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Mechanisms of Human Papillomavirus DNA Replication

人乳头瘤病毒 DNA 复制机制

基本信息

批准号：
8460134
负责人：
LOUISE T CHOW
金额：
$ 36.73万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8460134
关键词：
3-Dimensional Anogenital venereal warts Antiviral Agents Anus Benign Biochemical Carcinoma Cell Line Cells Cervical Complement Cutaneous DNA DNA Viruses DNA amplification DNA biosynthesis Disease Down-Regulation Dysplasia E6-target protein 1 Epithelial Epithelial Cells Epithelium ErbB Receptor Family Protein Exhibits Family Foundations Genetic Genetic Recombination Genital system Genome Genomics Genotype HPV-High Risk Head and Neck Cancer Head and Neck Squamous Cell Carcinoma Human Human Papillomavirus Human papillomavirus 11 Human papillomavirus 16 Human papillomavirus 18 Human papillomavirus 6 In Situ Infection Infection prevention Integral Membrane Protein Lead Length Lesion Life Cycle Stages Malignant Neoplasms Mediating Methods Mitogen-Activated Protein Kinases Mutation Oncogene Proteins Oncogenes Oral Papillomaviridae Papillomavirus Pathway interactions Patients Pattern Pharmaceutical Preparations Phase Phenotype Phosphorylation Plasmids Productivity Protein Tyrosine Kinase Protein p53 Proteins Receptor Protein-Tyrosine Kinases Recurrence Reporting Resistance Respiratory System Respiratory tract structure Retinoblastoma Protein Risk Role S Phase Signal Transduction Skin Squamous Epithelium Squamous Papilloma of the Larynx Staging System Testing Therapeutic Agents Time Tumor Suppressor Proteins Vaccines Viral Viral Physiology Virion Virus Woman drug development drug discovery genetic analysis high risk immortalized cell in vivo insight keratinocyte member mutant novel pathogen prevent programs prophylactic respiratory small hairpin RNA therapeutic target tumor progression tumorigenesis viral DNA

项目摘要

DESCRIPTION (provided by applicant): Human papillomaviridae comprise a large family of DNA viruses tropic for mucosal or cutaneous epithelium. Infections by these extremely prevalent pathogens can result in epithelial hyperproliferation, including ano-genital warts and laryngeal papillomas. Infections by the high-risk (HR) mucosal genotypes such as HPV-16 and HPV-18 and closely related types can progress to high grade dysplasias and cancers, notably cervical, penile, anal, and a fraction of head and neck carcinomas. Globally, over 1000 people, primarily women, die each day of HPV diseases. Prophylactic vaccines to HPV-6, -11, -16 and -18 effectively prevent new infections by these four virus types, but there are no therapeutic agents that are consistently efficacious and specific against papillomaviruses. HPVs infect basal keratinocytes, while the productive phase occurs only in the differentiated strata of squamous epithelia. We demonstrated that E7 destabilizes p130 (a member of the pRB family of pocket proteins) in the differentiated keratinocytes, enabling the S phase reentry and progression necessary for viral DNA amplification. The HR HPV E6 is known to degrade the major tumor suppressor p53 and a number of additional host proteins. The transmembrane protein E5 up-regulates the signal transduction of the ErbB family of receptor tyrosine kinases. Thus, HR HPV E7 and E6 together can immortalize primary human keratinocytes (PHKs), and E5 increases the efficiency. But how these E5 and E6 activities are utilized to promote the viral productive program remains elusive. This is because genetic analyses of HR HPVs have been conducted for well over a decade in organotypic raft cultures of immortalized epithelial cells that support viral productive program inefficiently. The limitation is compounded by the inability of E6 mutants to persist in PHKs or to immortalize them. We recently established a simple, effective, and reproducible method to generate HPV genomic plasmids in PHKs by using Cre-loxP mediated excisional recombination in vivo. A robust productive program was recapitulated by the wild type HPV-18 genome in PHK raft cultures, and progeny virions were passaged for the first time in raft cultures of naive PHKs. We validated the utility of our system for HPV genetic analyses by successfully examining an E6 mutant genome, not previously possible. Our results showed that a full-length E6 protein is necessary for efficient viral DNA amplification. Importantly, this approach circumvents the need for the immortalization functions of the HR HPVs or for pre-immortalized epithelial cell lines in order to perform genetic analyses of HPVs in PHK raft cultures. Preliminary studies revealed that E5 is also critical. We have formulated specific hypotheses on the possible roles of E5 and E6 proteins and propose to exploit this PHK raft culture system to test our hypotheses and elucidate their functions in the viral productive program. Complementary genetic and biochemical approaches will be used throughout. With a thorough understanding of the pathways which HPVs dysregulate and to which HPVs are addicted, targets of therapeutic agents will be identified to serve as the foundation for antiviral drug discovery.

描述（由申请人提供）：人乳头瘤病毒科包括一个大的DNA病毒家族，嗜粘膜或皮肤上皮。这些极为普遍的病原体感染可导致上皮过度增殖，包括肛门生殖器疣和喉乳头状瘤。由高危（HR）粘膜基因型如HPV-16和HPV-18以及密切相关的类型引起的感染可进展为高度发育不良和癌症，特别是宫颈癌、阴茎癌、肛门癌和一部分头颈癌。在全球范围内，每天有超过1000人，主要是女性，死于HPV疾病。针对HPV-6、-11、-16和-18的预防性疫苗有效地预防了这四种病毒类型的新感染，但是没有针对乳头瘤病毒的持续有效和特异性的治疗剂。HPV感染基底角质形成细胞，而生产阶段仅发生在鳞状上皮的分化层中。我们证明了E7使分化的角质形成细胞中的p130（口袋蛋白pRB家族的成员）不稳定，从而使病毒DNA扩增所必需的S期再入和进展成为可能。已知HR HPV E6降解主要肿瘤抑制因子p53和许多其他宿主蛋白。跨膜蛋白E5上调受体酪氨酸激酶的ErbB家族的信号转导。因此，HR HPV E7和E6一起可以使原代人角质形成细胞（PHK）永生化，并且E5增加了效率。但是如何利用这些E5和E6活性来促进病毒生产程序仍然是难以捉摸的。这是因为HR HPV的遗传分析已经在永生化上皮细胞的器官型筏培养物中进行了十多年，这些细胞低效地支持病毒生产程序。E6突变体不能在PHK中持续存在或使其永生化，这使得限制更加复杂。我们最近建立了一种简单、有效、可重复的方法，通过使用Cre-loxP介导的体内切除重组在PHK中产生HPV基因组质粒。在PHK筏培养物中通过野生型HPV-18基因组再现了稳健的生产程序，并且首次在幼稚PHK的筏培养物中传代子代病毒体。我们通过成功检查E6突变基因组验证了我们的系统用于HPV遗传分析的实用性，这在以前是不可能的。我们的研究结果表明，全长E6蛋白是必要的有效的病毒DNA扩增。重要的是，这种方法避免了对HR HPV的永生化功能或对预永生化上皮细胞系的需要，以便在PHK筏培养物中进行HPV的遗传分析。初步研究表明，E5也很关键。我们已经制定了具体的假设E5和E6蛋白可能的作用，并建议利用这个PHK筏培养系统来测试我们的假设，并阐明其功能的病毒生产程序。互补的遗传和生物化学方法将在整个过程中使用。随着对HPV失调和HPV成瘾的途径的深入了解，将确定治疗剂的靶点，作为抗病毒药物发现的基础。