T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus

Abstract

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.

Retinoblastoma gene mutations detected by whole exome sequencing of Merkel cell carcinoma

“…Merkel cell polyomavirus has a large T antigen LXCXE domain that, when expressed, binds directly to retinoblastoma protein. 45 Several lines of evidence have suggested We found that polyomavirus-negative cases with little or no detectable polyomavirus by sensitive real-time polymerase chain reaction had truncating, nonsense RB1 mutations. Even though two of the five polyomavirus-positive cases showed RB1 deletions (one case with a deletion and one with copy number variation), there were no single-nucleotide variation truncating nonsense mutations within polyomavirus-positive cases.…”

Protein-mediated viral latency is a novel mechanism for Merkel cell polyomavirus persistence

“…It was the first human pathogen discovered through nondirected transcriptome sequencing using an approach called digital transcriptome subtraction. MCV infection is nearly ubiquitous among human adults, and MCC tumors arise as rare biological accidents after prolonged infection in which the MCV genome becomes integrated into the host cell genome and mutations to the 3′ MCV LT gene clonally occur that eliminate the carboxyl terminus T antigen helicase domain (12)(13)(14). The viral integration and T antigen mutations that occur in tumors are incompatible with transmissible MCV replication, and tumors represent a biological dead end for the virus.…”

Cytokeratin 20-negative Merkel cell carcinoma is infrequently associated with the Merkel cell polyomavirus

“…Merkel cell polyomavirus in Merkel cell carcinoma displays clonal integration, high viral copy number, and tumorspecific mutations of large T antigen resulting in incapacity to replicate. 5,17,18,32 Furthermore, in vitro studies support a role for Merkel cell polyomavirus T antigens, especially small T antigen, in transformation and cell survival. 5 Most studies have reported that a significant minority of Merkel cell carcinoma lack detectable Merkel cell polyomavirus, 5 raising the question of how Merkel cell polyomavirus-negative tumors might differ from Merkel cell polyomavirus-positive tumors.…”

“…5 Merkel cell polyomavirus associated with Merkel cell carcinoma displays tumor-specific mutations or deletions in large T antigen which render the virus replication deficient, a common characteristic of oncogenic viruses. 5,18 Although numerous assays for Merkel cell polyomavirus detection have been reported, PCR targeting small T antigen and/or the 5' region of large T antigen is commonly used and highly sensitive. [19][20][21] Although present in the majority of Merkel cell carcinoma, Merkel cell polyomavirus has not been commonly observed in other malignancies.…”