MODULATION OF HUMAN PAPILLOMAVIRUSES IN CELL CULTURE

细胞培养中人乳头瘤病毒的调节

• 批准号: 3548108
• 负责人: THOMAS R BROKER
• 金额: $ 19.91万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3548108
• 关键词: antiviral agents; athymic mouse; cell differentiation; collagen; cooperative study; cytokine; drug screening /evaluation; fibroblasts; histology; human papillomavirus; human subject; immunocytochemistry; in situ hybridization; keratinocyte; messenger RNA; nucleoside analog; plasmids; suramin; tissue /cell culture; transfection; transmission electron microscopy; virion; virus DNA; virus replication

Human papillomavirus type 16 (HPV-16) is the most prevalent high risk virus associated with cervical and penile intraepithelial neoplasia and cancers, and HPV-11 is a common low risk genotype primarily causing benign anogenital condylomata and recurrent respiratory (laryngeal) papillomatosis. We propose to use infected keratinocyte culture systems developed in our laboratories to identify and evaluate specific pharmacological drugs that have differential effects on the mRNA transcription, episomal DNA replication, protein function and virion assembly of both virus types. These in vitro systems are also ideal for assessing agents that modulate epithelial proliferation and differentiation and thereby may affect viral activity indirectly. The basic strategy is to grow keratinocytes that contain episomal HPV DNA or are infected with HPV virus on dermal equivalents consisting of collagen matrices and fibroblasts. When the culture is grown at the medium:air interface mimicking the environment of normal skin, the keratinocytes stratify and differentiate. In such organotypic cultures, the complete HPV productive program is recapitulated, exactly as observed in vivo with patient lesions. The cell line W12, which was isolated in the lab of Co-PI MAS and contains episomal HPV-16, will be used as one the test systems. A second system uses primary human cervical and foreskin keratinocytes (PHK) infected with HPV-16 viruses generated from the W12 cells. The third system involves PHK transfected with HPV-11 DNA with high efficiency procedures developed in labs of the PI TRB and Co-PI LTC. Agents effective in modulating epithelial differentiation or viral activities will then be tested directly on raft cultures developed from fresh patient biopsies known to contain HPV DNAs or in grafts in nude mice. Potential modifiers to be tested include cytokines, nucleoside analogs, topoisomerase poisons, antisense or triplex forming oligonucleotides, and suramin, a potent multi-target drug which inhibits the binding of growth factors to cell surface receptors and interferes with host protein kinase C, DNA polymerases and topoisomerase, among others. Additional reagents that are identified by the Antivirals Program Director will also be tested. The assays include histology to monitor epithelial differentiation and to assess possible cellular toxicity, viral pathogenesis, in situ hybridization to measure viral DNA replication and the synthesis of early and late region viral mRNAs as well as host messenger RNAs for which we have constructed specific probes, immunocytochemistry to detect the synthesis of viral and host proteins, and transmission electron microscopy to visualize virions. Additional refinements to the procedures for tissue culturing and DNA transfection into keratinocytes and new probes will be developed as part of this project. In the future, transcription and replication assays in transiently transfected cells or cell-free systems can be used to complement the assays performed on raft cultures when appropriate. The combined results should elucidate the mechanisms of action of promising pharmacological modifiers of viral infection.

人乳头瘤病毒16型（HPV-16）是最普遍的高危型。 与宫颈和阴茎上皮内瘤变相关的病毒， HPV-11是一种常见的低风险基因型，主要引起良性肿瘤。 肛门生殖器尖锐湿疣和复发性呼吸道（喉） 乳头状瘤病我们建议使用感染的角质细胞培养系统 在我们的实验室开发，以识别和评估特定的 对mRNA具有不同作用的药理学药物 转录、附加体DNA复制、蛋白质功能和病毒体 两种病毒的组合。这些体外系统也是理想的， 评估调节上皮增殖的试剂， 分化，从而可能间接影响病毒活性。的 基本的策略是培养含有附加型HPV DNA的角质形成细胞， 在由胶原蛋白组成的皮肤等同物上感染HPV病毒 基质和成纤维细胞。当培养物在培养基中生长时：空气 界面模仿正常皮肤的环境， 分层和区分。 在这种器官型培养中，完整的 HPV的生产程序是概括，完全一样，在体内观察， 患者病变。在Co-PI实验室分离的细胞系W12， MAS并含有游离型HPV-16，将用作试验系统之一。一 第二种系统使用原代人宫颈和包皮角质形成细胞（PHK） 用W12细胞产生的HPV-16病毒感染。第三 系统涉及高效转染HPV-11 DNA的PHK PI TRB和Co-PI LTC实验室开发的程序。有效的试剂 在调节上皮分化或病毒活性方面， 直接在新鲜患者活检的筏式培养物上进行测试 已知含有HPV DNA或在裸鼠移植物中。电位改性剂 待测试的包括细胞因子，核苷类似物，拓扑异构酶毒物， 反义或三链形成寡核苷酸，以及苏拉明，一种有效的 抑制生长因子与细胞结合的多靶点药物 表面受体并干扰宿主蛋白激酶C，DNA 聚合酶和拓扑异构酶等。其他试剂， 由抗病毒药物项目主任确定的药物也将进行检测。的 分析包括组织学以监测上皮分化， 原位评估可能的细胞毒性、病毒发病机制 杂交来测量病毒DNA复制和早期 和晚期区域病毒mRNA以及宿主信使RNA， 构建了特异性探针，免疫细胞化学检测 病毒和宿主蛋白的合成，以及透射电子显微镜 to visualize可视化virions病毒粒子.对组织手术的额外改进 培养和DNA转染到角质形成细胞和新的探针将 作为这个项目的一部分。在未来，转录和 瞬时转染细胞或无细胞系统中的复制测定 可用于补充在筏培养物上进行的测定， 适当综合结果应阐明的机制， 病毒感染的有前途的药理学修饰剂的作用。