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Cellular and viral factors regulating Merkel cell polyomavirus replication

Feng, H and Kwun, HJ and Liu, X and Gjoerup, O and Stolz, DB and Chang, Y and Moore, PS (2011) Cellular and viral factors regulating Merkel cell polyomavirus replication. PLoS ONE, 6 (7).

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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. © 2011 Feng et al.


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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
Feng, H
Kwun, HJhyk14@pitt.eduHYK14
Liu, X
Gjoerup, O
Stolz, DBdonna.stolz@pitt.eduDSTOLZ
Chang, Yyc70@pitt.eduYC70
Moore, PSpsm9@pitt.eduPSM9
Centers: Other Centers, Institutes, Offices, or Units > Center for Biologic Imaging
Other Centers, Institutes, Offices, or Units > Pittsburgh Cancer Institute
Date: 28 July 2011
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 6
Number: 7
DOI or Unique Handle: 10.1371/journal.pone.0022468
Schools and Programs: School of Medicine > Cell Biology and Molecular Physiology
Refereed: Yes
MeSH Headings: Antigens, Polyomavirus Transforming--chemistry; Antigens, Polyomavirus Transforming--genetics; Antigens, Polyomavirus Transforming--metabolism; Cloning, Molecular; Conserved Sequence; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins--metabolism; Merkel cell polyomavirus--genetics; Merkel cell polyomavirus--immunology; Merkel cell polyomavirus--metabolism; Merkel cell polyomavirus--physiology; Mutation; Protein Structure, Tertiary; Vesicular Transport Proteins--metabolism; Virus Replication--genetics
Other ID: NLM PMC3142164
PubMed Central ID: PMC3142164
PubMed ID: 21799863
Date Deposited: 03 Aug 2012 15:49
Last Modified: 04 Feb 2019 16:55


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