Characterization of functional domains in the Merkel cell polyoma virus Large T antigen

Merkel cell polyomavirus (MCPyV)-positive Merkel cell carcinoma (MCC) tumor cell growth is dependent on the expression of a viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. This RB-binding domain in MCPyV-LT isin contrast to other polyomavirus LTs (e.g., SV40)embedded between two large MCPyV unique regions (MUR1 and MUR2). To identify elements of the MCPyV-LT necessary for tumor cell growth, we analyzed the rescue activity of LT variants following knockdown of the endogenous LT in MCC cells. These experiments demonstrate that nuclear localization is essential for LT function, but that a motif previously described to be a nuclear localization sequence is neither required for nuclear accumulation of truncated MCPyV-LT nor for promotion of MCC cell proliferation. Furthermore, large parts of the MURs distal to the RB binding domain as well as ALTOa second protein encoded by an alternative reading frame in the MCPyV-LT mRNAare completely dispensable for MCPyV-driven tumor cell proliferation. Notably, even MCPyV-LTs in which the entire MURs have been removed are still able to promote MCC cellular growth although rescue activity is reduced which may be due to MUR1 being required for stable LT expression in MCC cells. Finally, we provide evidence implying thatwhile binding to Vam6p is not essentialHSC-70 interaction is significantly involved in mediating MCPyV-LT function in MCC cells including growth promotion and induction of E2F target genes. What's new? The Merkel cell polyomavirus (MCPyV) is the first virus of the polyomavirus family to be established as a causal factor of a human cancer. Merkel cell carcinoma is a rare but very aggressive skin cancer with high mortality rates. Here, the authors describe experiments defining certain domains and molecular functions of the Large T antigen encoded by MCPyV, which are either essential or dispensable for its growth-promoting function in Merkel cell carcinoma cells.