Human papillomaviruses (HPV) are causative agents in ano-genital and head and neck cancers. Our long-term goal is to identify, and develop, novel approaches for targeting these viral diseases; to do this we must enhance our understanding of the viral life cycle and how it interacts with the host. HPV activates the DNA damage response (DDR) during the viral life cycle and uses homologous recombination (HR) to replicate its genome. The Morgan lab has identified several HR factors involved in HPV replication including TopBP1, BRD4, SIRT1 and WRN. We have demonstrated that these factors regulate the levels and fidelity of HPV16 E1-E2 DNA replication in C33a cells. Following WRN depletion, E1-E2 replication switches from high fidelity mechanisms to break induced replication (BIR). BIR is highly mutagenic and occurs when a paused fork is unable to be resolved by WRN. In the absence of WRN there is excess recruitment of MUS81 to E1-E2 replicating DNA, an endonuclease that causes DNA double strand breaks at paused forks in the absence of WRN. This resolves the fork and allows BIR to continue replication, albeit with low fidelity. We present data demonstrating that WRN is a restriction factor for the HPV16 life cycle; in the absence of WRN there is increased cell proliferation, DNA damage and viral replication in organotypic raft cultures. We observed a similar phenotype following SAMHD1 depletion, another HR factor. WRN and SAMHD1 are in the same cellular complex along with other DDR factors involved in HPV life cycles. Our first objective is to determine the roles of the WRN-SAMHD1 complex in controlling HPV life cycles. The central hypothesis is that this complex controls high fidelity replication during the viral life cycle, and that targeting components of this complex along with MUS81 may block HPV replication. Using a novel HPV16 genome, we will investigate the levels and the fidelity of viral replication during the viral life cycle. Our second objective is to determine whether there are altered host DNA replication forks in HPV16 positive cells. The central hypothesis is that differences can be exploited for differential targeting of HPV positive cells. This will be done in association with Pietro Pichierri, a WRN and DNA replication and repair expert; Morgan and Pichierri have already published together. Our third objective is to investigate the activation of the innate immune response (IIR) in HPV16 cells depleted for WRN and SAMHD1. The central hypothesis is that depletion of WRN or SAMHD1 in the presence of the active DDR in HPV16 cells results in excess cytoplasmic DNA fragments that activate the IIR. The outcomes are crucial for achieving our long-term goals. For example, targeting of WRN enzyme activities could boost the IIR in HPV16 positive cells, assisting the adaptive immune response (and immunotherapy) to eliminate HPV positive cells. If there are different factors replicating host DNA in the presence of HPV, these could be targeted to...