Type of Thesis
Molecular, Cellular, & Developmental Biology
Polyomaviruses are small, DNA tumor viruses that can infect multiple species, including humans. In recent years, the number of identified human polyomaviruses (huPyV) has grown. To identify possible therapeutic targets for huPyVs, a better understanding of virus infection at the structural level is needed. Confocal microscopy allows for the visualization of fluorescently-tagged proteins but without seeing the cell’s subcellular structures, functional information can be lost. Electron microscopy provides structural insight but does not always provide biological or biochemical details. Combining these two approaches can relate biological function to structural information about virus replication. Polyomaviruses (PyV) co-opt host proteins that function during normal DNA damage repair to aid in replication of viral genomes. Here we describe a mouse embryonic cell line (MEF) that stably expresses a fluorescently-tagged component of the replication protein complex A (RPA). RPA is made up of three proteins (RPA70/RPA32/RPA14) and is an important host component for PyV vDNA replication. MEFs stably-expressing GFP-human RPA32 or RPA70 (GFP-huRPA32/70) were infected with murine polyomavirus (MuPyV) to study viral DNA (vDNA) replication by correlative light and electron microscopy (CLEM). Light microscopy studies found that GFP-huRPA32/70 localized to sites of vDNA replication along with other host DNA damage response (DDR) proteins, which have been previously characterized. We attempted to correlate the immunofluorescence of GFPhuRPA proteins at viral replication centers to the ultrastructure of virus factories that have been observed by electron microscopy (EM).
Henderson, Christina, "Correlative Light and Electron Microscopy on Mouse Polyomavirus-Infected Fibroblasts" (2017). Undergraduate Honors Theses. 1363.