Human papillomaviruses (HPV) have genotype-specific disease associations, with high-risk alpha types causing at least 5% of all human cancers. Despite these conspicuous differences, our data show that high- and low- risk HPV types use similar approaches for genome maintenance and persistence. During the maintenance phase, viral episomes and the host cell genome are replicated synchronously, and for both the high- and low-risk HPV types, the E1 viral helicase is non-essential. During virus genome amplification, replication switches from an E1-independent to an E1-dependent mode, which can uncouple viral DNA replication from that of the host cell. It appears that the viral E2 protein, but not E6 and E7, is required for the synchronous maintenance-replication of both the high and the low-risk HPV types. Interestingly, the ability of the high-risk E6 protein to mediate the proteosomal degradation of p53 and to inhibit keratinocyte differentiation, was also seen with low-risk HPV E6, but in this case was regulated by cell density and the level of viral gene expression. This allows low-risk E6 to support genome amplification, while limiting the extent of E6-mediated cell proliferation during synchronous genome maintenance. Both high and low-risk E7s could facilitate cell cycle re-entry in differentiating cells and support E1-dependent replication. Despite the well-established differences in the viral pathogenesis and cancer risk, it appears that low- and high-risk HPV types use fundamentally similar molecular strategies to maintain their genomes, albeit with important differences in their regulatory control. Our results provide new insights into the regulation of high and low-risk HPV genome replication and persistence in the epithelial basal and parabasal cells layers. Understanding the minimum requirement for viral genome persistence will facilitate the development of therapeutic strategies for clearance. Human papillomavirus research has been prioritised towards understanding the progression of high-risk HPV infections to malignant cancer, rather than the regulation of the HPV productive cycle. Low-risk HPV types are often considered as a ‘less effective’ version of their high-risk counterparts. To date, they have been inadequately investigated and have generally served as reference viruses that lack oncogenic HPV functions. Here we carried out a comparative analysis using the most prominent high-risk (HPV16) and low-risk (HPV11) HPV papillomaviruses, focusing on viral genome replication and maintenance in the epithelial basal layer. During the maintenance phase, the E1 viral helicase of both HPV types are dispensable. Neither E6 nor E7 proteins are required for the maintenance replication. Once cells become confluent, replication switches from an E1-independent to an E1-dependent mode and requires p53 degradation by E6. Interestingly, the characteristic ability of high-risk E6 to degrade p53 and inhibit keratinocyte differentiation was also seen with low-risk HPV E6, but in this case was regulated by cell density and the extent of viral gene expression. Despite the differences in the pathogenicity of low- and high-risk HPV types, they appear to follow similar molecular strategies to complete viral life cycle.

NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop. IMPORTANCE A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent genome replication of HPV16. These results suggest that NF-κB mediates a negative feedback loop to regulate HPV replication and that this feedback loop could be associated with control of the viral copy numbers. We could thus show for the first time that NF-κB activity is involved in the establishment and maintenance of persistent HPV infection.

The success rate of establishing human cancer cell lines is not satisfactory and the established cell lines often do not preserve the molecular and histological features of the original tissues. In this study, we developed a novel culture method which can support proliferation of almost all primary epithelial ovarian cancer cells, as well as primary normal human oviductal epithelial cells. Cancer cells from fresh or frozen specimens were enriched by the anti-EpCAM antibody-conjugated magnetic beads, plated on Matrigel-coated plate and cultivated under the optimized culture conditions. Seventeen newly established ovarian cancer cell lines, which included all four major histotypes of ovarian cancer, were confirmed to express histotype-specific markers in vitro. Some of the cell lines from all the four histotypes, except mucinous type, generated tumors in immune-deficient mice and the xenograft tumor tissues recapitulated the corresponding original tissues faithfully. Furthermore, with poorly tumorigenic cell lines including mucinous type, we developed a novel xenograft model which could reconstruct the original tissue architecture through forced expression of a set of oncogenes followed by its silencing. With combination of the novel culture method and cell-derived xenograft system, virtually every epithelial ovarian cancer can be reconstituted in mice in a timely fashion.

Cervical adenocarcinoma is the second most common pathological subtype of cervical cancer after squamous cell carcinoma. It accounts for approximately 20% of cervical cancers, and the incidence has increased in the past few decades, particularly among young patients. The persistent infection of high-risk HPV is responsible for most cervical adenocarcinoma. However, almost all available in vitro models are designed to study the carcinogenesis of cervical squamous cell carcinoma. To gain better insights into molecular background of adenocarcinoma, we aimed to establish an in vitro carcinogenesis model of adenocarcinoma. We previously reported the establishment of an in vitro model for cervical squamous cell carcinoma by introducing defined viral and cellular oncogenes, HPV16 E6 and E7, c-MYC and activated RAS to human cervical keratinocytes. In this study, the expression of potential lineage-specifying factors and/or SMAD4 reduction was introduced in addition to the defined four oncogenes to direct carcinogenesis toward adenocarcinoma and the cell properties associated with the cell lineage were analyzed in monolayer and organoid cultures and the tumors in mouse xenografts. In the cells expressing FOXA2, apparent changes in the cell property were observed, such as elevated expression of columnar cell markers and decreased expression of squamous cell markers. Strikingly, the histopathology of tumors expressing FOXA2 resembled cervical adenocarcinoma, proposing that FOXA2 plays a vital role in dictating the histopathology of cervical cancers.

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3‐9‐16, that recognized a linear epitope located at the N‐terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Mα) whose 3‐9‐16 epitope is hidden, and an N‐terminal‐exposed epitope‐positive form (Mβ). The Mβ protein accounted for about 25–30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mβ antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mβ antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb‐dependent complement‐mediated cytolysis. Similarly, the Mβ antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mβ) of which exposes the 3‐9‐16 epitope located in the N‐terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Mα), probably through its intimate association with the Golgi area and/or the cell membrane.