Revealing the structural mechanisms of microtubule manipulation by polyomaviruses

Polyomaviruses infect a variety of animals and in humans they cause various illnesses in immunocompromised individuals. For example, Trichodysplasia spinulosa polyomavirus causes a skin condition, BK polyomavirus causes kidney disease and is a leading cause of kidney transplant failure and JC polyomavirus is associated with a fatal neurodegenerative disease. Furthermore, Merkel cell polyomavirus causes the aggressive and quickly spreading skin cancer, Merkel cell carcinoma. A viral initiator of cancer is of immense scientific interest and in this respect a tumorigenic mouse polyomavirus (MPyV) has become an important research model into cancer-causing biochemical pathways. Animal cells have an internal ‘cytoskeleton’ composed of filaments that can grow and shrink and are further organised into various macro-architectures, in order to mediate vital cellular processes such as changing shape, migration and cell division. Furthermore, cell components are moved along these filaments for delivery to cellular subregions. In the late stages of MPyV infection, the viral protein VP1 has been observed to coat a vital cytoskeletal filament type known as microtubules, preventing their growth and shrinkage. This coating causes issues with cell functions, cell division and causes increased cell death. This manipulation of the host cellular cytoskeleton by the virus may be a key strategy in its replicative cycle and/or be involved in causing illness including cancer. However, it is unknown how MPyV VP1 attaches to microtubules and why this prevents their growth and shrinkage. Furthermore, it is unknown whether evolutionarily equivalent VP1 proteins from human polyomaviruses associate with microtubules and regulate their dynamics in the same way. This project’s core goals are to decipher how polyomavirus VP1 proteins attach to microtubules to regulate their growth and shrinkage and how generalisable this is to human polyomaviruses linked to diseases including cancer. We aim to detail at the 3D nanoscale how MPyV VP1 associates with microtubules, both as purified components and in the cellular environment during infection, including, in order to understand VP1’s inhibition of dynamics, at the ends of microtubules where shrinkage and growth occurs. We will also determine if VP1 accessory proteins (VP2 and VP3) are involved in the interaction with microtubules. Finally, we will further investigate the crucial question of whether VP1 proteins of human disease-causing polyomaviruses have the same evolutionarily conserved properties. Towards these aims, we will chiefly use the recent Nobel prize-winning technique of cryo-electron microscopy (cryo-EM), alongside supporting cutting-edge biochemical and imaging methodologies. Cryo-EM allows the artefact-free visualisation of proteins (including viral and cytoskeletal proteins) at up to atomic level detail, when purified or even within their cellular context. Recently, rapid improvements in cryo-EM hardware and software, including artificial intelligence (AI)-based image processing approaches, have produced an incredibly effective and versatile technique suited to the objectives of this work. Uncovering how polyomaviruses manipulate their host cells’ cytoskeletons will better our grasp of the mechanisms behind polyomavirus-related medical conditions. Furthermore, given the tumour-causing nature of polyomavirus species, it will also bolster our understanding of biochemical pathways leading to cancer. Structural studies of protein interactions such as this give us vital information towards drug and vaccine design, conferring this project significant therapeutic potential.