The Minimum Replication Origin of Merkel Cell Polyomavirus Has a Unique Large T-Antigen Loading Architecture and Requires Small T-Antigen Expression for Optimal Replication

Abstract

Merkel cell polyomavirus (MCV) is a recently discovered human polyomavirus causing the majority of human Merkel cell carcinomas. We mapped a 71-bp minimal MCV replication core origin sufficient for initiating eukaryotic DNA replication in the presence of wild-type MCV large T protein (LT). The origin includes a poly(T)-rich tract and eight variably oriented, GAGGC-like pentanucleotide sequences (PS) that serve as LT recognition sites. Mutation analysis shows that only four of the eight PS are required for origin replication. A single point mutation in one origin PS from a naturally occurring, tumor-derived virus reduces LT assembly on the origin and eliminates viral DNA replication. Tumor-derived LT having mutations truncating either the origin-binding domain or the helicase domain also prevent LT-origin assembly. Optimal MCV replication requires coexpression of MCV small T protein (sT), together with LT. An intact DnaJ domain on the LT is required for replication but is dispensable on the sT. In contrast, PP2A targeting by sT is required for enhanced replication. The MCV origin provides a novel model for eukaryotic replication from a defined DNA element and illustrates the selective pressure within tumors to abrogate independent MCV replication.Unlike human cellular DNA replication origins, polyomavirus replication origins are discrete and well defined and yet retain many features of eukaryotic cellular origins. For this reason, polyomavirus replication origins, particularly the simian virus 40 (SV40) origin, have been used as easily tractable models to define eukaryotic replication requirements (1).Polyomaviruses are small, double-stranded DNA viruses with circular genomes functionally divided into coding and noncoding regions (10). The early coding region for all polyomaviruses encodes large tumor (LT) and small T (sT) antigens that serve as viral oncoproteins and a late coding region that produces viral structural proteins. Aside from LT and sT, other T-antigen isoforms, such as middle T antigen (MT) and 17kT/57kT, may be present and are virus specific. LT has pleiotropic functions that include initiation and maintenance of viral DNA replication, regulation of early and late genes transcription, and virion assembly (11,21,36,43,51,52,54). Expression of LT also leads to the transformation of susceptible cell lines mediated in part by functional regions such as the DnaJ, pocket protein binding, and p53 binding domains that target growth-suppressing and cell cycle regulatory proteins (53). In addition, sT has been shown to play an important role in LT mediated cell transformation in SV40 (3,6,7,24,38) and has been reported to increase virus replication efficiency in JC virus (JCV) (39).Merkel cell polyomavirus (MCV) was recently identified by digital transcriptome subtraction (23) as a new human polyomavirus present in ϳ80% of Merkel cell carcinoma (MCC) (22). Preferential detection of MCV in MCC has subsequently been confirmed in a variety of different settings (4,17,29,58). Similar to JCV and BK virus, this n...

Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

“…Signal intensities were analyzed at 700 or 800 nm by using the Odyssey IR Imaging System (Li-Cor). The following primary antibodies were used: anti-MCV sT CM5E1 (33), CM8E6 (40), and Xt7 (http://home.ccr.cancer .gov/lco/BuckLabAntibodies.htm, kindly provided by Christopher Buck); anti-SV40 sT (pAb419, sc-58665; Santa Cruz); anti-␣ tubulin (B-5-1-2, T5168; Sigma-Aldrich); anti-HA (16B12; Covance); anti-FLAG (M2, F-3165; Sigma-Aldrich); anti-PP2A A-alpha (6F9; Covance), anti-PP2A C-alpha (1D6; EMD Millipore); and anti-4E-BP1 S65 (Cell Signaling).…”

“…The MCV replication origin assay was described previously (40). Briefly, 293 cells were transfected with T antigen expression vector (LT/sT, 0.3 g) and pMCV-Ori339(97) (0.…”

“…Unlike SV40 sT, however, MCV sT is a transforming oncoprotein in immortalized rodent fibroblast cells (38,39) and has unique functions that have not been identified in other polyomavirus small T antigens. For example, MCV sT enhances LT-mediated MCV viral genome replication by stabilizing LT through sequestration of Fbw7, an E3 ligase that ubiquitinates LT for rapid turnover (40,41). This MCV sT function was mapped to amino acids 91 to 95, defined as the large T stabilization domain (LSD) (42), and determined to be essential for rodent cell transformation (42).…”

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

“…MCV gene expression and DNA replication have been investigated using origin replicon assays and by transfection of the molecular viral clone DNA into cells (17,20). These assays have revealed that LT sequesters Vps39 (21), which also inhibits MCV replication through an unknown mechanism (15,17,18).…”

“…Each of the 15 potentially phosphorylated residues was individually mutated to alanine (A), and LT protein half-life and LTdependent replicon replication was determined (20). LT turnover was markedly reduced by alanine mutations at three residues (S147, S220, and S239), and LT-dependent replication was increased over wild-type by mutations at S220 and S239 (Fig.…”