FC28 Characterization of the small T Antigen in Merkel cell-associated polyomavirus in Merkel cell carcinogenesis

Abstract

Background Merkel cell carcinoma (MCC) is a highly aggressive skin malignancy affecting neuroectodermal cells. Recently it was discovered that MCC have an infectious origin and a novel polyomavirus, the Merkel cell associated polyomavirus (MCPyV), was identified in MCC. MCPyV expresses two ‘early’ proteins, namely the Large T (LT) and Small t (St) antigens [1,2]. Hypothesis Based on the research data on other polyomaviruses, it is suggestive that LT and St antigens are involved in viral replication and host cell transformation. However the LT antigen is found truncated in MCC losing helicase activity and putatively inactivating p53 tumor suppressor binding capacity. Therefore, in this study, we propose to investigate the role of the intact St antigen in MCC carcinogenesis. Aim We planned to express and purify St protein. Polyclonal antibody will be developed against specific St oligopeptide and will be used in the verification of the produced St protein. Methods The full St gene was PCR amplified using DNA extracted from a MCPyV positive MCC case. Prokariotic expression system including pEXP5-CT/TOPO vector and BL21 Star(tm)(λDE3)pLysS E. coli cells (Invitrogen Co.) was utilized. Results Small t protein was successfully expressed and verified with His-Tag antibody and St specific antibody. Conclusion The expressed St protein will be purified and utilized in further assays to find the cellular protein binding partners. These experiments intended to reveal the interference of St antigen with cell regulatory proteins. This information can lead to understanding of MCC carcinogenesis and to developing better therapy to combat this malignant cancer. Key Words: MCC, MCPyV, small t antigen protein expression Funding: NIAID: ‘Molecular Basis of Infectious Disease’ (1 T32 AI055449-01A2) Co-Investigator (i.e. Mentor), 2005-2010.