Murine cytomegalovirus stimulates cellular thymidylate synthase gene expression in quiescent cells and requires the enzyme for replication.
Herpesviruses accomplish DNA replication either by expressing their own deoxyribonucleotide biosynthetic genes or by stimulating the expression of the corresponding cellular genes. Cytomegalovirus (CMV) has adopted the latter strategy to allow efficient replication in quiescent cells. In the present report, we show that murine CMV (MCMV) infection of quiescent fibroblasts induces both mRNA and protein corresponding to the cellular thymidylate synthase (TS) gene, which encodes the enzyme that catalyzes the de novo synthesis of thymidylic acid. The increase in TS gene expression was due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse TS promoter was induced following MCMV infection. Mutagenesis of the potential E2F-responsive element immediately upstream from the TS essential promoter region abolished the virus-mediated stimulation of the TS promoter, suggesting that the transactivating activity of MCMV infection was E2F dependent. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in TS promoter activity. Finally, MCMV replication and viral DNA synthesis were found to be inhibited by ZD1694, a quinazoline-based folate analog that inhibits TS activity. These results demonstrate that upregulation of cellular TS expression is required for efficient MCMV replication in quiescent cells.
Gribaudo
G
G
Department of Public Health and Microbiology, University of Turin, Turin, Italy. gribaudo@molinette.unito.it
Riera
L
L
Lembo
D
D
De Andrea
M
M
Gariglio
M
M
Rudge
T L
TL
Johnson
L F
LF
Landolfo
S
S
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018146:Muromegalovirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011799:Quinazolines / Q000494:pharmacology; D056465:Retinoblastoma-Binding Protein 1; D013876:Thiophenes / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics*; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014764:Viral Proteins; D014779:Virus Replication / Q000502:physiology*
C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D011799:Quinazolines; D056465:Retinoblastoma-Binding Protein 1; D013876:Thiophenes; D050680:Transcription Factor DP1; D014157:Transcription Factors; D014764:Viral Proteins; D013940:Thymidylate Synthase; C068874:raltitrexed