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.

Lecompte maneuver for compressed left coronary artery after pulmonary artery banding.

A one-month-old baby boy with a complete atrioventricular septal defect underwent pulmonary artery banding. A high take-off of the left coronary artery, overlooked on the echocardiogram, was identified. It was compressed by the right pulmonary artery that was dilated owing to pulmonary artery banding. The patient developed severe heart failure, and a Lecompte maneuver was performed. The procedure helped effectively treat this congenital heart disease with a high take-off coronary artery compressed by the right pulmonary artery.

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.

Total arch replacement for re-coarctation of the aorta after a Norwood procedure.

This is the first report of total arch replacement to repair re-coarctation. A 14-year-old boy with hypoplastic left heart syndrome developed re-coarctation, severe stenosis of neck vessels, and right ventricle dysfunction after a Norwood procedure. We performed total arch replacement; the postoperative course was unremarkable. He was followed up until 18 years of age and did not need re-intervention. Using artificial blood vessels in total arch replacement is rarely indicated but can be safely achieved when required. Mismatch between patient and graft size may be an issue in the future.

Aortic valve replacement using Konno procedure for prior Yasui procedure.

A 2-year-old girl underwent conversion to the Konno procedure by removing the Damus-Kaye-Stansel anastomosis after the neonatal Yasui procedure for an interrupted aortic arch with left ventricular outflow tract stenosis. Her postoperative course was uneventful. However, left ventricular outflow tract restenosis occurred due to narrowed ventricular septal defect and moderate neoaortic regurgitation from the old pulmonary valve. The Konno procedure was performed by removing the Damus-Kaye-Stansel anastomosis for left ventricular outflow tract restenosis and neoaortic regurgitation and performing right ventricular outflow tract reconstruction and ventricular septal defect closure. Left ventricular outflow tract restenosis was not observed.

A valuable echocardiographic indicator for the optimal tightness of bilateral pulmonary artery banding.

OBJECTIVES: The optimal tightness of bilateral pulmonary artery banding (BPAB) is considered to balance not only systemic-to-pulmonary blood flow but also each pulmonary blood flow, which is still challenging. To achieve them, we adopt the end-diastolic velocity (EDV) to the peak systolic velocity (PSV) ratio at BPAB with intraoperative epicardial echocardiography. We evaluated the usefulness of the EDV to PSV ratio and the patient outcomes. METHODS: 34 patients underwent BPAB with this indicator and using a looped polytetrafluoroethylene suture. The PSV and the EDV to PSV ratio with echocardiography were measured in the intraoperative, early postoperative and late postoperative period. Lung perfusion scintigraphy was performed to quantify flow to each lung. RESULTS: There were 3 early deaths (< 30 days). Two patients required re-BPAB due to hypoxia. The intraoperative EDV to PSV ratios in the right and left were almost equal (0.50 ± 0.07 versus 0.51 ± 0.06, P = 0.73). There was no significant difference in the right and left EDV to PSV ratios throughout the postoperative course. The right PSV was smaller than the left PSV due to the Doppler angle intraoperatively (2.78 ± 0.57 versus 3.02 ± 0.50, P = 0.030). In addition, the PSV changed significantly until the late postoperative period (P < 0.001). Lung perfusion scintigraphy revealed only two patients had perfusion abnormalities. CONCLUSIONS: Our clinical outcomes are satisfactory with low early mortality and a low rate of re-BPAB. The EDV to PSV ratio can be a reliable indicator to assess flow distribution to each lung and may be a valuable adjunct to achieve balanced systemic to pulmonary flow.

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.

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.

Infected pulmonary aneurysm following pulmonary artery banding.

A 1-month-old girl, diagnosed with a common atrioventricular canal, moderate atrioventricular valvular regurgitation, and pulmonary hypertension, underwent pulmonary artery banding. Postoperatively, methicillin-resistant Staphylococcus aureus wound infection was treated with antibiotics. One month later, emergency surgery was performed for oozing rupture of an infected pulmonary aneurysm. The pulmonary aneurysm was completely resected, the banding tape was removed, and pulmonary angioplasty was performed to create pulmonary stenosis without using foreign material. Methicillin-resistant Staphylococcus aureus was cultured from the resected tissues and banding tape. The patient was discharged after antibiotic administration. Correction was performed at 1 year of age, and she remains well.

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.

A case of biatrial drainage of the right superior vena cava.

Biatrial drainage of the right superior vena cava resulting from a sinus venosus defect is a rare congenital malformation. It can result in severe desaturation although a sinus venosus defect usually presents as a left-to-right shunt. A male baby was noted to have cyanosis while nursing and was referred to us for medical treatment on his second day of life. Echocardiography showed that most of the blood flowing through the superior vena cava drained into the left atrium. He underwent successful surgery to correct this defect at the age of 2 years.

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.

Postoperative atypical hemolytic uremic syndrome associated with complement c3 mutation.

Atypical hemolytic uremic syndrome (aHUS) can be distinguished from typical or Shiga-like toxin-induced HUS. The clinical outcome is unfavorable; up to 50% of affected patients progress to end-stage renal failure and 25% die during the acute phase. Multiple conditions have been associated with aHUS, including infections, drugs, autoimmune conditions, transplantation, pregnancy, and metabolic conditions. aHUS in the nontransplant postsurgical period, however, is rare. An 8-month-old boy underwent surgical repair of tetralogy of Fallot. Neurological disturbances, acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia developed 25 days later, and aHUS was diagnosed. Further evaluation revealed that his complement factor H (CFH) level was normal and that anti-FH antibodies were not detected in his plasma. Sequencing of his CFH, complement factor I, membrane cofactor protein, complement factor B, and thrombomodulin genes was normal. His ADAMTS-13 (a disintegrin-like and metalloprotease with thrombospondin-1 repeats 13) activity was also normal. However, he had a potentially causative mutation (R425C) in complement component C3. Restriction fragment length polymorphism analysis revealed that his father and aunt also had this mutation; however, they had no symptoms of aHUS. We herein report a case of aHUS that developed after cardiovascular surgery and was caused by a complement C3 mutation.

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.

Use of the Konno procedure in an 80-year-old woman with aortic stenosis, a narrow left ventricular outflow tract, and a small aortic annulus.

This report describes a technique for repairing an aortic stenosis in an elderly patient with a small aortic annulus and a narrow left ventricular outflow tract. Preoperative echocardiography in an 80-year-old woman showed severe aortic stenosis with a narrow outflow tract: the aortic valve area was 0.48 cm(2), the aortic annular diameter was 14 mm, and the left ventricular outflow tract diameter was 14 mm. The Konno procedure was used to enlarge both the small aortic annulus and the left ventricular outflow tract, and a 19-mm Carpentier-Edwards bioprosthetic valve was implanted. The patient's postoperative course was uneventful. The left ventricular mass decreased from a preoperative value of 236 g to 96 g, 3 years after surgery. Only a few reports have described the use of the Konno operation in adult patients. In the present case, the Konno operation was demonstrated to be a good option for aortic stenosis accompanied by a small aortic annulus and a narrow left ventricular outflow tract, even in an elderly patient.