Cellular and Viral Factors Regulating Merkel Cell Polyomavirus Replication

Abstract

Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, 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 large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.

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

“…To determine the role of these SCF E3 recognition sites in MCV replication, we examined a whole virus genome clone (MCV-HF) (17). Individual alanine substitution mutations at S147, S220, S239, and a double mutation (S220A/S239A) were engineered into the MCV-HF genome and transfected into 293 cells.…”

“…In support of this idea, MCV infection persists throughout the lifespan, and yet even under conditions of severe immune suppression, no disease syndrome has been described for actively replicating virus (16). In addition, transfection of plasmid viral DNA in cell culture produces scant viral particles even though the viral genome is maintained (15,17,18). It has not been possible to achieve serial virion transmission using a variety of cell lines, even when the MCV miRNA locus is mutated (10).…”

“…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).…”

“…These assays have revealed that LT sequesters Vps39 (21), which also inhibits MCV replication through an unknown mechanism (15,17,18). In contrast, MCV replication is enhanced by expression of the MCV small T (sT) protein (17), an alternatively spliced form of the T antigen locus that inhibits the cellular Skp-Cullin-F box (SCF) RING Fbw7 (F-box and WD repeat domain-containing 7) E3 ligase and promotes stabilization of the MCV LT protein (22). SCF E3 ligases comprise a large family of multisubunit proteins that bind specific phosphorylated peptide motifs (phosphodegron sites) to initiate substrate degradation after serine/threonine kinasemediated phosphorylation (23).…”

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

“…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).…”