The Role of The Fanconi Anemia Pathway in Human Papillomavirus Infection

 描述(申请人提供)：人乳头瘤病毒(HPV)是性传播的，与口咽和肛门恶性肿瘤的发展有关，包括宫颈癌，它是这些疾病的病原体。HPV感染上皮的基底层，在那里它以低拷贝数建立其基因组作为上皮体。在细胞分化时，病毒将其基因组放大到每个细胞数千个拷贝，并完成其病毒生命周期。虽然预防性疫苗可以预防感染，但没有治愈或现有的HPV感染，因此揭示其在分化上皮细胞中调节其复制的机制对于开发抗病毒治疗和限制HPV相关癌症至关重要。HPV在分化过程中激活ATM(共济失调-毛细血管扩张突变)DNA损伤反应(DDR)途径，用于病毒基因组扩增；然而，ATM激活的Fanconi贫血(FA)途径被报道限制了病毒在分化细胞中的复制。此外，有报道称FA患者易患HPV相关癌症，他们在FA途径中涉及的一个或多个基因发生突变，这表明该途径降低了病毒的致癌潜力。初步实验表明，该途径的关键调控蛋白之一FANCD2直接与病毒基因组结合，并在病毒感染过程中与ATM途径组件共存。此外，FANCD2的敲除扰乱了病毒在未分化细胞中的胞体维持，这表明FA途径在调节HPV复制方面发挥了更复杂的作用。本研究旨在明确FA通路在HPV分化依赖的生命周期中的作用，并研究FA患者对HPV相关恶性肿瘤易感性增加的机制。为了确定FA途径在HPV感染中的作用，将使用shRNA敲除和FANCD2的定点突变来确定其对病毒基因组维持和扩增的影响。该提案还将调查病毒调节FA途径以完成其生命周期的机制。这将通过在存在野生型或突变病毒蛋白的情况下评估FA途径组件的表达和激活来实现。最后，将通过使用体外致瘤性分析来分析FA缺乏对病毒转化的长期影响。这项研究的结果将有助于深入了解FA途径调控HPV病毒感染的机制，并更好地理解HPV与宿主DDR途径在病毒感染和转化过程中的关系。

 DESCRIPTION (provided by applicant): Human papillomaviruses (HPVs) are sexually transmitted and linked to the development of oropharyngeal and anogenital malignancies, including cervical cancer, of which it is the etiological agent. HPV infects the basal layer of epithelium where it establishes its genome as episomes at low copy numbers. Upon cellular differentiation, the virus amplifies its genomes to thousands of copies per cell and completes its viral life cycle. While prophylactic vaccines are available to prevent infection, there is no cure or existing HPV infections, so uncovering the mechanisms regulating its replication in differentiating epithelia is critical for the development of anti-viral therapies and the restrictin of HPV-associated carcinomas. HPV activates the ATM (ataxia- telangiectasia mutated) DNA damage response (DDR) pathway for viral genome amplification upon differentiation; however, the Fanconi Anemia (FA) pathway, which is activated by ATM, was reported to limit viral replication in differentiated cells. Additionally, a predisposition for HPV-associated cancers was reported in FA patients, who have a mutation in one or more of genes involved in the FA pathway, suggesting that this pathway lessens the oncogenic potential of the virus. Preliminary experiments reveal that one of the pathway's key regulatory proteins, FANCD2, directly binds the viral genome and colocalizes with ATM pathway components during viral infection. Additionally, the knockdown of FANCD2 disrupts the maintenance of viral episomes in undifferentiated cells, suggesting a more complex role for the FA pathway in regulating HPV replication. This research proposal aims to define the role of the FA pathway in the differentiation-dependent life cycle of HPV and examine the mechanism behind the increased susceptibility to HPV-associated malignancies seen in FA patients. To identify the role of the FA pathway in HPV infection, shRNA knockdown and site-directed mutagenesis of FANCD2 will be used to determine its impact on viral genome maintenance and amplification. The proposal also will investigate mechanisms through which the virus modulates the FA pathway for completion of its life cycle. This will be accomplished by assessing the expression and activation of FA pathway components in the presence of wild-type or mutated viral proteins. Lastly, an analysis of the long- term effects of an FA deficiency on viral transformation will be performed through the use of in vitro tumorigenicity assays. Results from this study will provide insight into the mechanism through which the FA pathway regulates HPV viral infection and a better understanding of the relationship between HPV and host DDR pathways during viral infection and transformation.