Merkel cell carcinoma (MCC) is a rare but aggressive human skin cancer that typically affects elderly and immunosuppressed individuals, a feature suggestive of an infectious origin. We studied MCC samples by digital transcriptome subtraction and detected a fusion transcript between a previously undescribed virus T antigen and a human receptor tyrosine phosphatase. Further investigation led to identification and sequence analysis of the 5387-base-pair genome of a previously unknown polyomavirus that we call Merkel cell polyomavirus (MCV or MCPyV). MCV sequences were detected in 8 of 10 (80%) MCC tumors but only 5 of 59 (8%) control tissues from various body sites and 4 of 25 (16%) control skin tissues. In six of eight MCV-positive MCCs, viral DNA was integrated within the tumor genome in a clonal pattern, suggesting that MCV infection and integration preceded clonal expansion of the tumor cells. Thus, MCV may be a contributing factor in the pathogenesis of MCC.

Merkel cell polyomavirus (MCV) is a virus discovered in our laboMerkel cell carcinoma ͉ pRB interaction ͉ viral integration ͉ virus replication ͉ helicase M erkel cell carcinoma (MCC) is an aggressive skin cancer associated with sun exposure and immunosuppression (1, 2). Using digital transcriptome subtraction, we recently identified Merkel cell polyomavirus (MCV) as a novel polyomavirus integrated into the genome of MCC tumors (3, 4). The close association between MCV and MCC has been confirmed by others (5). Polyomaviruses are small circular DNA viruses encoding a T antigen oncoprotein locus. T antigens are expressed from variably spliced viral transcripts that target tumor suppressor and cell cycle regulatory proteins, including retinoblastoma tumor suppressor protein (Rb) (6), p53 (7, 8), protein phosphatase 2A (9), and Bub1 (10). Murine polyomavirus (MuPyV) middle T (MT) antigen, a membrane-bound protein, is particularly potent in initiating cell transformation through interactions with phosphatidylinositol 3-kinase, protein phosphatase 2A, Src, and Shc proteins (11,12). MCV large T (LT) antigen retains conserved domains, such as pocket Rb binding LXCXE and DnaJ motifs, present across virus species (13). LT not only encodes tumor suppressor targeting domains but also origin binding and helicase/ATPase functions required for viral genome replication. MCV integration in MCC tumors is incompatible with transmissible virus and likely represents a rare biological accident in which the tumor cell is a dead-end host. The monoclonal pattern of MCV integration into MCC tumors suggests that virus integration occurs before tumor cell expansion and that MCV is a contributing factor in a portion of MCC (3).We have isolated MCV T antigen sequences from both tumor cases and nontumor cases to characterize their abilities to act as a viral DNA replicase. Sequence analysis demonstrates that LT protein is prematurely truncated in all MCC cases, whereas the Rb-interacting domain is preserved. Viruses from nontumor sources do not possess these mutations. We also describe here an MCC cell line stably harboring MCV and an origin replication assay to assess MCV replication. We show that wild type (WT) LT from nontumorous sources activates MCV replication of integrated tumor virus, suggesting that MCV-associated MCC arises from a two-step process in which viral genome integrates into the host genome and develops T antigen mutations to prevent autonomous viral genome replication. Failure to truncate the viral T antigen may lead to DNA damage responses or immune recognition that hinders nascent tumor cell survival.

Merkel cell polyomavirus (MCV) is the recently discovered cause of most Merkel cell carcinomas (MCCs), an aggressive form of nonmelanoma skin cancer. Although MCV is known to integrate into the tumor cell genome and to undergo mutation, the molecular mechanisms used by this virus to cause cancer are unknown. Here, we show that MCV small T (sT) antigen is expressed in most MCC tumors, where it is required for tumor cell growth. Unlike the closely related SV40 sT, MCV sT transformed rodent fibroblasts to anchorageand contact-independent growth and promoted serum-free proliferation of human cells. These effects did not involve protein phosphatase 2A (PP2A) inhibition. MCV sT was found to act downstream in the mammalian target of rapamycin (mTOR) signaling pathway to preserve eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) hyperphosphorylation, resulting in dysregulated cap-dependent translation. MCV sT-associated 4E-BP1 serine 65 hyperphosphorylation was resistant to mTOR complex (mTORC1) and mTORC2 inhibitors. Steady-state phosphorylation of other downstream Akt-mTOR targets, including S6K and 4E-BP2, was also increased by MCV sT. Expression of a constitutively active 4E-BP1 that could not be phosphorylated antagonized the cell transformation activity of MCV sT. Taken together, these experiments showed that 4E-BP1 inhibition is required for MCV transformation. Thus, MCV sT is an oncoprotein, and its effects on dysregulated cap-dependent translation have clinical implications for the prevention, diagnosis, and treatment of MCV-related cancers.

Merkel cell carcinoma (MCC) is the most aggressive skin cancer. Recently, it was demonstrated that human Merkel cell polyomavirus (MCV) is clonally integrated inMerkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer. Although it is rare, its reported incidence is increasing (19). MCC is associated with UV exposure and affects primarily elderly and immune-suppressed patients (5,11,17,26). The susceptibility of MCC to immune surveillance is similar to that of known virus-induced cancers and suggests that MCC has an infectious trigger (9). Recently, a new human polyomavirus, termed Merkel cell polyomavirus (MCV), was discovered to be clonally integrated into MCC tumor genomes (14). While MCV integration occurs at distinct sites in MCC tumors from different individuals, primary tumors and corresponding metastases have identical integration sites, consistent with the occurrence of MCV infection and integration prior to clonal expansion and metastasis (14,37). A number of studies have confirmed that MCV is present in 69 to 85% of MCC tumors collected from Europe and the United States (4, 15, 21, 41). Surveys of control non-MCC skin, hematolymphoid, and neuroendocrine tumors are generally negative for MCV, although incidental low-level infection can be detected (4,14,22,33,34,39,42,44).All polyomaviruses encode alternatively spliced large T (LT) and small T (sT) antigen transcripts that share exon 1 of the T-antigen (TA) locus. Additional multiply spliced TA transcripts have been described for different polyomaviruses, including the 17kT and 57kT antigens in simian virus 40 (SV40) and MCV, respectively (40,46). Research on viral proteins encoded by the TA locus has been central to uncovering cell signaling networks important in cancer biology (10, 38). The targeting of cellular proteins, such as retinoblastoma protein (Rb), p53, and protein phosphatase 2A (PP2A), by TAs contributes to polyomavirusinduced cell transformation (for reviews, see references 1 and 2). MCV TAs that are expressed in MCC tumors lack a putative p53 binding domain because of tumor-associated T-antigen deletion mutations (37,40). Other conserved tumor suppressor-targeting motifs, including the Rb binding domain (LXCXE motif), the J domain (HPDK) in LT/57kT, and a putative PP2A interaction domain in sT, remain intact (40).Current data point toward MCV as the infectious cause for most Merkel cell cancers: the virus is associated with MCC tumors and, when present, expresses T antigen in tumor cells but not in healthy surrounding tissues (7,20,39). MCV is specific to MCC and is not detected at significant levels in other cancers or in healthy skin examined to date, despite widespread circulation of MCV among human populations (8,23,29,42). Clonal analysis of MCC tumors also supports the correct temporal relationship for causality; i.e., MCV infection occurs prior to MCC tumor development (18). If MCV is a direct cause of MCC tumorigenesis, it is expected that MCC tumors will require MCV protein expression to maintain the tumor ...

Merkel cell polyomavirus (MCV) is a recently discovered human virus closely related to African green monkey lymphotropic polyomavirus. MCV DNA is integrated in 80% of Merkel cell carcinomas (MCC), a neuroendocrine skin cancer linked to lymphoid malignancies such as chronic lymphocytic leukemia (CLL). To assess MCV infection and its association with human diseases, we developed a monoclonal antibody that specifically recognizes endogenous and transfected MCV large T (LT) antigen. We show expression of MCV LT protein localized to nuclei of tumor cells from MCC having PCR quantified MCV genome at an average of 5.2 (range 0.8-14.3) T antigen DNA copies per cell. Expression of this putative viral oncoprotein in tumor cells provides the mechanistic underpinning supporting the notion that MCV causes a subset of MCC. In contrast, although 2.2% of 325 hematolymphoid malignancies surveyed also showed evidence for MCV infection by DNA PCR, none were positive at high viral copy numbers, and none of 173 lymphoid malignancies examined on tissue microarrays expressed MCV LT protein in tumor cells. As with some of the other human polyomaviruses, lymphocytes may serve as a tissue reservoir for MCV infection, but hematolymphoid malignancies associated with MCC are unlikely to be caused by MCV. ' 2009 UICC