Ubiquitin-dependent proteolysis of cyclin D1 is associated with coxsackievirus-induced cell growth arrest.
Coxsackievirus group B3 (CVB3) replication is influenced by host cell cycle status. However, the effect of CVB3 infection on cell cycle regulation and the mechanisms involved are not precisely defined. In this study, we examined cell cycle progression and regulation when the infection was initiated in late G(1) phase of the cell cycle. Analysis of cellular DNA synthesis in infected cells by thymidine incorporation assays showed a significant reduction in [(3)H]thymidine uptake compared to that of sham-infected cells. To further clarify the effects of CVB3 on the host cell cycle, we examined the cell cycle regulatory proteins involved in G(1) progression and G(1)/S transition. Infection resulted in dephosphorylation of retinoblastoma protein and reduced G(1) cyclin-dependent kinase activities, accompanied by decreased levels of G(1) cyclin protein expression (cyclin D1 and cyclin E). We further investigated the mechanisms by which CVB3 infection down-regulates cyclin D1 expression. Northern blotting showed that cyclin D1 mRNA levels were modestly increased following CVB3 infection, suggesting that cyclin D1 regulation occurs by a posttranscriptional mechanism. Viral infection resulted in only a 20 to 30% inhibition of cyclin D1 protein synthesis 3 h postinfection. However, the proteasome inhibitors MG132 and lactacystin prevent CVB3-induced cyclin D1 reduction, indicating that CVB3-induced down-regulation of cyclin D1 is facilitated by ubiquitin-proteasome proteolysis. Finally, using GSK3beta pathway inhibitors, we showed that the reduction of cyclin D1 is GSK3beta independent. Taken together, our results demonstrate that CVB3 infection disrupts host cell homeostasis by blocking the cell cycle at the G(1)/S boundary and induces cell cycle arrest in part through an increase in ubiquitin-dependent proteolysis of cyclin D1.
Luo
Honglin
H
McDonald Research Laboratories/The iCAPTUR4E Center, Department of Pathology and Laboratory Medicine, St. Paul's Hospital/Providence Health Care-University of British Columbia, Vancouver, Canada.
Zhang
Jingchun
J
Dastvan
Frank
F
Yanagawa
Bobby
B
Reidy
Michael A
MA
Zhang
Huifang M
HM
Yang
Decheng
D
Wilson
Janet E
JE
McManus
Bruce M
BM
D042846:CDC2-CDC28 Kinases; D002455:Cell Division; D019938:Cyclin D1 / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000032:analysis; D003546:Cysteine Endopeptidases / Q000502:physiology; D004247:DNA / Q000096:biosynthesis; D029822:Enterovirus B, Human / Q000502:physiology*; D016193:G1 Phase; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology; D000071679:Glycogen Synthase Kinase 3 beta; D006367:HeLa Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000502:physiology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases / Q000032:analysis; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
D009097:Multienzyme Complexes; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D025801:Ubiquitin; D019938:Cyclin D1; D004247:DNA; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D038362:Glycogen Synthase Kinase 3; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex