How does a viral pathogen manipulate DNA Damage Responses to promote infection?

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SUMMARY DNA viruses elicit a cellular DNA damage response (DDR) in infected cells, either in response to incoming viral genomes, or to the large number of foreign genomes generated during virus replication. The DDR forms an innate, critical barrier that can impede or facilitate virus replication. Following host cell infection, replication of the parvovirus minute virus of mice (MVM) induces a sustained DDR, which is then exploited to enhance its replication. Parvovirinae, small non-enveloped icosahedral viruses, are important pathogens in many animal species including humans. These are the only known viruses of vertebrates that contain single-stranded linear DNA genomes, presenting novel replicative DNA structures to the host cell nucleus during infection while relying on cellular processes to replicate. Recent studies by the Pintel lab has shown that MVM interacts with sites of DNA damage to initiate and amplify its infection. This application proposes to examine how MVM exploits the cellular DDR to prepare the nuclear environment for effective parvovirus takeover. Aim 1: How does MVM initiate and activate the host cell’s DNA damage signaling pathway? MVM infection recruits the cellular DDR sensor MRE11 in an MRN (MRE11-RAD50-NBS1) holocomplex- independent manner, but activates a downstream ATM signaling cascade. This project will examine how MRE11 localizes to MVM, and initiates a downstream DDR without its canonical intermediate adaptor complex. Aim 2: How does MVM utilize DNA- and protein- bridging molecules to establish replication centers at sites of DNA damage? MVM associates with cellular DDR sites bound by the architectural protein CTCF, and the DDR-chromatin binding protein MDC1. This study will decipher the mechanism by which CTCF and MDC1 drive the formation of viral replication centers, and how they influence the cellular chromatin architecture. Aim 3: Determine how MVM infection induces additional host-cell DNA damage. MVM infection leads to the production of cellular reactive oxygen species (ROS), which contributes to cellular DNA damage. This research will characterize how MVM induces ROS, and how it utilizes the resulting stress signals to amplify downstream DDR signals, thereby expanding infection. Characterization of the interaction between the incoming viral genome and cellular DDR sensors will provide important insight into how cellular DDR cascades are initiated. Secondly, it will elucidate the mechanisms of trans-interaction between MVM and host chromosome at sites of DNA damage. Finally, studies on ROS induction will help in the understanding of how virus replication induces additional DNA breaks, allowing it to expand further. Examining these processes will be essential for understanding how parvoviruses interact with cells, how they program successful infection and ultimately how they cause disease or persist as gene therapy vehicles. Findings from these studies will be extrapolatable to other DNA viruses and pathogenic viral systems.