Detection of Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma cell lines: Cell morphology and growth phenotype do not reflect presence of the virus

Abstract

The recently discovered human polyomavirus (MCPyV) is frequently found in Merkel cell carcinoma (MCC) tissue and is believed to be causally linked to MCC pathogenesis. While cell lines established from MCC represent a valuable tool to study the contribution of MCPyV to MCC pathogenesis, hitherto only 1 MCPyV‐positive line has been described. We have analyzed 7 MCC cell lines for the presence, integration pattern and copy number of MCPyV. In 5 cell lines, MCPyV specific sequences were detected. In 3 of these lines, multiple copies of viral genomes per cell were detected, and sequencing of PCR amplificates identified distinct mutations predicted to lead to the expression of a truncated large T‐Antigen (LT‐Ag). In 1 cell line, clonal integration of concatamerized viral genomes was confirmed by Southern Blotting. MCC cell lines are conventionally categorized as “classic” or “variant” and further divided into 4 subtypes, based on expression of neuroendocrine markers and morphology. While it has been suggested that the presence of MCPyV might promote a classic phenotype, such a notion is not supported by our data. Instead, we find MCPyV‐positive as well as ‐negative lines of the classic variety, indicating that the distinguishing features are either inherently independent of viral infection or have become so in the course of tumorigenesis and/or cell line establishment. We therefore suggest a novel classification scheme based on MCPyV presence, integration patterns and T‐Ag mutations. The cell lines described here extend the repertoire of available MCPyV‐positive MCC‐lines and should aid in the elucidation of the role of MCPyV in the pathogenesis of MCC.

High-Affinity Rb Binding, p53 Inhibition, Subcellular Localization, and Transformation by Wild-Type or Tumor-Derived Shortened Merkel Cell Polyomavirus Large T Antigens

“…A number of findings suggest a causative role of MCPyV in Merkel cell carcinoma tumorigenesis: the virus can be identified in the majority of MCC biopsy specimens (3,4,(6)(7)(8)(9), MCPyV DNA is monoclonally integrated in the tumor cells (3,9), the viral T-Ags are expressed in tumor cells (5,10), and LT-Ag sequences isolated from tumor cells harbor tumor-specific muta- tions that lead to premature truncation of LT-Ag, presumably due to a selection pressure to abrogate viral replication (9,18,64,65). It is striking that all truncated LT proteins retain the LxCxE motif, a fact that would seem to provide a strong argument for a dominant requirement for LT-Ag-mediated Rb inactivation during MCC pathogenesis.…”

“…Coding sequences for truncated SV40 LT and MCPyV LT were PCR amplified using pZIPTEX (33) or cDNA from the MCPyV-positive MCC cell line 12 (18) as the template, and PCR fragments were sequenced and subcloned into pRSETA (Invitrogen). Nterminal His-tagged proteins were expressed in BL21 Star in LB medium supplemented with 1% glucose at 37°C for 4 h using 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG; for SV40 LT) or at 16°C for 18 h using 0.5 mM IPTG (for MCPyV LT).…”

“…These truncation products unequivocally preserve the Rb binding motif, but they lack a functional origin binding domain (OBD) and ATPase/helicase region (10,18,19). It is thought that these truncations are the result of a selection pressure to abrogate untimely firing of viral origins from integrated viral genomes (9,20).…”

“…Given the above, we have performed a side-by-side comparison of a consensus, full-length MCPyV LT-Ag (28) and several truncated LT protein genes from previously characterized MCCderived cell lines (18). Here, we demonstrate the characterization of the encoded LT-Ag proteins with regard to their subcellular localization, steady-state expression, transformation potential, and capacity to bind to Rb and p53.…”

Involvement of Merkel Cell Polyomavirus in the Etiology and Pathogenesis of Merkel Cell Carcinoma: A Systematic Review

“…The classification of MCC cell lines is made under the name of "classical" and "variant" phenotypes. These phenotypes are further divided into subtypes based on morphology and expression of genes taken neuroendocrine markers [62]. It has been suggested that the classical phenotype is for the MCC MCV-positive strains, and the MCV-negative strains are the variant [43].…”