Project Summary/Abstract Polyomaviruses (PyVs) are small DNA tumor viruses that cause debilitating disease in humans, including the often-fatal Merkel cell carcinoma by Merkel cell polyomavirus (MCPyV). To cause infection, these non-enveloped viruses must transport through the crowded host cellular environment to reach the nucleus where transcription and replication of the viral genome leads to lytic infection or cellular transformation. During entry, PyV, is endocytosed and trafficked to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol. Once in the cytosol, the virus is disassembled and transported to the nucleus where it is thought to enter the nucleus through the nuclear pore complex (NPC). How PyV reaches the nuclear membrane and is subsequently imported into the nucleus have yet to be determined. Intracellular transport is mediated largely through the actions of the kinesin and cytoplasmic dynein host motor proteins that transport cellular cargo towards the periphery and center of the cell, respectively. Studies of the archetype PyV, simian virus 40 (SV40), revealed that the virus exploits these motors in escaping the ER to reach the cytosol and for virus disassembly in this compartment. This K99/R00 proposal seeks to understand the role of cellular motors in the subsequent transport and nuclear entry of PyVs. During the mentored phase of this award, Dr. Spriggs will determine how kinesin-1 and dynein coordinate the transport of disassembled SV40 to the host nucleus and the mechanism by which it enters the nucleus through the NPC. At this time, she will receive training in high-resolution microscopy techniques, including correlative light and electron microscopy (CLEM), that when used in combination with both traditional and state-the-art biochemical approaches, will lead to impactful discoveries in viral and cellular nuclear import mechanisms. While much of our knowledge of human PyV infection has come from the study of SV40, key differences have been observed between it and MCPyV, the only PyV definitely associated with human cancer. One prominent distinction lies in its proposed mechanism of nuclear entry, which may instead require mitotic nuclear envelope breakdown. Despite its impact on public health, little is known of the basic biology of MCPyV infection. During the independent phase, and with her K99 training, Dr. Spriggs will delineate the entry pathway of this distinct human pathogen, culminating in a mechanistic understanding of its nuclear entry. When combined with her strong background in virology and cell biology, learning high-resolution microscopy should fully equip Dr. Spriggs to run a successful independent research program studying virus-host interactions. Further, along with her highly motivated Advisory Committee, the University of Michigan provides an ideal environment for her training, given its exceptional research facilities and professional development resources.

Project Narrative Polyomaviruses (PyVs) cause debilitating human diseases, including the often-fatal Merkel cell skin carcinoma. How these small DNA viruses transport to the nuclear membrane and then into the nucleus, where genome replication leads to infection and transformation, is not well understood. This innovative study aims to elucidate the nuclear entry mechanisms of two distinct PyVs and the role of host motor proteins in this process.