Abstract PR5: Human Merkel cell polyomavirus causes Merkel cell carcinoma: Implication of viral etiology in human cancers

Over 20% of human cancers worldwide are associated with infection. We developed digital transcriptome subtraction (DTS) to identify a previously unregcognized virus, Merkel cell polyomavirus (MCV), in 80% of Merkel cell carcinomas (MCC). MCV is a double-strand DNA virus belonging to the Polyomaviridae family, having conserved early, late and regulatory regions. The polyomavirus early viral tumor (T) antigens play key roles in viral genome replication as well as tumorigenesis. For most polyomaviruses, Large T (LT) stimulate progressions of the cell cycle by binding to a number of cellular control proteins, such as tumor suppressor proteins p53 and the retinoblastoma (pRB). The small T (sT) antigen protein also targets the protein phosphatase 2A (PP2A) family of serine-threonine phosphatases pathway, which may contribute to viral neoplasia. The late region encodes viral capsid proteins (VP1 and VP2) are responsible for the viral particle composition. MCV was found as clonally-integrated virus into MCC and metastasis genomes. We sequenced full-length MCV genomes from 5 of the 8 virus-positive tumors as well as from 1 MCV positive cell line. All tumor-derived sequences have a T antigen truncation mutation that prematurely truncate the MCV LT helicase, rendering the virus nonpermissive. In contrast, full-length MCV genomes isolated from a peripheral blood sample can activate unlicensed replication from an integrated origin—suggesting that secondary mutations to T antigen are required for the integrated virus to survive. We reconstructed a consensus MCV clone, MCV-HF, from 7 MCV genomes. Three days after MCV-HF transfection into 293 cells, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical −40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which LT and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. MCV replication and encapsidation are increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. As a new human tumor virus, MCV can be expected to induce cell transformation in unexpected ways. Knockdown of all T antigen isoforms leads to cell cycle arrest and cell death of MCV-positive MCC cells, showing that the virus is absolutely required for these tumor cells. In transformation studies, overexpression of MCV sT, but not wild-type or tumor-derived MCV LT, causes rodent fibroblast transformation and human fibroblast serum-independent growth. In distinct contrast to SV40, MCV sT-induced cell transformation is independent of its ability to bind and interact with PP2A and heat-shock protein. Instead, MCV sT acts downstream in the mammalian target of rapamycin (mTOR) signaling pathway to target the eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1) and maintain its hyperphosphorylation. Our studies suggest that sT is an new viral oncoprotein that targets dysregulation of cap-dependent translation. In three years since its discovery using human genome project data, new diagnostics based on the Merkel cell polyomavirus have been developed for this enigmatic and severe cancer, and a basic understanding of how MCV promotes MCC has been uncovered. These findings hold promise for development of rational chemotherapeutic targeting of this enigmatic and severe cancer in the near future. This abstract is also presented as Poster B31. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR5.