Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen



Merkel cell polyomavirus (MCV) is a newly discovered human cancer virus encoding a small T (sT) oncoprotein. We performed MCV sT FLAG-affinity purification followed by mass spectroscopy (MS) analysis, which identified several protein phosphatases (PP) including PP2A A and C subunits and PP4C as potential cellular interacting proteins. PP2A targeting is critical for the transforming properties of non-human polyomaviruses, such as simian virus 40 (SV40), but is not required for MCV sT-induced rodent cell transformation. We compared similarities and differences in PP2A binding between MCV and SV40 sT. While SV40 sT co-immunopurified with subunits PP2A AI? and PP2A C, MCV sT co-immunopurified with PP2A AI?, PP2A AI? and PP2A C. Scanning alanine mutagenesis at 29 sites across the MCV protein revealed that PP2A-binding domains lie on the opposite molecular surface from a previously-described large T stabilization domain (LSD) loop that binds E3 ligases, such as Fbw7. MCV sT-PP2A interactions can be functionally distinguished by mutagenesis from MCV sT LSD-dependent 4E-BP1 hyperphosphorylation and viral DNA replication enhancement. MCV sT has a restricted range for PP2A B subunit substitution, inhibiting only the assembly of B56I? into the phosphatase holoenzyme. In contrast, SV40 sT inhibits the assembly of B55I?, B56I? and B56I? into PP2A. We conclude that MCV sT is required for Merkel cell carcinoma growth but its in vitro transforming activity depends on LSD interactions rather than PP2A targeting.

IMPORTANCE: Merkel cell polyomavirus is a newly-discovered human cancer virus that promotes cancer, in part, through expression of its small T (sT) oncoprotein. Animal polyomavirus sT oncoproteins have been found to cause experimental tumors by blocking the activities of a group of phosphatases called PP2A. Our structural analysis reveals that MCV sT also displaces the B subunit of PP2A to inhibit PP2A activity. MCV sT, however, only displaces a restricted subset of PP2A B subunits, which is insufficient to cause tumor cell formation in vitro. MCV sT in stead transforms tumor cells through another region called the large T stabilization domain. The PP2A targeting and transforming activities lie on opposite faces of the MCV sT molecule and can be genetically separated from each other.

Kwun HJ, Shuda M, Camacho CJ, Gamper AM, Thant M, Chang Y, Moore PS

PMID: 25631078