The Role of ALTO in the MCPyV Lifecycle and Tumorigenicity

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DESCRIPTION (provided by applicant): Murine and simian polyomaviruses (MCPyV and SV40) have been extraordinarily useful models to study transformation and tumorigenicity, processes that are attributable to proteins encoded in the early region (ER) of their genomes by the T antigen genes. To date eleven human polyomaviruses have been discovered but only one, Merkel Cell polyomavirus (MCPyV) is clearly associated with a human tumor, Merkel Cell Carcinoma (MCC). Little is known about the mechanism by which MCPyV contributes to tumorigenesis, nor is much known about the viral lifecycle. Like all polyomaviruses, MCPyV encodes large and small T antigens (LT and ST). Interestingly we discovered that the +1 reading frame relative to MCPyV LT exon 2 encodes another protein that we have called ALTO (Alternate to LT antigen Open reading frame). ALTO is evolutionarily related to the middle T antigen of mouse polyomavirus through a conserved hydrophobic C-terminus and defines a new clade of mammalian polyomaviruses that includes PyVs from mouse, hamster, raccoon, bat gorilla, chimp and human. Our goals are to understand the role of ALTO in the MCPyV lifecycle and how it affects tumorigenicity. Transfection of a circularized MCPyV genome results in transient amplification for several days. We have constructed WT and ALTO-mutated genomes and will compare the levels and kinetics of viral replication, expression of the T antigens, virion production and infectivity. The mechanism by which MCPyV transforms cells and contributes to tumorigenicity is not well understood. We will examine the effects of LT, ST and ALTO on cell proliferation, immortalization and transformation. Preliminary data indicates that ALTO induces markers of autophagy and apoptosis. We will determine more fully how ALTO affects these pathways. The T antigens of PyVs exert their effects through binding to cellular proteins, thus it is likely that ALTO also influences the virus lifecycle and tumorigenicity through specific partners. We will take both an unbiased and a biased approach to identify the partners. First we will use tandem affinity purification and mass spectrometry to identify ALTO associated proteins. Secondly, we will specifically test the hypothesis that ALTO is regulated by tyrosine phosphorylation.