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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.

Simple Summary Transcription factor homeobox D9 (HOXD9) was previously reported to bind to the P97 promoter of HPV16 to induce viral E6/E7 oncogenes. In this article, we investigated whether HOXD9 regulated the P105 promoter of HPV18 and examined the role of HOXD9 in intracellular signaling of cervical cancer (CC). HOXD9 was directly bound to the P105 promoter and regulated the expression of E6/E7 genes of HPV18. The HOXD9 knockdown suppressed the E6/E7 gene expression in HPV18-positive cervical cancer cells. It decreased the expression of E6, activated the p53 pathway, and induced apoptosis. In addition, downregulation of the E7 gene expression activated the Rb pathway, causing G1 arrest in the cell cycle and markedly suppressing cell proliferation. Our results indicate that HOXD9 has pivotal roles in the proliferation and immortalization of HPV18-positive cervical cancer cells through activating the P105 promoter. Abstract 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.

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.

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.

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-β1, IL-1α, IL-1β, 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-1β, PDGF-AA, TGF-α, TGF-β1, 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-β1, 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.

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.

金沢大学医薬保健研究域医学系, 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. © 2020 The Author(s).

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.

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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13027-017-0155-4) contains supplementary material, which is available to authorized users.

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.

We have newly developed the mutual-capacitive touch screen using double layered metal mesh electrodes with separated floating electrodes in borders between X- and Y-directional electrodes. We have defined the effect of the floating electrode with an electric field simulation and improved the sensitivity and the response time of the touch screen by separating the floating electrode into several small patterns.

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&ndash, 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.

We have developed a new high performance projected capacitive touch screen which has double layered metal mesh electrodes. By using high conductive metal electrodes and optimizing the mesh design, high sensitive 15-inch diagonal self capacitance touch screen and 8-inch diagonal mutual capacitance touch screen, both of which are optically bonded to LCD modules, have been developed.

We report the prevalence and genotype distribution of human papillomaviruses (HPVs) among Japanese women with abnormal cervical cytology using the PGMY-CHUV assay, one of PGMY-PCR-based lineblot assays that was validated and shown to be suitable for the detection of multiple HPV types in a specimen with minimum bias. Total DNA was extracted from cervical exfoliated cells collected from 326 outpatients with abnormal Pap smears. Overall, 307 specimens (94%) were HPV-positive, 30% of which contained multiple genotypes. The prevalence of HPV DNA was 83% (49/59 samples) in atypical squamous cells of undetermined significance (ASC-US); 91% (20/22 samples) in atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H); 97% (130/134 samples) in low-grade squamous intraepithelial lesion (LSIL); and 99% (85/86 samples) in high-grade squamous intraepithelial lesion (HSIL). Three most frequent HPV types detected in HSIL were HPV16 (36%), HPV52 (24%), and HPV58 (14%). Our results suggest that multiple HPV infections are more prevalent in Japanese women than previously reported, and confirm that HPV52 and 58 are more dominant in their cervical precancerous lesions when compared to those reported in Western countries.

A VeraCode-allele-specific primer extension (ASPE) method was applied to the detection and genotyping of human papillomavirus (HPV)-DNA. Oligonucleotide primers containing HPV-type-specific L1 sequences were annealed to HPV-DNA amplified by PGMY-PCR, followed by ASPE to label the DNA with biotinylated nucleotides. The labeled DNA was captured by VeraCode beads through hybridization, stained with a streptavidin-conjugated fluorophore, and detected by an Illumina BeadXpress® reader. By using this system, 16 clinically important HPV types (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) were correctly genotyped in a multiplex format. The VeraCode-ASPE genotyping of clinical DNA samples yielded identical results with those obtained by validated PGMY-reverse blot hybridization assay, providing a new platform for high-throughput genotyping required for HPV epidemiological surveys.

Rabbit anti-HPV16 L2 serum (anti-P56/75) neutralizes multiple oncogenic human papillomaviruses (HPVs). We inoculated HeLa cells with HPV16 pseudovirus (16PV) and with anti-P56/75-bound 16PV (16PV-Ab). Both 16PV and 16PV-Ab attached equally well to the cell surface. However, the cell-attached L1 protein of 16PV became trypsin-resistant after incubation at 37°C, whereas approximately 20% of the cell-attached 16PV-Ab L1 remained trypsin-sensitive. Confocal microscopy of HeLa cells inoculated with 16PV revealed packaged DNA in the nucleus at 22h after inoculation; however, nuclear DNA was not detected in cells inoculated with 16PV-Ab. Electron microscopy of HeLa cells inoculated with 16PV showed particles located in multivesicular bodies, lamellar bodies, and the cytosol after 4h; no cytosolic particles were detected after inoculation with 16PV-Ab. These data suggest that anti-P56/75 inhibits HPV infection partly by blocking viral entry and primarily by blocking the transport of the viral genome to the nucleus.

The deduced amino acid ( aa ) sequence of L2 of the newly sequenced HPV51 strain, isolated by Matsukura and Sugase (Ma-strain), was markedly different from that of the prototype HPV51 isolated by Nuovo et al. (Nu-strain) (GenBank M62877 ) in two regions: aa 95–99 (region I) and aa 179–186 (region II). The two regions of Ma-strain were homologous to those of the other mucosal HPVs. The aa sequences of the N-terminal and C-terminal regions of Ma-L2 and Nu-L2 were identical and contained the nuclear localizing signal (NLS). When expressed in HEK293 cells, Ma-strain L2 (Ma-L2) was located in the nucleus but Nu-strain L2 (Nu-L2), in the cytoplasm. The chimeric L2s having both Nu-L2 regions I and II were located in the cytoplasm, and those having one of them were located both in the nucleus and cytoplasm, suggesting that Nu-L2 regions I and II inhibit the NLS function. For a better understanding of a role of L2 in infection, pseudovirion (PV) preparations were produced with a reporter, Ma-strain L1, and various L2s (Ma-L2, Nu-L2, or the chimeric L2s). These PV preparations contained structurally similar particles composed of L1 and L2 and the packaged reporter plasmid at a similar level. The reporter expression was not induced in HEK293 cells after inoculation with PVs containing the L2s that are incapable of localizing in the nucleus when expressed alone. Among PVs containing L2s capable of localizing in the nucleus, the reporter expression was induced only by PVs containing Ma-L2 region I. Thus, the results indicate that the expression of the reporter in the HPV51 PV requires the nuclear localizing ability of L2 and another unknown function associated with region I.

Human papillomavirus type 16 (HPV16) DNA replication, which requires two viral proteins E1 and E2, occurs only in the differentiating epithelium. Besides the general factors necessary for cellular DNA synthesis, other unidentified cellular factors are assumed to be involved in the regulation of HPV DNA replication. In the present study, we found that the POU-domain transcription factor human Skn-1a, which induces the terminal differentiation of keratinocytes and activates the HPV16 late promoter, enhanced the transient replication of a plasmid containing the HPV16 replication origin in HEK293 cells when co-transfected with a plasmid expressing E1 and E2. An electrophoretic mobility shift assay with a bacterially expressed human Skn-1a or an extract of HeLa cells over-expressing human Skn-1a revealed the presence of two human Skn-1a binding sites that are distinct from the known three sites, near the replication origin. A chromatin immunoprecipitation analysis showed that human Skn-1a bound to these sites in cells. Nucleotide substitutions in the sites abolished the binding of human Skn-1a and the human Skn-1a-mediated replication enhancement. The data strongly suggest that, through the binding to the two sites, human Skn-1a enhances HPV DNA replication.

In host animals, adeno-associated virus (AAV) is detectable mainly in the lymphoid tissue, which appears to be a target in natural infection. We used the human monocytic cell lines THP-1 and U937 to study the effect of mouse anti-AAV2 antiserum on infection with an AAV2 vector having the luciferase gene (AAV2/Luc). AAV2/Luc was found to infect THP-1 and U937 cells much less efficiently than HeLa cells, as monitored with the induced enzyme activity. Pre-incubation of AAV2/Luc with anti-AAV2 antiserum at a sub-neutralizing concentration enhanced by 2-to-10 fold infection of THP-1 and U937 with AAV2/Luc, but not of HeLa. Similarly, anti-AAV10 serum at a low level enhanced infection of THP-1 with AAV10/Luc. Sera of two cynomolgus monkeys, which had been probably infected with an AAV2-like virus, enhanced infection of THP-1 with AAV2/Luc. The enhancement was reduced with blocking the IgG-receptors Fcgamma-RI and Fcgamma-RII, which were displayed on the surface of THP-1 and U937 but not HeLa cells, with anti-Fcgamma-RI antibody or anti-Fcgamma-RII antibody. The data indicate that infection of Fcgamma receptor-bearing cells with AAV is enhanced by anti-AAV IgG antibodies at a sub-neutralizing concentration that play a role in linking AAV particles and Fcgamma receptors.

Adeno-associated virus (AAV) is used in gene-therapy studies, but its tissue distribution is unknown in natural infection. We examined cynomolgus AAVs (previously isolated AAV10 and AAV11 and novel AAVcy.7) for their tissue distribution in 14 cynomolgi by type-specific PCR. We found AAV10, AAV11, and AAVcy.7 in 6, 10, and 14 monkeys, respectively, and two or three types in 11 monkeys, showing that these AAVs are widespread in the monkeys. We detected AAV at a higher level mainly in the lymphatic tissues and ileum, which suggests that AAV may invade the host through Peyer's patches in the ileum and infect immune cells.

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.

Production of human papillomavirus type 16 major capsid protein L1 in undifferentiated cells is negatively regulated by several yet unidentified cis-acting inhibitory RNA elements, among which a major element is located within the first 514 nucleotides of the L1-mRNA. By Northern blotting we examined effect of the major element on the steady-state level of mRNA transiently transcribed in 293T cells from the firefly luciferase (Fluc) gene combined with the L1 DNA fragment encoding the major element. As reported previously, the element down-regulated steady-state level of the mRNA. The most efficient down-regulation was achieved by insertion of the element near the 5' end of mRNA, resulting in an undetectable level of the mRNA. The longer the distance from the 5' end of the mRNA to the element, the weaker the down-regulation. The half-life of the mRNA having the element was similar to that of normal Fluc-mRNA. When the element near the 5' end was removed by splicing, the steady-state level of the resultant mRNA was raised to a readily detectable level. The steady-state level of RNA synthesized by RNA polymerase-I was not influenced by the presence of the element. Taken together, it is suggested that DNA region encoding the major inhibitory element does not disturb transcription and that the pre-mRNA is degraded by an RNA element-mediated mechanism after the splicing step in the course of mRNA maturation.

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.

After trauma or excision of malignant tumor, it is difficult to achieve satisfactory results when reconstructing deformed eyelids and the socket for an ocular prosthesis. The authors demonstrate examples of successful reconstruction for a prosthetic eye that provided adequate and aesthetic soft-tissue support achieved by applying a three-step surgical procedure of reconstruction of the eye socket, the eyelids, and the tarsus and eyelid margin. Because it is highly vascularized and its distal end can be divided into two or three portions for easy three-dimensional reconstruction, the expanded forehead flap alone, with a galea flap, or with a free rectus abdominis muscle perforator flap was used. The expanded forehead flap also provides excellent thin upper lid contour and good color-matching with a recipient site. For the eye socket, sufficient volume of tissue was provided from the expanded forehead flap with or without a galea or a free rectus abdominis muscle perforator flap, and a deep and convex fornix was formed. This resulted in a good fit and in stability of the ocular prosthesis. The surface and the inner lining of the eyelids were reconstructed using portions of the expanded forehead flap. For the tarsus and eyelid margin, conventional reconstruction techniques use cartilage of the concha, which has limitations of length and which does not fit the shape of the tarsal margin. The authors used the scapha composite graft, and a natural shape and good elasticity resulted.

To achieve a higher take rate for epithelial grafts, this study investigated grafting techniques. Seventy-seven nude mice received flap grafting in which cultured human epithelium was grafted inside the flap, and 55 nude rats received transplantation of epithelium to a full-thickness skin defect. In each group, four models were studied, including model 1, in which epithelium was cultured with the conventional method; model 2, in which epithelium was cultured with fibrin gel to avoid sheet damage, then absorptive mesh was incorporated into the epithelium for anchoring to the graft bed; model 3, in which epithelium was cultured with fibrin gel and combined with absorptive mesh and artificial dermis containing fibroblasts; and model 4, in which the model 2 epithelium was grafted after artificial dermis was transplanted. The take for these models was evaluated grossly and histologically. The results show that the take percentage of models 2 and 3 was significantly higher than that of model 1 (conventional epithelium) and that there was no significant difference between model 3 (simultaneous grafting) and model 4 (two-step grafting). The difference in the take percentages of the grafts to the flap and to the full-thickness skin defect was also insignificant. In immunohistochemistry, human keratin appeared in all epidermis layers and diversification of the layer was observed in models 2, 3, and 4. In these three models, type IV collagen appeared in the basal layer and the formation of basal membrane was confirmed. These findings suggest that epithelia cultured on fibrin gel and combined with absorptive mesh could be used in a new technique for better, more stable take.

We previously observed that human cytomegalovirus (CMV) infection induced a massive production of a chemokine with potent neutrophil chemotactic activity, interleukin-8 (IL-8). Hence, we examined the effect of CMV immediate early (IE) gene products on IL-8 production by the human astrocytoma cell line, U373MG. Transient or stable transfection with a CMV IE1 gene expression vector, but not with a IE2 gene expression vector, significantly augmented IL-8 protein secretion and IL-8 mRNA expression. Luciferase activity was enhanced in U373MG cells when the cells were cotransfected with CMV IE1 and chimeric firefly luciferase reporter genes driven by the transcriptional regulatory region of the human IL-8 gene. Moreover, IE1 gene-mediated enhancement of luciferase activity was abolished by the introduction of mutations into the AP-1 or NF-kappa B factor binding elements in the regulatory region of the IL-8 promoter. Furthermore, electrophoretic mobility shift assays demonstrated that CMV IE1 gene products induced the formation of NF-kappa B or AP-1 complexes. Finally, Western blotting analysis demonstrated that the CMV IE1 gene product increased the amount of NF-kappa B complexes translocated into the nucleus. Collectively, CMV IE1 gene expression may be sufficient to activate AP-1 and NF-kappa B, resulting in IL-8 gene expression.

GaAs/AlGaAs quantum dots were grown in tetrahedral-shaped recesses (TSRs) on GaAs ( 1 1 1 ) B substrates at 850°C by metalorganic vapor-phase epitaxy on the bottom ( 1 1 1 ) B regions of the TSRs. As the AlGaAs barrier layer became thicker, a trench-like region comprised of {11 2 } and { 1 1 2} vertical sidewalls formed at the bottom region of the TSR. A 780 A -wide and 96 A -thick GaAs quantum dot was formed at the bottom of the trench-like region. The luminescence from GaAs layer at only the ( 1 1 1 ) B bottom region of the TSR was confirmed by cathodoluminescence image observation. By micro-photoluminescence observation a narrow line from the bottom region was observed at 1.5511 eV and the luminescence from higher-energy states was observed. The calculation for a 780 A ×780 A ×96 A quantum box revealed that these energy states reflected both the lateral and vertical quantum confinements.

Previously, we found that a ganciclovir (GCV)-resistant clinical human cytomegalovirus (HCMV) isolate had an amino acid substitution at codon 501 (Leu→Phe) in the δ-region C of the DNA polymerase gene. DNA polymerases have now been (partially) purified from both the GCV-resistant and sensitive parental strains and the activity of DNA polymerase and 3′-5′ exonuclease compared. With respect to DNA polymerase activity, the Michaelis constant ( K m ) and maximum velocity ( V max ) of the GCV-resistant strain for the DNA template were lower than those of the GCV-sensitive strain. With respect to 3′-5′ exonuclease activity, the K m and V max of the GCV-resistant strain for the DNA substrate in the presence of ammonium sulfate were lower than those of the GCV-sensitive strain, while being similar in the absence of ammonium sulfate. Although the polymerase activity of the two strains showed almost the same sensitivity for the different polymerase inhibitors, the 3′-5′ exonuclease activity of the GCV-resistant strain was more resistant to these inhibitors than that of the GCV-sensitive strain.

The 30-base pair (bp) deletion of the cytoplasmic carboxy-terminal domain of the latent membrane protein 1 (LMP-1) gene was analyzed in 37 frozen tissues from patients with Epstein-Barr virus (EBV)-related gastric cancer and 18 throat washings from healthy adults in southern Japan. The 30-bp deletion was identified in 33 (91.7%) of 36 specimens of EBV-related gastric cancers and in 15 (83.3%) of 18 throat washings from healthy adults. In one case of gastric cancer, an additional 9-bp deletion was identified downstream of the 30-bp deletion. From the last transmembane domain to the end of the carboxy terminal of LMP-1, mutations were examined in 37 cases of gastric cancers and in three cases of throat washings. Twenty-eight nonsilent mutations were identified in this region of EBV-related gastric cancer and throat washings. Five nonsilent mutations at positions 168,755, 168,746, 168,687, 168,357, and 168,355 were identified in all 30-bp-deleted cases of EBV-related gastric cancers and throat washings. However, these nonsilent mutations were not identified in three patients without the 30-bp deletion. Although the deletion and single-base mutations of the LMP-1 gene in gastric cancers and throat washings were similar to those of nasopharyngeal carcinoma in Taiwan and China, more single-base mutations were found in southern Japan. These data indicate that high prevalence of the 30-bp deletion of the LMP-1 gene in gastric cancers may reflect the prevalence of the deletion variant in the normal population in southern Japan. J. Med. Virol. 57:152–158, 1999. © 1999 Wiley-Liss, Inc.

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.

Human papillomavirus (HPV) is a non-enveloped virus composed of a circular DNA genome and two capsid proteins, L1 and L2. Multiple interactions between its capsid proteins and host cellular proteins are required for infectious HPV entry, including cell attachment and internalization, intracellular trafficking and viral genome transfer into the nucleus. Using two variants of HPV type 51, the Ma and Nu strains, we have previously reported that MaL2 is required for efficient pseudovirus (PsV) transduction. However, the cellular factors that confer this L2 dependency have not yet been identified. Here we report that the transport protein particle complex subunit 8 (TRAPPC8) specifically interacts with MaL2. TRAPPC8 knockdown in HeLa cells yielded reduced levels of reporter gene expression when inoculated with HPV51Ma, HPV16, and HPV31 PsVs. TRAPPC8 knockdown in HaCaT cells also showed reduced susceptibility to infection with authentic HPV31 virions, indicating that TRAPPC8 plays a crucial role in native HPV infection. Immunofluorescence microscopy revealed that the central region of TRAPPC8 was exposed on the cell surface and colocalized with inoculated PsVs. The entry of Ma, Nu, and L2-lacking PsVs into cells was equally impaired in TRAPPC8 knockdown HeLa cells, suggesting that TRAPPC8-dependent endocytosis plays an important role in HPV entry that is independent of L2 interaction. Finally, expression of GFP-fused L2 that can also interact with TRAPPC8 induced dispersal of the Golgi stack structure in HeLa cells, a phenotype also observed by TRAPPC8 knockdown. These results suggest that during viral intracellular trafficking, binding of L2 to TRAPPC8 inhibits its function resulting in Golgi destabilization, a process that may assist HPV genome escape from the trans-Golgi network.

Antisera induced by immunization of rabbits with the synthetic peptide P56/75, which has the amino acid (aa) sequence from aa56 to aa75 of HPV16 L2, neutralize pseudovirions and raft-virions of multiple high-risk HPV types, indicating that cross-neutralization epitopes are present in the aa56–75 region. We generated two mouse monoclonal antibodies (MAb): MAb13B and MAb24B recognizing the regions of aa64–73 and aa58–64, respectively. The neutralization assay using pseudovirions of HPV16, 18, 31, 33, 35, 51, 52 and 58 showed that MAb13B neutralized HPV16, 18, and 51, and MAb24B neutralized all the types tested. The mixture of MAb13B and MAb24B neutralized HPV16, 18, and 51 pseudovirions more efficiently than each of the MAbs alone. The data indicate that there are at least two cross-neutralization epitopes in the aa56–75 region and that an antigen capable of presenting the two cross-neutralization epitopes would be a good vaccine candidate for a broad-spectrum of HPVs.

A VeraCode-allele-specific primer extension (ASPE) method was applied to the detection and genotyping of human papillomavirus (HPV)-DNA. Oligonucleotide primers containing HPV-type-specific L1 sequences were annealed to HPV-DNA amplified by PGMY-PCR, followed by ASPE to label the DNA with biotinylated nucleotides. The labeled DNA was captured by VeraCode beads through hybridization, stained with a streptavidin-conjugated fluorophore, and detected by an Illumina BeadXpress® reader. By using this system, 16 clinically important HPV types (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) were correctly genotyped in a multiplex format. The VeraCode-ASPE genotyping of clinical DNA samples yielded identical results with those obtained by validated PGMY-reverse blot hybridization assay, providing a new platform for high-throughput genotyping required for HPV epidemiological surveys.

Mouse 3T3-Swiss albino cells are widely used as feeder cells to culture the human epidermis for the treatment of burns. To minimize the risk of xenoinfection, quality control of the feeder cells is required in the Japanese guidelines on regenerative medicine using feeder cells. We characterized three lots of 3T3-Swiss albino cells that are publicly or commercially available in Japan. One lot, which propagated more rapidly than the other two without showing typical contact inhibition, was found to release endogenous murine leukemia virus upon iododeoxyuridine-treatment. Southern blotting of restriction fragments showed that the rapidly growing lot consisted of genetically altered cells that had probably emerged during the passages. The data support the guidelines that recommend the quality control of each lot of 3T3-Swiss albino cells if they are to be used clinically.

In gene therapy trials, adeno-associated virus (AAV) vectors are injected directly into target tissues such as muscle and liver. Direct injection can lead to the introduction of a low level of the vector into blood circulation. To determine the systemic effects of the vector released in the blood, we extensively examined the biodistribution of intravenously administered AAV serotype 2 (AAV2) vector in cynomolgus monkeys. Although the vector distribution pattern varied from monkey to monkey, the vector DNA was maintained in the various tissues beyond 7 months post-inoculation (pi). The vector DNA was detected in the lymphoid tissues, particularly in the spleen, more frequently and at a much higher level than in the other tissues tested (i.e., brain, lung, liver, heart, gallbladder, pancreas, colon, kidney, ovary, uterus, etc.). The expression of a transgene was detected in the lymph nodes at 3 months pi. The distribution of two pseudotyped vectors, AAV2/10 and AAV2/11, was similar to that of the AAV2 vector. The present results suggest that when introduced intravenously, the AAV vector DNA persists and may induce transgene expression in various monkey tissues. Thus, the possibility of inadvertent gene transfer to various non-target tissues should be considered in a gene therapy strategy with an AAV vector.