The Role of The Fanconi Anemia Pathway in Human Papillomavirus Infection

范可尼贫血途径在人乳头瘤病毒感染中的作用

• 批准号: 8838374
• 负责人: Chelsey Cierra Spriggs
• 金额: $ 3.44万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2017-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8838374
• 关键词: Antiviral Agents; Binding; Biological Assay; Carcinoma; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Regulation; Cells; ChIP-seq; Complex; Cutaneous; DNA; DNA Damage; DNA Interstrand Crosslinking; Development; Double Stranded DNA Virus; Episome; Epithelial; Epithelium; Fanconi' s Anemia; Genes; Genetic; Genome; Genomic Instability; Human Papilloma Virus-Related Carcinoma; Human Papillomavirus; Human papilloma virus infection; Immune response; In Situ Hybridization; In Vitro; Indium; Infection prevention; Life Cycle Stages; Link; Long-Term Effects; Maintenance; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Measures; Mediating; Molecular; Mutate; Mutation; Oncogene Proteins; Oncogenic; Oropharyngeal; Pathway interactions; Patients; Play; Predisposition; Premalignant Cell; Process; Proteins; Reporting; Research Proposals; Role; S Phase; Site-Directed Mutagenesis; Southern Blotting; Stratified Epithelium; Stratum Basale; Syndrome; Testing; Tumorigenicity; Undifferentiated; Vaccines; Viral; Viral Genome; Viral Proteins; Virus; Virus Diseases; Woman; ataxia telangiectasia mutated protein; genetic regulatory protein; high risk; insight; keratinocyte; men; prophylactic; public health relevance; repaired; research study; response; restrictin; small hairpin RNA; virus episome maintenance

 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.

 描述（由申请方提供）：人乳头瘤病毒（HPV）通过性传播，并与口咽和肛门生殖器恶性肿瘤（包括宫颈癌）的发生有关，HPV是宫颈癌的病原体。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附加体。在细胞分化时，病毒将其基因组扩增到每个细胞数千个拷贝，并完成其病毒生命周期。虽然预防性疫苗可用于预防感染，但没有治愈或现有的HPV感染，因此揭示其在分化上皮中复制的调节机制对于开发抗病毒疗法和限制HPV相关癌至关重要。HPV激活ATM（共济失调-毛细血管扩张突变）DNA损伤反应（DDR）途径，用于分化后的病毒基因组扩增;然而，据报道，由ATM激活的范可尼贫血（FA）途径限制分化细胞中的病毒复制。此外，在FA患者中报告了HPV相关癌症的易感性，这些患者在FA途径中涉及的一个或多个基因中存在突变，这表明该途径降低了病毒的致癌潜力。初步实验表明，该途径的关键调节蛋白之一FANCD 2直接结合病毒基因组，并在病毒感染期间与ATM途径组分共定位。此外，FANCD 2的敲低破坏了未分化细胞中病毒附加体的维持，表明FA途径在调节HPV复制中的作用更复杂。这项研究计划旨在确定FA途径在HPV分化依赖性生命周期中的作用，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FANCD 2的shRNA敲低和定点诱变来确定其对病毒基因组维持和扩增的影响。该提案还将研究病毒调节FA途径以完成其生命周期的机制。这将通过评估存在野生型或突变病毒蛋白时FA途径组分的表达和活化来实现。最后，将通过使用体外致瘤性试验分析FA缺乏对病毒转化的长期影响。这项研究的结果将提供深入了解FA途径调节HPV病毒感染的机制，并更好地了解病毒感染和转化过程中HPV与宿主DDR途径之间的关系。