MODULATION OF HUMAN PAPILLOMAVIRUSES IN CELL CULTURE

人乳头瘤病毒16型(HPV-16)是最流行的高危病毒 与宫颈和阴茎上皮内瘤变相关的病毒 癌症，而HPV-11是一种常见的低危基因型，主要导致良性 肛门生殖器尖锐湿疣与反复呼吸道(喉) 乳头状瘤病。我们建议使用感染角质形成细胞培养系统 在我们的实验室中开发，以识别和评估特定的 对信使核糖核酸有不同影响的药物 转录、DNA复制、蛋白质功能与病毒粒子 两种病毒的组合。这些体外系统也是理想的 评估调节上皮细胞增殖和 分化，从而可能间接影响病毒活性。这个 基本策略是培养含有上体HPV DNA或 在含有胶原蛋白的真皮上感染HPV病毒 基质和成纤维细胞。当培养在空气中生长时 界面模拟正常皮肤的环境，角质形成细胞 层次化、差异化。在这种器官类型的培养中，完整的 HPV的生产程序被概括，与在体内观察到的完全相同 病人的损伤。Co-PI实验室分离的W12细胞株 MAS和含有异构体的HPV-16，将被用作测试系统之一。一个 第二个系统使用原代人宫颈和包皮角质形成细胞(PHK) 感染W12细胞产生的HPV-16病毒。第三 高效导入HPV-11DNA的PHK系统 在PI TRB和Co-PI LTC的实验室中开发的程序。有效的代理 在调节上皮分化或病毒活性的过程中 直接在新鲜患者活检组织培养的RAFT上进行检测 已知含有HPVDNA或在裸鼠的移植物中。潜在的修饰语 要检测的包括细胞因子、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 表面受体和对宿主蛋白激酶C、DNA的干扰 聚合酶和拓扑异构酶等。其他试剂是 由反病毒计划主任确定的也将进行测试。这个 检测包括组织学以监测上皮细胞分化和 评估可能的细胞毒性、病毒致病机制、原位 用杂交法检测病毒DNA复制和早期合成 和晚期区域病毒RNA以及宿主信使RNA，我们对其 已经构建了特异性探针，免疫细胞化学来检测 病毒和宿主蛋白的合成及透射电子显微镜 使病毒粒子可视化。对组织程序的额外改进 角质形成细胞的培养和DNA转染和新的探针将被 作为该项目的一部分开发。在未来，转录和 瞬时转染细胞或无细胞系统中的复制分析 可用于补充在浮筏培养上进行的分析，当 恰如其分。这些综合的结果应该可以阐明 有希望的病毒感染药物修饰剂的作用。

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.