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.
Highlights
Merkel cell polyomavirus (MCV) was identified by digital transcriptome subtraction from Merkel cell carcinoma (MCC), a rare but aggressive human skin cancer [1,2]
We originally found 8 of 10 (80%) MCC tumors to be positive for MCV DNA [1]
A 200-bp deletion is present in the VP1 locus of MCC352, generates a truncated VP1 protein that is likely to lead to incomplete viral assembly for this strain (Fig. 1A)
Summary
Merkel cell polyomavirus (MCV) was identified by digital transcriptome subtraction from Merkel cell carcinoma (MCC), a rare but aggressive human skin cancer [1,2]. Large T (LT) antigenencoded helicase activity, for example, unwinds the viral replication origin [3,4] and enhances the polyomavirus late promoter leading to an early-to-late switch in gene expression. For murine polyomavirus, this switch has been shown to depend on LT-initiated viral DNA replication [5]. The late region encodes viral capsid proteins (VP1 and VP2) that self-assemble into virus-like particles (VLP) when expressed in cells [6,7,8,9,10]. The concerted regulation and interaction of both early and late polyomavirus proteins are necessary to produce viral particles
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