Gabrielė Stakaitytė, Nnenna Nwogu, Samuel J. Dobson, Laura M. Knight, Christopher W. Wasson, Francisco J. Salguero, David J. Blackbourn, G. Eric Blair, Jamel Mankouri, Andrew Macdonald, Adrian Whitehouse

Cell motility and migration is a complex, multi-step, and multi-component process, intrinsic to progression and metastasis. Motility is dependent on the activity of integrin receptors and Rho-family GTPases resulting in the remodelling of the actin cytoskeleton and formation of various motile actin-based protrusions. Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high likelihood of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is associated with the majority of MCC cases, and MCPyV-induced tumourigenesis largely depends on the expression of the small tumour antigen (ST). Since the discovery of MCPyV, a number of mechanisms have been suggested to account for replication and tumourigenesis, but to date, little is known about potential links between MCPyV T antigen expression and the metastatic nature of MCC. Previously, we have described the action of MCPyV ST on the microtubule network and how this impacts on cell motility and migration. Here we demonstrate that MCPyV ST affects the actin cytoskeleton, to promote the formation of filopodia, through a mechanism involving the catalytic subunit of protein phosphatase 4 (PP4C). We also show that MCPyV ST-induced cell motility is dependent upon the activity of Rho-family GTPases Cdc42 and RhoA. In addition, our results indicate that the MCPyV ST-PP4C interaction results in the dephosphorylation of β1 integrin, likely driving the cell motility pathway. These findings describe a novel mechanism by which a tumour virus induces cell motility, which may ultimately lead to cancer metastasis and provides opportunities and strategies for targeted interventions for disseminated MCC.IMPORTANCE Merkel Cell Polyomavirus (MCPyV) is the most recently discovered human tumour virus. It causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer. However, the molecular mechanisms implicating MCPyV-encoded proteins in cancer development are yet to be fully elucidated. This manuscript builds upon our previous observations which demonstrate that the MCPyV ST antigen enhances cell motility, providing a potential link between MCPyV protein expression and the highly metastatic nature of MCC. Here we show that MCPyV ST remodels the actin cytoskeleton, promoting the formation of filopodia which is essential for MCPyV ST-induced cell motility and we also implicate the activity of specific Rho-family GTPases, cdc42 and RhoA in these processes. Moreover, we describe a novel mechanism for the activation of Rho GTPases and the cell motility pathway due to the interaction between MCPyV ST and the cellular phosphatase catalytic subunit PP4C, which leads to the specific dephosphorylation of β1 integrin. These findings may therefore provide novel strategies for therapeutic intervention for disseminated MCC.