The Role of The Fanconi Anemia Pathway in Human Papillomavirus Infection

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 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.