Mechanisms of Human Papillomavirus DNA Replication

描述(申请人提供)：人乳头瘤病毒科包括一个DNA病毒大家族，嗜性于粘膜或皮肤上皮。感染这些极其普遍的病原体可导致上皮过度增殖，包括无生殖器疣和喉乳头状瘤。感染HPV-16和HPV-18等高危(HR)粘膜基因型别和密切相关的类型可进展为高度不典型增生和癌症，特别是子宫颈癌、阴茎癌、肛门癌和部分头颈癌。在全球范围内，每天有1000多人，主要是妇女，死于HPV疾病。HPV-6、-11、-16和-18的预防性疫苗有效地预防了这四种病毒的新感染，但目前还没有对乳头瘤病毒始终有效和特异的治疗剂。HPV感染基底层角质形成细胞，而生产期仅发生在鳞状上皮的分化层。我们证明了E7破坏了分化角质形成细胞中p130(pRB家族的成员)的稳定性，使S期重新进入和进展是病毒DNA扩增所必需的。众所周知，HR HPVE6可以降解主要的肿瘤抑制因子P53和一些额外的宿主蛋白。跨膜蛋白E5上调ErbB受体酪氨酸激酶家族的信号转导。因此，人乳头瘤病毒E7和E6共同作用可使原代人角质形成细胞永生化，而E5可提高其永生化效率。但如何利用这些E5和E6活动来促进病毒生产计划仍然难以捉摸。这是因为HR HPV的遗传分析已经在器官移植培养的永生化上皮细胞中进行了十多年，这些细胞支持病毒生产程序的效率低下。E6突变体无法在PHKs中持续存在或使其永生，这加剧了这种限制。我们最近建立了一种简单、有效和可重复性的方法，利用Cre-loxP介导的体内切除重组在PHKs中产生HPV基因组质粒。野生型HPV-18基因组在PHK RAFT培养中重现了一个强大的生产程序，并首次在幼稚PHK的RAFT培养中传代了后代病毒粒子。我们通过成功地检查了E6突变基因组，验证了我们的系统在HPV基因分析中的实用性，这在以前是不可能的。我们的结果表明，一个全长的E6蛋白是有效扩增病毒DNA所必需的。重要的是，这种方法避免了为了在PHK RAFT培养中进行HPV的遗传分析而需要HR HPV的永生化功能或永生化前的上皮细胞系。初步研究表明，E5也是至关重要的。我们已经就E5和E6蛋白的可能作用提出了具体的假设，并建议开发PHK RAFT培养系统来验证我们的假设，并阐明它们在病毒生产程序中的功能。将在整个过程中使用互补的遗传和生化方法。随着对HPV失控和HPV成瘾途径的深入了解，治疗药物的靶点将被确定为抗病毒药物开发的基础。

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.