PROJECT SUMMARY An emerging concept in virology is that viral replication and virion assembly are often mechanistically coupled in specific locations within the cell at sites termed virus “factories”. The spatial and temporal coordination of these processes permit assembly line production of virions in a manner that is energetically favorable while allowing for quality control of the final structure. Although factories have been described in some detail for viruses replicating in the cytoplasm, most DNA viruses replicate/assemble in the nucleus where morphological features of factories are problematic to identify and characterize due to the dense chromatin network. There are also indications that the factories usurp essential nuclear domains otherwise used for such functions as DNA damage repair (DDR), and indeed many cellular proteins involved in DDR are found at replication sites for DNA viruses. We propose to characterize the structure and composition of virion assembly factories for the well-studied murine polyomavirus using a combined approach employing iPOND protein identification, high resolution imaging, and virus/host cell genetics. Our goal is to determine the molecular architecture of these factories at nanometer resolution as well as the spatial and functional relationships of their protein components. With the recent identification of ten new human polyomaviruses, and the marked increase in immunomodulatory therapies for a variety of diseases, polyomaviruses have emerged as important human pathogens that at present have no specific therapy. Characterization of the assembly factories not only will identify new therapeutic targets for affecting polyomavirus replication, as well as other DNA viruses, but also provide insight as to their function in normal cellular processes.