Iwao Kukimoto

Enhanced oncogenicity of human papillomavirus type 16 (HPV16) variants in Japanese population

To investigate whether HPV16 E6 variants carry an elevated risk for cervical cancer in Japanese population, we investigated the E6 sequence variation in 40 cervical intraepithelial neoplasias (CINs) I-III and 43 invasive cervical cancers (ICCs), all positive for HPV16. HPV16 E6 variants were frequently found in ICCs than in CINs (88 vs. 65%, P=0.01). The E6 D25E, a rare variant in Western countries, was most frequently observed in ICC (44%). CIN I/II lesions with HPV16 variants were less likely to regress than those with HPV16 prototype (P=0.048). The finding that HPV16 E6 variants represent a significant risk factor is common between Western and Japanese women despite the different distribution of each variant.

APOBEC3 deaminases induce hypermutation in human papillomavirus 16 DNA upon beta interferon stimulation

ABSTRACT 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.

Biased amplification of human papillomavirus DNA in specimens containing multiple human papillomavirus types by PCR with consensus primers

Genotyping human papillomavirus (HPV) in clinical specimens is important because each HPV type has different oncogenic potential. Amplification of HPV DNA by PCR with the consensus primers that are derived from the consensus sequences of the L1 gene has been used widely for the genotyping. As recent studies have shown that the cervical specimens often contain HPV of multiple types, it is necessary to confirm whether the PCR with the consensus primers amplifies multiple types of HPV DNA without bias. We amplified HPV DNA in the test samples by PCR with three commonly used consensus primer pairs (L1C1/L1C2+C2M, MY09/11, and GP5+/6+), and the resultant amplicons were identified by hybridization with type‐specific probes on a nylon membrane. L1C1/L1C2+C2M showed a higher sensitivity than the other primers, as defined by the ability to detect HPV DNA, on test samples containing serially diluted one of HPV16, 18, 51, 52, and 58 plasmids. L1C1/L1C2+C2M failed to amplify HPV16 in the mixed test samples containing HPV16, and either 18 or 51. The three consensus primers frequently caused incorrect genotyping in the selected clinical specimens containing HPV16 and one or two of HPV18, 31, 51, 52, and 58. The data indicate that PCR with consensus primers is not suitable for genotyping HPV in specimens containing multiple HPV types, and suggest that the genotyping data obtained by such a method should be carefully interpreted. (Cancer Sci 2011; 102: 1223–1227)

Displacement of YY1 by differentiation-specific transcription factor hSkn-1a activates the P(670) promoter of human papillomavirus type 16

ABSTRACT Transcription from human papillomavirus type 16 (HPV16) P670, a promoter in the E7 open reading frame, is repressed in undifferentiated keratinocytes but becomes activated upon differentiation. We showed that the transient luciferase expression driven by P670 was markedly enhanced in HeLa cells cotransfected with an expression plasmid for human Skn-1a (hSkn-1a), a transcription factor specific to differentiating keratinocytes. The hSkn-1a POU domain alone, which mediates sequence-specific DNA binding, was sufficient to activate the expression of luciferase. Electrophoretic mobility shift assay revealed the presence of two binding sites, sites 1 and 2, upstream of P670, which were shared by hSkn-1a and YY1. Site 1 bound more strongly to hSkn-1a than site 2 did. YY1 complexing with a short DNA fragment having site 1 was displaced by hSkn-1a, indicating that hSkn-1as affinity with site 1 was stronger than YY1s. Disrupting the binding sites by nucleotide substitutions raised the basal expression level of luciferase and decreased the enhancing effect of hSkn-1a. In HeLa cells transfected with circular HPV16 DNA along with the expression plasmid for hSkn-1a, the transcript from P670 was detectable, which indicates that the results obtained with the reporter plasmids are likely to have mimicked the regulation of P670 in authentic HPV16 DNA. The data strongly suggest that the transcription from P670 is repressed primarily by YY1 binding to the two sites, and the displacement of YY1 by hSkn-1a releases P670 from the repression.

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.

Genetic variation of human papillomavirus type 16 in individual clinical specimens revealed by deep sequencing.

Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.

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

BackgroundCo-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.MethodsReplication 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.ResultsThe 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.ConclusionsCo-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.

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. J. Med. Virol. 87:1754–1760, 2015.

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.

Human Papillomavirus Genotype Distribution in Cervical Intraepithelial Neoplasia Grade 2/3 and Invasive Cervical Cancer in Japanese Women

OBJECTIVEnHuman 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.nnnMETHODSnCervical 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.nnnRESULTSnOverall, 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.nnnCONCLUSIONSnThe 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.