De novo protein synthesis is required for lytic cycle reactivation of Epstein-Barr virus, but not Kaposi's sarcoma-associated herpesvirus, in response to histone deacetylase inhibitors and protein kinase C agonists.
The oncogenic human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are latent in cultured lymphoma cells. We asked whether reactivation from latency of either virus requires de novo protein synthesis. Using Northern blotting and quantitative reverse transcriptase PCR, we measured the kinetics of expression of the lytic cycle activator genes and determined whether abundance of mRNAs encoding these genes from either virus was reduced by treatment with cycloheximide (CHX), an inhibitor of protein synthesis. CHX blocked expression of mRNAs of EBV BZLF1 and BRLF1, the two EBV lytic cycle activator genes, when HH514-16 Burkitt lymphoma cells were treated with histone deacetylase (HDAC) inhibitors, sodium butyrate or trichostatin A, or a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine. CHX also inhibited EBV lytic cycle activation in B95-8 marmoset lymphoblastoid cells by phorbol ester phorbol-12-myristate-13-acetate (TPA). EBV lytic cycle induction became resistant to CHX between 4 and 6 h after application of the inducing stimulus. KSHV lytic cycle activation, as assessed by ORF50 mRNA expression, was rapidly induced by the HDAC inhibitors, sodium butyrate and trichostatin A, in HH-B2 primary effusion lymphoma cells. In HH-B2 cells, CHX did not inhibit, but enhanced, expression of the KSHV lytic cycle activator gene, ORF50. In BC-1, a primary effusion lymphoma cell line that is dually infected with EBV and KSHV, CHX blocked EBV BRLF1 lytic gene expression induced by TPA and sodium butyrate; KSHV ORF50 mRNA induced simultaneously in the same cells by the same inducing stimuli was resistant to CHX. The experiments show, for the cell lines and inducing agents studied, that the EBV BZLF1 and BRLF1 genes do not behave with "immediate-early" kinetics upon reactivation from latency. KSHV ORF50 is a true "immediate-early" gene. Our results indicate that the mechanism by which HDAC inhibitors and TPA induce lytic cycle gene expression of the two viruses differs and suggest that EBV but not KSHV requires one or more proteins to be newly synthesized between 4 and 6 h after application of an inducing stimulus.
Ye
Jianjiang
J
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Gradoville
Lyndle
L
Daigle
Derek
D
Miller
George
G
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015152:Blotting, Northern; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000254:growth & development*; D019288:Herpesvirus 8, Human / Q000254:growth & development*; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000235:genetics; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D014775:Virus Activation / Q000187:drug effects*
C068382:BRLF1 protein, Human herpesvirus 4; C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D017874:Immediate-Early Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012367:RNA, Viral; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D014764:Viral Proteins; C012589:trichostatin A; C016658:phorbolol myristate acetate; D000077209:Decitabine; D003513:Cycloheximide; D011493:Protein Kinase C; D001374:Azacitidine; D013755:Tetradecanoylphorbol Acetate