ABSTRACT A fundamental question in cancer virology is, how do some viruses persist as chronic viral infections that can lead to cancer? Virus latency–in which the virus does not replicate independently of the host cell or produce active viral progeny–is required for long term viral escape from immune surveillance. This leads to prolonged persistence and chronic infection. Latency is well-defined for herpesviruses and retroviruses but not for other viruses such as Merkel cell polyomavirus (MCPyV). This proposal seeks to investigate how MCPyV controls its own replication to achieve persistence/latency with new technologies developed in our laboratory. The MCPyV Large T (LT) oncoprotein is a replication helicase needed for MCPyV genome replication. Other MCPyV viral factors including small T protein, miRNA, circRNA, as well as cellular elements (e.g. Skip-Cullen-F box (SCF) E3 ligases, Vam6p, USP7) regulate MCPyV replication by changing LT protein accumulation at the viral origin or by changing LT functional activity. In Aim 1, we will characterize LT binding and helicase activity by single molecule visualization of LT protein binding to MCPyV genome DNA using LUMICKS C-Trap. These studies will allow us to directly determine in situ LT binding, multimerization kinetics, initiation of helicase activity, and, potentially, initiation of DNA synthesis. This allows us to separate molecular LT binding to DNA from its transcriptional activities that feedback to regulate LT. Non-origin LT binding sites that may serve as transcription factor binding sites will also be investigated. Accessory factors regulating LT-origin binding will be determined in subsequent aims. In Aim 2, we will extend our findings on proteostatic viral latency for MCPyV using CRISPR-Cas9 library screening of 72 clonal cellular Skip-Cullen-F box (SCF) E3 ligase knockouts to search for novel LT-E3 ligase interactors regulating MCPyV replication by targeting LT for degradation. Confirmation of these factors will be performed by gene knockout and mutation of putative LT phosphodegron sites. In Aim 3, we will use live cell imaging of a novel MCPyV fluorescent reporter virus in a single round replication system to monitor cellular factors, such as cell cycle stage using FUCCI stable cells, and viral factors found in Aims 1 and 2 to determine when and how these factors regulate viral replication at the single cell level. Completion of these aims will build a fundamental understanding on how a small DNA tumor virus regulates its own replication. These studies make use of new technologies applied by our laboratory to examine MCPyV replication. Taken together, these aims will define whether a programmed MCPyV latency lifecycle exists and, if so, how this lifecycle impacts virus persistence. These studies will have direct importance to the understanding of the genesis of Merkel cell carcinoma and may point toward targeted therapies to control persistent MCPyV, as well as other polyomaviruses causing diseases among cancer and immune suppressed patients.

PROJECT NARRATIVE How does Merkel cell polyomavirus (MCPyV) infect most children and young adults, and remain quiescent for decades, but then re-emerge as an active infection to be transmitted to other individuals years later? During MCPyV reactivation, mutations may occur that drive the virus to cause Merkel cell carcinoma, one of the most severe skin cancers, in susceptible people. Understanding MCPyV quiescence and replication is critical to developing therapeutic and preventive measures for this cancer virus.