ß1, IL-1α, IL-1ß, PDGF-AA, VEGF, KGF, IL-6, b-FGF, as well as metalloprotease-1 (MMP-1) and HGF, which are noted to have scarring suppression effects, were measured before grafting. RESULTS: Grafting of cryopreserved allo-CEGs in 50 cases of childhood DDB resulted in early epithelialization (9.32 ± 3.63 days on the average) and an almost 100% take rate. Also, pain relief (pain reduction or elimination, reduced need for anesthetics) was seen in all cases. Although 15-23 years have now elapsed, adverse events have not been observed. Cryopreserved allo-CEG contains IL-1α, IL-1ß, PDGF-AA, TGF-α, TGF-ß1, VEGF, and IL-6 have wound healing effects. The concentration of IL-1α was higher than the concentrations of other components, and this was followed by TGF-α, TGF-ß1, b-FGF and VEGF. Although the concentration of MMP-1, which has a scarring suppression effect, was high, HGF was not detected. CONCLUSION: Cryopreserved allo-CEG contains growth factors that promote wound healing and factors that suppress scarring. Three effects, namely (1) early wound closure, (2) scarring suppression, and (3) pain relief were seen with grafts of cryopreserved allo-CEG in cases of childhood DDB. These observations show that cryopreserved allo-CEG is clinically useful and effective for the treatment of childhood DDB.

Hypermutation in the E2 gene of human papillomavirus type 16 in cervical intraepithelial neoplasia.

Persistent infection with oncogenic human papillomavirus (HPV) causes cervical cancer. However, viral genetic changes during cervical carcinogenesis are not fully understood. Recent studies have revealed the presence of adenine/thymine-clustered hypermutation in the long control region of the HPV16 genome in cervical intraepithelial neoplasia (CIN) lesions, and suggested that apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) proteins, which play a key role in innate immunity against retroviral infection, potentially introduce such hypermutation. This study reports for the first time the detection of adenine/thymine-clustered hypermutation in the E2 gene of HPV16 isolated from clinical specimens with low- and high-grade CIN lesions (CIN1/3). Differential DNA denaturation PCR, which utilizes lower denaturation temperatures to selectively amplify adenine/thymine-rich DNA, identified clusters of adenine/thymine mutations in the E2 gene in 4 of 11 CIN1 (36.4%), and 6 of 27 CIN3 (22.2%) samples. Interestingly, the number of mutations per sample was higher in CIN3 than in CIN1. Although the relevance of E2 hypermutation in cervical carcinogenesis remains unclear, the observed hypermutation patterns strongly imply involvement of APOBEC3 proteins in editing the HPV16 genome during natural viral infection.

Identification of APOBEC3B promoter elements responsible for activation by human papillomavirus type 16 E6.

Recent cancer genomics studies have identified mutation patterns characteristic of APOBEC3B (A3B) in multiple cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. A3B expression is upregulated by HPV E6/E7 oncoproteins, implying a crucial role for A3B upregulation in HPV-induced carcinogenesis. Here, we explored the molecular mechanisms underlying the activation of the A3B promoter by E6. Luciferase reporter assays with a series of deleted fragments of the human A3B promoter in normal immortalized human keratinocytes (NIKS) identified two functional regions in the promoter: the distal region (from -200 to -51), which is required for basal promoter activity, and the proximal region (from +1 to +45), which exerts an inhibitory effect on gene expression. Each promoter region was found to contain an E6-responsive element(s). Disruption of an AT-rich motif located between +10 and +16 abrogated the proximal-region-mediated activation of the A3B promoter by E6. DNA pull-down assays revealed that a cellular zinc-finger protein, ZNF384, binds to the AT-rich motif in the A3B promoter, and chromatin immunoprecipitation assays confirmed that ZNF384 binds to the A3B promoter in cells. ZNF384 knockdown reduced the A3B mRNA levels in NIKS expressing E6, but not in the parental NIKS, indicating that ZNF384 contributes to A3B upregulation by E6, but not to basal A3B expression. The exogenous expression of ZNF384 led to the activation of the A3B promoter in NIKS. Collectively, these results indicate that E6 activates the A3B promoter through the distal and proximal regions, and that ZNF384 is required for the proximal-region-mediated activation of A3B.

APOBEC3A and 3C decrease human papillomavirus 16 pseudovirion infectivity.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) proteins are cellular DNA/RNA-editing enzymes that play pivotal roles in the innate immune response to viral infection. APOBEC3 (A3) proteins were reported to hypermutate the genome of human papillomavirus 16 (HPV16), the causative agent of cervical cancer. However, hypermutation did not affect viral DNA maintenance, leaving the exact role of A3 against HPV infection elusive. Here we examine whether A3 proteins affect the virion assembly using an HPV16 pseudovirion (PsV) production system, in which PsVs are assembled from its capsid proteins L1/L2 encapsidating a reporter plasmid in 293FT cells. We found that co-expression of A3A or A3C in 293FT cells greatly reduced the infectivity of PsV. The reduced infectivity of PsV assembled in the presence of A3A, but not A3C, was attributed to the decreased copy number of the encapsidated reporter plasmid. On the other hand, A3C, but not A3A, efficiently bound to L1 in co-immunoprecipitation assays, which suggests that this physical interaction may lead to reduced infectivity of PsV assembled in the presence of A3C. These results provide mechanistic insights into A3s' inhibitory effects on the assembly phase of the HPV16 virion.

In vivo and in vitro studies suggest a possible involvement of HPV infection in the early stage of breast carcinogenesis via APOBEC3B induction.

High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction.

Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases.

BACKGROUND: Co-infection of multiple genotypes of human papillomavirus (HPV) is commonly observed among women with abnormal cervical cytology, but how different HPVs interact with each other in the same cell is not clearly understood. A previous study using cultured keratinocytes revealed that genome replication of one HPV type is inhibited by co-existence of the genome of another HPV type, suggesting that replication interference occurs between different HPV types when co-infected; however, molecular mechanisms underlying inter-type replication interference have not been fully explored. METHODS: Replication interference between two most prevalent HPV types, HPV16 and HPV18, was examined in HPV-negative C33A cervical carcinoma cells co-transfected with genomes of HPV16 and HPV18 together with expression plasmids for E1/E2 of both types. Levels of HPV16/18 genome replication were measured by quantitative real-time PCR. Physical interaction between HPV16/18 E1s was assessed by co-immunoprecipitation assays in the cell lysates. RESULTS: The replication of HPV16 and HPV18 genomes was suppressed by co-expression of E1/E2 of heterologous types. The interference was mediated by the heterologous E1, but not E2. The oligomerization domain of HPV16 E1 was essential for HPV18 replication inhibition, whereas the helicase domain was dispensable. HPV16 E1 co-precipitated with HPV18 E1 in the cell lysates, and an HPV16 E1 mutant Y379A, which bound to HPV18 E1 less efficiently, failed to inhibit HPV18 replication. CONCLUSIONS: Co-infection of a single cell with both HPV16 and HPV18 results in replication interference between them, and physical interaction between the heterologous E1s is responsible for the interference. Heterooligomers composed of HPV16/18 E1s may lack the ability to support HPV genome replication.