Raft Culture Research Articles

In Vitro Organotypic Systems to Model Tumor Microenvironment in Human Papillomavirus (HPV)-Related Cancers.

Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors (i.e., all cervical cancers and other human malignancies from the mucosal squamous epithelia, including anogenital and oropharyngeal cavity), its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia (anogenital and oropharynx), as well as rare cutaneous non-melanoma associated cancer.

Targeting DNA Damage Response as a Strategy to Treat HPV Infections.

Mucosotropic human papillomaviruses (HPVs) cause prevalent anogenital infections, some of which can progress to cancers. It is imperative to identify efficacious drug candidates, as there are few therapeutic options. We have recapitulated a robust productive program of HPV-18 in organotypic raft cultures of primary human keratinocytes. The HPV E7 protein induces S phase reentry, along with DNA damage response (DDR) in differentiated cells to support viral DNA amplification. A number of small molecule inhibitors of DDR regulators are in clinical use or clinical trials to treat cancers. Here, we used our raft culture system to examine effects of inhibitors of ATR/Chk1 and ATM/Chk2 on HPV infection. The inhibitors impaired S-phase reentry and progression as well as HPV DNA amplification. The Chk1 inhibitor MK-8776 was most effective, reducing viral DNA amplification by 90–99% and caused DNA damage and apoptosis, preferentially in HPV infected cells. We found that this sensitivity was imparted by the E7 protein and report that MK-8776 also caused extensive cell death of cervical cancer cell lines. Furthermore, it sensitized the cells to cisplatin, commonly used to treat advanced cervical cancer. Based on these observations, the Chk1 inhibitors could be potential effective agents to be re-purposed to treat the spectrum of HPV infections in single or combination therapy.

Influence of mild saline stress and growing season on yield and leaf quality of baby lettuce grown in floating system

Consumer demand has been increasing and many vegetable farmers have oriented their production plans towards baby leaf vegetables such as lettuce. The management of the nutrient solution as well as the growing conditions could significantly affect the yield and quality of leafy vegetable crops. The aim of the present work was to determine the effects of increasing the level of salinity in the nutrient solution (1, 10 or 20 mM NaCl) during two consecutive growing seasons (spring and autumn) on the yield and quality of Lactuca sativa L. var. acephala grown in floating raft culture. Increased salinity in the nutrient solution decreased marketable yield and dry biomass with more detrimental effect during the warm season. The percentage of yield reduction in comparison to the non-saline control was 7 and 20% in the spring season and 4 and 13% in the autumn growing season at 10 and 20 mM NaCl, respectively. Plants grown in the spring season exhibited higher yield and dry biomass but lower leaf quality than those grown in the autumn season. When averaged over salinity level the lipophylic antioxidant activity, total ascorbic acid, phenols and nitrate contents were significantly increased by 7.1, 20.1, 19.4 and 24.4%, respectively, in plants grown in spring as compared to autumn growing season. The floating system could be considered an efficient and low-cost tool to improve quality aspects through proper management of the salt concentration in the nutrient solution with an acceptable yield reduction.

Risk of Human Pathogen Internalization in Leafy Vegetables During Lab-Scale Hydroponic Cultivation

Controlled environment agriculture (CEA) is a growing industry for the production of leafy vegetables and fresh produce in general. Moreover, CEA is a potentially desirable alternative production system, as well as a risk management solution for the food safety challenges within the fresh produce industry. Here, we will focus on hydroponic leafy vegetable production (including lettuce, spinach, microgreens, and herbs), which can be categorized into six types: (1) nutrient film technique (NFT), (2) deep water raft culture (DWC), (3) flood and drain, (4) continuous drip systems, (5) the wick method, and (6) aeroponics. The first five are the most commonly used in the production of leafy vegetables. Each of these systems may confer different risks and advantages in the production of leafy vegetables. This review aims to (i) address the differences in current hydroponic system designs with respect to human pathogen internalization risk, and (ii) identify the preventive control points for reducing risks related to pathogen contamination in leafy greens and related fresh produce products.

Gracilaria debilis cultivation, agar characterization and economics: bringing new species in the ambit of commercial farming in India

Gracilaria debilis (Forsskal) Borgesen, an economically important red alga, was cultivated for 2 years by a floating bamboo raft method from November 2014 to October 2016 along the southeastern coast of India in order to evaluate possibilities for its year-round cultivation. This was achieved by analyzing biomass yield, growth rate, and agar properties from each harvest. Bench-scale agar characterization and economics were calculated for the first time for G. debilis. Higher biomass yields were recorded during first harvest (November–December) of both the first (11.02 ± 2.08 kg fr.wt m−2) and second (7.17 ± 3.95 kg fr.wt m−2) years. Similarly, higher growth rate was recorded during the first harvest of the first (3.59 ± 0.4% day−1) and second (4.17 ± 0.96% day−1) year, respectively. Biomass yield and DGR were at their minima during monsoon season (i.e., July–August). There was no clear trend pertaining to yield and gel strength of the extracted agar which were in the range of 14–32.6% and 300–866 g cm-2, respectively. This study confirmed that year-round cultivation of G. debilis is possible in Indian waters using the raft culture method with six harvest cycles per annum. The annual income for a single operator was estimated to be US$141, while break-event point per hectare could be achieved after 126 days.

The prevalence of abalone herpesvirus in two Haliotis species in South China during 2002–2013

Abalones (Haliotis spp.) are economically important seafoods. However, the appearance of lethal diseases since 1999, such as those caused by low-temperature viruses, have led to the near collapse of the aquaculture industry of small abalone (H. diversicolor) in southern China. The abalone herpesvirus (AbHV) is one of these suspected low-temperature viral pathogens, but the absence of epidemic data of AbHV in abalone species in China has hampered in-depth studies of AbHV and the identification of the pathogen(s) causing abalone viral disease(s). Here, we performed the first epidemiological study on AbHV in China, using quantitative PCR. The first batch of abalone individuals (n = 1082) was collected monthly from April 2012 to October 2013 at seven sites along the coast of the South China Sea. Analyses revealed the presence of AbHV in cultured abalone in China, with a detection rate of 27.73%. Most individuals were healthy and showed a viral DNA load below 103 copies per ng host DNA. No effect of the culture method (artificial pond culture or open sea raft culture), shell length, or host species (H. diversicolor and H. discus hannai) on the viral load was detected. In the second batch of samples (n = 500), collected between 2002 and 2012, AbHV was detected at a detection rate of 29.20%. Ultra-high viral DNA loads (105 to 107 copies/ng host DNA) were detected in the diseased abalone samples. Artificial infection results confirmed the pathogenicity of AbHV to H. diversicolor, suggesting that AbHV is probably the pathogen responsible for the abalone low-temperature viral disease. These findings will benefit the abalone aquaculture industry, as it provides data that can be used to improve the control of viral diseases, and facilitate further AbHV-related studies.

Coastal Aquaculture Mapping from Very High Spatial Resolution Imagery by Combining Object-Based Neighbor Features

Coastal aquaculture plays an important role in the provision of seafood, the sustainable development of regional and global economy, and the protection of coastal ecosystems. Inappropriate planning of disordered and intensive coastal aquaculture may cause serious environmental problems and socioeconomic losses. Precise delineation and classification of different kinds of aquaculture areas are vital for coastal management. It is difficult to extract coastal aquaculture areas using the conventional spectrum, shape, or texture information. Here, we proposed an object-based method combining multi-scale segmentation and object-based neighbor features to delineate existing coastal aquaculture areas. We adopted the multi-scale segmentation to generate semantically meaningful image objects for different land cover classes, and then utilized the object-based neighbor features for classification. Our results show that the proposed approach effectively identified different types of coastal aquaculture areas, with 96% overall accuracy. It also performed much better than other conventional methods (e.g., single-scale based classification with conventional features) with higher classification accuracy. Our results also suggest that the multi-scale segmentation and neighbor features can obviously improve the classification performance for the extraction of cage culture areas and raft culture areas, respectively. Our developed approach lays a solid foundation for intelligent monitoring and management of coastal ecosystems.

Growth rates of Eucheuma denticulatum and Kappaphycus alvarezii (Rhodophyta; Gigartinales) cultured using modified off-bottom and floating raft techniques on the Kenyan coast

The study compared relative growth rates (RGR) of Eucheuma denticulatum and Kappaphycus alvarezii under modi- fied off-bottom (MB) and floating raft (FR) culture techniques on the southern coast of Kenya. Seasonal variability in RGRs was evident over 10 months in both techniques and sites. RGRs were in the range of 0.9 – 10.2 % d 1 for E. denticulatim and 0.3 – 5.7 % d 1 for K. alvarezii at Mkwiro, and -0.2 – 7.3 % d 1 for E. denticulatum and -1.7 – 4 % d 1 for K. alvarezii at Kibuyuni. The RGR of 4.3 ± 0.4 % d 1 from the FR technique was significantly higher than the 3.2 ± 0.4 % d 1 from the MB technique (P ˂ 0.05). Strong oceanic waves which were accompanied by the loss of seaweed thalli in the MB technique at Mkwiro between April and June led to significantly higher RGR in the FR than in the MB (P ˂ 0.05). The higher percentage herbivory, epiphytes, and susceptibility to ‘ice-ice’ (white and soft thallus) associated with K. alvarezii than E. denticulatum in both techniques led to the former having significantly lower RGRs than the latter . Further research on the stability of MB in deep water and accessibility of FR techniques is recommended prior to commercial adoption.

Impacts of maricultural activities on characteristics of dissolved organic carbon and nutrients in a typical raft-culture area of the Yellow Sea, North China

Ailian Bay is an integrated multi-trophic aquaculture bay with approximately 60 years maricultural activities in North China. The floating raft culture of kelp and shellfish is the unique mariculture mode. In this study, the impacts of intensive mariculture activities on seasonal carbon and nutrient dynamics in Ailian Bay were systematically analysed via seasonal surveys between 2015 and 2016. The dissolved inorganic nitrogen and silicon reached the maximum concentrations during summer, which were mainly attributed to the release of shellfish metabolic by-products and their filter-feeding effects on diatoms. Dissolved organic carbon (DOC) concentrations were significantly elevated when kelps were rotting in summer and kelp seeding were occurring in winter. Meanwhile, the fluorescence intensity of humic-like chromophoric dissolved organic matter was relatively high in kelp mariculture zone. As most humic-like DOC are potentially refractory substances, we propose that kelp mariculture would contribute importantly to the increase of refractory DOC pool in oceans.

CDK9 Inhibitor FIT-039 Suppresses Viral Oncogenes E6 and E7 and Has a Therapeutic Effect on HPV-Induced Neoplasia.

Purpose: Cervical cancer is one of the leading causes of cancer-related deaths among women worldwide. The purpose of this study is to assess the therapeutic effect of the newly developed cyclin-dependent kinase 9 (CDK9) inhibitor FIT-039 on cervical neoplasia induced by human papillomavirus (HPV) infection.Experimental Design: We examined FIT-039 for its effect on HPV gene expression in HPV+ cervical cancer cells. Primary keratinocytes monolayer and organotypic raft culture models were used to evaluate HPV viral replication and cervical intraepithelial neoplasia (CIN) phenotypes. Preclinical pharmacokinetics and toxicity tests for FIT-039 were also conducted. Finally, the anti-HPV effect of FIT-039 was further examined in vivo, using HPV+ cervical cancer xenografts.Results: FIT-039 inhibits HPV replication and expression of E6 and E7 viral oncogenes, restoring tumor suppressors p53 and pRb in HPV+ cervical cancer cells. The therapeutic effect of FIT-039 was demonstrated in CIN model of an organotypic raft culture, where FIT-039 suppressed HPV18-induced dysplasia/hyperproliferation with reduction in viral load. FIT-039 also repressed growth of HPV16+, but not HPV- cervical cancer xenografts without any significant adverse effects. Safety and pharmacokinetics of FIT-039 were confirmed for systemic and topical routes.Conclusions: The CDK9 inhibitor FIT-039 showed potent anti-HPV activity without significant toxicity in preclinical studies. Thus, FIT-039 is expected to be a novel therapeutic for CIN to prevent cervical cancer. Clin Cancer Res; 24(18); 4518-28. ©2018 AACR.

ΔNp63γ/SRC/Slug Signaling Axis Promotes Epithelial-to-Mesenchymal Transition in Squamous Cancers.

Purpose: To investigate the regulation of epithelial-to-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC) and its importance in tumor invasion.Experimental Design: We use a three-dimensional invasive organotypic raft culture model of human foreskin keratinocytes expressing the E6/E7 genes of the human papilloma virus-16, coupled with bioinformatic and IHC analysis of patient samples to investigate the role played by EMT in invasion and identify effectors and upstream regulatory pathways.Results: We identify SNAI2 (Slug) as a critical effector of EMT-activated downstream of TP63 overexpression in HNSCC. Splice-form-specific depletion and rescue experiments further identify the ΔNp63γ isoform as both necessary and sufficient to activate the SRC signaling axis and SNAI2-mediated EMT and invasion. Moreover, elevated SRC levels are associated with poor outcome in patients with HNSCC in The Cancer Genome Atlas dataset. Importantly, the effects on EMT and invasions and SNAI2 expression can be reversed by genetic or pharmacologic inhibition of SRC.Conclusions: Overexpression of ΔNp63γ modulates cell invasion by inducing targetable SRC-Slug-evoked EMT in HNSCC, which can be reversed by inhibitors of the SRC signaling. Clin Cancer Res; 24(16); 3917-27. ©2018 AACR.

Extraction of coastal raft cultivation area with heterogeneous water background by thresholding object-based visually salient NDVI from high spatial resolution imagery

ABSTRACTThe development of high spatial resolution satellite imaging has enabled the acquisition of mariculture area information. This data could play an important role in mariculture investigations, ocean disaster evaluations, and coastal management. Because chlorophyll is concentrated in the widely distributed raft culture (a major kind of mariculture), the Normalized Difference Vegetation Index (NDVI) can be used for extraction. However, extensive coastal raft culture is easily confused with the heterogeneous water background. This results in unsatisfactory extraction when surveying a large water area with heterogeneous water background. By combining object-based image analysis and the centre-surround mechanism of a visual attention model, we propose an object-based visually salient NDVI (OBVS-NDVI) feature. Comparison experiments using Gaofen-2 spectral imagery of Luoyuan Bay, Fuzhou, China, indicate that OBVS-NDVI can effectively discriminate raft cultivation areas over large areas with a heterogeneous water background.

Global Trends in Rural People Livelihood Practice of Raft Culture of Red Seaweed Kappaphycus Alvarezii in Gujarat, India

Global Trends in Rural People Livelihood Practice of Raft Culture of Red Seaweed Kappaphycus Alvarezii in Gujarat, India

E5 can be expressed in anal cancer and leads to epidermal growth factor receptor-induced invasion in a human papillomavirus 16-transformed anal epithelial cell line.

We created the first human papillomavirus (HPV)-16-immortalized anal epithelial cell line, known as AKC2 cells to establish an in vitro model of HPV-16-induced anal carcinogenesis. Consistent with detection of E6, E7 and E5 expression in anal cancer biopsies, AKC2 cells expressed high levels of all three HPV oncogenes. Also, similar to findings in anal cancer biopsies, epidermal growth factor receptor (EGFR) was overexpressed in AKC2 cells. AKC2 cells exhibited a poorly differentiated and invasive phenotype in three-dimensional raft culture and inhibition of EGFR function abrogated AKC2 invasion. Reducing E5 expression using E5-targeted siRNAs in AKC2 cells led to knockdown of E5 expression, but also HPV-16 E2, E6 and E7 expression. AKC2 cells treated with E5-targeted siRNA had reduced levels of total and phosphorylated EGFR, and reduced invasion. Rescue of E6/E7 expression with simultaneous E5 knockdown confirmed that E5 plays a key role in EGFR overexpression and EGFR-induced invasion.

The current situation and environmental conditions of green mussel farming in the gulf of Thailand

This study investigated the current situation and environmental conditions of green mussel Perna viridis farming in Surat Thani, Phetchaburi and Samut Prakan provinces where were located in the gulf of Thailand. Samples were collected during April, August and November in 2014. Based on the results of this study, there were three major types of mussel culture patterns in Thailand: bouchot culture, longline culture (rope culture), and raft culture. Mussel farmers encountered problems on decreasing of larvae mussel in the water column, water quality degradation, high production cost and farmer’s lack of negotiation power to control the price. The water quality of the mussel farms, such as dissolved oxygen (DO), pH, salinity, electrical conductivity (EC), fecal coliforms bacteria and the total coliforms bacteria met the standard for aquaculture. However, the nutrient, such as ammonium (NH4-N), nitri-nitrogen (NO2-N), nitrate-nitrogen (NO3-N) and soluble reactive phosphorus (PO4-P) were higher than the value of marine aquaculture standard. The results of the contamination of Salmonella spp. bacteria were not found in the mussel’s tissue, however Escherichia coli was found at very low level.

In vitro activity of novel 1,3-oxazole derivatives against human papillomavirus

Background: Chemotherapeutic approaches to the control of HPV infection suffer from a lack of specificity. For most existing HPV inhibitors, the weak antiviral effects observed in cellular assays suggest that further improvements in selecting targets, in drug potency, and in bioavailability and cell uptake are required. Objective: To synthesize novel 1,3-oxazole derivatives and define their antiviral activities against the human papillomavirus (HPV) in vitro. Methods: Determination of transient replication of an HPV-11 in transfected HEK293 cells, and HPV-18 DNA amplification in an organotypic squamous epithelial raft culture of primary human keratinocytes (PHKs), and cytotoxicity assays were used. Results: Bioassays showed that the synthesized compounds 2, 4, 5, and 9 exhibited potent antiviral activity against low-risk HPV-11 (IC50 = 1.7–9.6 μM) in a transient DNA replication assay and exhibited low cytotoxicity in HEK293 cells compared to cidofovir (CDV), antiviral agent in clinical use. Selectivity indices of compounds 4 and 5 were 20–40 times greater than that of CDV. However, compounds 4 and 9 did not exhibit a significant antiviral effect against high-risk HPV-18 infections in organotypic epithelial raft cultures. Although prophylactic HPV vaccines are now available to protect against primary infections by the seven genotypes most commonly found in cervical, penile, anal and oro-pharyngeal cancers (HPV16, 18, 31, 33, 45, 52, and 58) and two genotypes (HPV6 and 11) that cause benign anogenital warts and laryngeal papillomas, they do not protect against infections by other HPV types. Moreover, individuals already infected with HPV will not benefit from the vaccines. Thus, the need for antiviral agents to treat HPV-associated diseases remains great, but few options currently exist. Conclusions: We show that substituted 1,3-oxazole derivatives are a promising structure class of chemical compounds for the development of antiviral drugs against HPV lesions.

Harvesting machine for kelp culture in floating raft

Kelp is not only a kind of food of sea vegetable for human, but also widely used as raw materials in processing food, chemical engineering, pharmaceutical and cosmetic industries. In China kelp is generally planted on floating raft by using horizontal kelp rope cultivation method and often harvested manually. Harvesting kelp by hand is a difficult process with low production efficiency and high labor intensity. In this paper, a harvesting machine was developed for floating raft culture of kelp. It consists of mainly two locomotion modules, a collection/transmission module and a power module. The structure and working mechanism of this machine was presented and analyzed in detail. The tests of experiments were conducted as well with the developed machine. The manual harvest efficiency is 16.7%; however the mechanized harvest efficiency average value is 35.8%. The harvest time could be reduced by half if the company takes the same people to work. It is proven that the design of the machine is feasible.

Modulation of basal cell fate during productive and transforming HPV-16 infection is mediated by progressive E6-driven depletion of Notch.

In stratified epithelia such as the epidermis, homeostasis is maintained by the proliferation of cells in the lower epithelial layers and the concomitant loss of differentiated cells from the epithelial surface. These differentiating keratinocytes progressively stratify and form a self‐regenerating multi‐layered barrier that protects the underlying dermis. In such tissue, the continual loss and replacement of differentiated cells also limits the accumulation of oncogenic mutations within the tissue. Inactivating mutations in key driver genes, such as TP53 and NOTCH1, reduce the proportion of differentiating cells allowing for the long‐term persistence of expanding mutant clones in the tissue. Here we show that through the expression of E6, HPV‐16 prevents the early fate commitment of human keratinocytes towards differentiation and confers a strong growth advantage to human keratinocytes. When E6 is expressed either alone or with E7, it promotes keratinocyte proliferation at high cell densities, through the combined inactivation of p53 and Notch1. In organotypic raft culture, the activity of E6 is restricted to the basal layer of the epithelium and is enhanced during the progression from productive to abortive or transforming HPV‐16 infection. Consistent with this, the expression of p53 and cleaved Notch1 becomes progressively more disrupted, and is associated with increased basal cell density and reduced commitment to differentiation. The expression of cleaved Notch1 is similarly disrupted also in HPV‐16‐positive cervical lesions, depending on neoplastic grade. When taken together, these data depict an important role of high‐risk E6 in promoting the persistence of infected keratinocytes in the basal and parabasal layers through the inactivation of gene products that are commonly mutated in non‐HPV‐associated neoplastic squamous epithelia. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract 4794: Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture

Abstract An epidemic rise in oropharyngeal squamous cell carcinoma (OSCC) infected with human papillomavirus has been observed over the past decade. The majority of HPV+OSCC arise from the tonsils and base of tongue (BOT), which are lymphoid-rich regions that are known to harbor Epstein-Barr virus (EBV). Indeed, we previously reported HPV/EBV co-infection in 25% of tonsillar and 70% of BOT tumors. HPV is a DNA tumor virus that specifically infects proliferating basal cells in stratified squamous epithelia. HPV replication is facilitated by viral oncoproteins E6 and E7, which create an S-phase-like environment via degradation of cell cycle regulators p53 and Rb, respectively. EBV is also an established DNA tumor virus that replicates in differentiated epithelia in its normal lifecycle. However, EBV exhibits a latent infection characterized by oncogene expression and lack of viral replication in EBV-associated carcinomas. E6/E7-mediated alteration of the cell cycle results in an interruption in epithelial differentiation, which is required for EBV replication in epithelial cells. Thus, we hypothesize that HPV immortalization of keratinocytes shifts the EBV life cycle from replication to latency. To test our hypothesis, we developed an EBV/HPV co-infection model utilizing organotypic raft culture to produce stratified epithelia in vitro. Tonsillar keratinocytes immortalized by HPV were EBV-infected by co-culture with IgG-induced EBV-positive Akata Burkitt’s lymphoma cells four days after differentiation induction. Raft tissues were harvested 6 days post-EBV infection for analysis of viral life cycles. EBV infection did not alter HPV replication or gene expression. In HPV-negative tonsillar keratinocytes, robust EBV replication was observed that was blocked by treatment with acyclovir, a nucleoside analog that inhibits EBV lytic replication. In contrast, a dramatic decrease in EBV DNA levels was observed in HPV-positive keratinocytes, which was not further reduced by acyclovir. Human telomerase-immortalized normal oral keratinocytes supported robust EBV replication in organotypic raft culture suggesting a specific effect of HPV rather than immortalization on EBV replication. Expression of E7 alone was sufficient to decrease EBV DNA levels. Preliminary data suggest no change in EBV infectivity between HPV-negative and HPV-positive rafts, and an increase in EBV latency-associated gene expression in HPV-positive cells. The ability of EBV to establish persistent latent infection in HPV-infected epithelial cells would contribute additional viral oncogene expression to explain the rapid development and progression of HPV+OSCC. Citation Format: Joseph T. Guidry, William K. Songock, Xiaohui Ma, Cherie-Ann O. Nathan, Jason M. Bodily, Rona S. Scott. Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2017-4794

HPV16 and 18 genome amplification show different E4-dependence, with 16E4 enhancing E1 nuclear accumulation and replicative efficiency via its cell cycle arrest and kinase activation functions.

To clarify E1^E4’s role during high-risk HPV infection, the E4 proteins of HPV16 and 18 were compared side by side using an isogenic keratinocyte differentiation model. While no effect on cell proliferation or viral genome copy number was observed during the early phase of either virus life cycle, time-course experiments showed that viral genome amplification and L1 expression were differently affected upon differentiation, with HPV16 showing a much clearer E4 dependency. Although E4 loss never completely abolished genome amplification, its more obvious contribution in HPV16 focused our efforts on 16E4. As previously suggested, in the context of the virus life cycle, 16E4s G2-arrest capability was found to contribute to both genome amplification success and L1 accumulation. Loss of 16E4 also lead to a reduced maintenance of ERK, JNK and p38MAPK activity throughout the genome amplifying cell layers, with 16E4 (but not 18E4) co-localizing precisely with activated cytoplasmic JNK in both wild type raft tissue, and HPV16-induced patient biopsy tissue. When 16E1 was co-expressed with E4, as occurs during genome amplification in vivo, the E1 replication helicase accumulated preferentially in the nucleus, and in transient replication assays, E4 stimulated viral genome amplification. Interestingly, a 16E1 mutant deficient in its regulatory phosphorylation sites no longer accumulated in the nucleus following E4 co-expression. E4-mediated stabilisation of 16E2 was also apparent, with E2 levels declining in organotypic raft culture when 16E4 was absent. These results suggest that 16E4-mediated enhancement of genome amplification involves its cell cycle inhibition and cellular kinase activation functions, with E4 modifying the activity and function of viral replication proteins including E1. These activities of 16E4, and the different kinase patterns seen here with HPV18, 31 and 45, may reflect natural differences in the biology and tropisms of these viruses, as well as differences in E4 function.