Analysis of Epstein-Barr virus gene expression upon phorbol ester and hydroxyurea treatment by real-time quantitative PCR.
Epstein-Barr virus (EBV) has the potential to undergo latent and lytic pathways during infection. However, expression of many of the viral genes during the lytic-latent transition remains unclear. In this study, we investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and hydroxyurea (HU), two commonly used modulators of EBV life cycle, on the expression profiles of the entire genome of EBV persistent infected in B95-8 cells. After treatment with TPA for 48 h, the copy number of EBV genome in the cells increased about 2.5 fold, whereas HU treatment resulted in a reduction to approximately two-thirds of the original level. Except a small set of genes, the amounts of EBV mRNA are generally less abundant than that of beta-actin. The expression of a large fraction of the 80 EBV genes was found to be activated after TPA treatment with a noticeable increase of 19 and 21 fold, respectively in BSLF1 and BBLF4. In contrast, treatment of the B95-8 cells with HU, a nucleotide synthesis inhibitor, dramatically suppressed the expression of EBV lytic genes. In summary, we have demonstrated that real-time quantitative PCR is a reliable method to monitor the influence of drug-treatment in EBV genes regulation. Our results also provide a basis for further investigation on how the virus coordinates its own gene expression during latent-lytic pathway transition.
Pan
Y-R
YR
Institute of Molecular Medicine, National Tsing Hua University, Hsin Chu, Taiwan, ROC.
Fang
C-Y
CY
Chang
Y-S
YS
Chang
H-Y
HY
D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D016679:Genome, Viral; D004854:Herpesvirus 4, Human / Q000187:drug effects / Q000235:genetics*; D006918:Hydroxyurea / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D013698:Templates, Genetic; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral; D013755:Tetradecanoylphorbol Acetate; D006918:Hydroxyurea