Cellular and viral factors regulating Merkel cell polyomavirus replication.
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...
Main Authors: | , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Public Library of Science (PLoS)
2011-01-01
|
Series: | PLoS ONE |
Online Access: | http://europepmc.org/articles/PMC3142164?pdf=render |
id |
doaj-e1782b045250401b96ddd5eca88fe96d |
---|---|
record_format |
Article |
spelling |
doaj-e1782b045250401b96ddd5eca88fe96d2020-11-24T22:17:06ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0167e2246810.1371/journal.pone.0022468Cellular and viral factors regulating Merkel cell polyomavirus replication.Huichen FengHyun Jin KwunXi LiuOle GjoerupDonna B StolzYuan ChangPatrick S MooreMerkel 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.http://europepmc.org/articles/PMC3142164?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Huichen Feng Hyun Jin Kwun Xi Liu Ole Gjoerup Donna B Stolz Yuan Chang Patrick S Moore |
spellingShingle |
Huichen Feng Hyun Jin Kwun Xi Liu Ole Gjoerup Donna B Stolz Yuan Chang Patrick S Moore Cellular and viral factors regulating Merkel cell polyomavirus replication. PLoS ONE |
author_facet |
Huichen Feng Hyun Jin Kwun Xi Liu Ole Gjoerup Donna B Stolz Yuan Chang Patrick S Moore |
author_sort |
Huichen Feng |
title |
Cellular and viral factors regulating Merkel cell polyomavirus replication. |
title_short |
Cellular and viral factors regulating Merkel cell polyomavirus replication. |
title_full |
Cellular and viral factors regulating Merkel cell polyomavirus replication. |
title_fullStr |
Cellular and viral factors regulating Merkel cell polyomavirus replication. |
title_full_unstemmed |
Cellular and viral factors regulating Merkel cell polyomavirus replication. |
title_sort |
cellular and viral factors regulating merkel cell polyomavirus replication. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2011-01-01 |
description |
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. |
url |
http://europepmc.org/articles/PMC3142164?pdf=render |
work_keys_str_mv |
AT huichenfeng cellularandviralfactorsregulatingmerkelcellpolyomavirusreplication AT hyunjinkwun cellularandviralfactorsregulatingmerkelcellpolyomavirusreplication AT xiliu cellularandviralfactorsregulatingmerkelcellpolyomavirusreplication AT olegjoerup cellularandviralfactorsregulatingmerkelcellpolyomavirusreplication AT donnabstolz cellularandviralfactorsregulatingmerkelcellpolyomavirusreplication AT yuanchang cellularandviralfactorsregulatingmerkelcellpolyomavirusreplication AT patricksmoore cellularandviralfactorsregulatingmerkelcellpolyomavirusreplication |
_version_ |
1725786587228274688 |