Gene expression and transcription factor profiling reveal inhibition of transcription factor cAMP-response element-binding protein by gamma-herpesvirus replication and transcription activator

Abstract

Herpesvirus replication involves the expression of over 80 viral genes in a well ordered sequence, leading to the production of new virions. Viral genes expressed during the earliest phases of replication often regulate both viral and cellular genes. Therefore, they have the potential to bring about dramatic functional changes within the cell. Replication and transcription activator (RTA) is a potent immediate early transcription activator of the gamma-herpesvirus family. This family includes Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus, human pathogens associated with malignancy. Here we combine gene array technology with transcription factor profiling to identify the earliest DNA promoter and cellular transcription factor targets of RTA in the cellular genome. We find that expression of RTA leads to both activation and inhibition of distinct groups of cellular genes. The identity of the target genes suggests that RTA rapidly changes the cellular environment to counteract cell death pathways, support growth factor signaling, and also promote immune evasion of the infected cell. Transcription factor profiling of the target gene promoters highlighted distinct pathways involved in gene activation at specific time points. Most notable throughout was the high level of cAMP-response element-binding protein (CREB)-response elements in RTA target genes. We find that RTA can function as either an activator or an inhibitor of CREB-response genes, depending on the promoter context. The association with CREB also highlights a novel connection and coordination between viral and cellular "immediate early" responses.

FIGURE 1.

Effect of C-terminal activation domain deletion on RTA transactivation. A, diagram of RTA showing the location of the deleted region and sequence alignment with four other γ-herpesvirus RTA homologues. RRV, rhesus rhadinovirus; MHV68, murine γ-herpesvirus 68; EBV, Epstein-Barr virus. Shading indicates amino acid conservation across several (gray) or all (black) RTA homologues shown. LZ, leucine zipper dimerization domain. B, Western blot showing expression of wild type and mutant RTA in transfected 293T cells. C and D, luciferase assays showing activity of wild type and mutant RTA proteins on reporters bearing the KSHV PAN promoter (pPAN−122) (C) or the KSHV vIL-6 promoter (K2p(−827)Luc) (D) upstream of firefly luciferase.

FIGURE 2.

Characterization of cell lines expressing mutant and wild type RTA proteins under a tetracycline-inducible promoter. A, Western blot showing expression of FLAG-tagged wild type and mutant RTA after cell treatment with 1 μg/ml tetracycline. p.i., postinduction. B, Western blot showing expression of RTA proteins with the indicated amounts of tetracycline for 24 h. C and D, luciferase assays showing inducible activity of reporters bearing RTA-responsive viral promoters in the RTA wild type and mutant cell lines. MJmulti is pPAN−122 carrying multiple mutations eliminating the RTA-response element (19). K2p(−414) is a deletion of the vIL-6 reporter K2p(−827), which is less responsive to RTA (35). Error bars, S.D.

FIGURE 3.

Consistency between duplicate RNA samples used in microarray analysis. Comparison plots of RNA expression levels in duplicate T-RExRTAwt RNA samples are shown, at 4, 8, and 12 h.

FIGURE 4.

Effect of RTA and CREB on promoter activity of PCSK1. A, diagram of the PCSK1 promoter showing location of full and half CREB-response elements and deletion mutants. B and C, luciferase assays showing activation of the proximal 769 nucleotides of the PCSK1 promoter by wild type but not mutant RTA in transfected 293 cells (B) and tetracycline-induced RTA-expressing cell lines (C). D and E, luciferase assays showing the effect of dominant negative CREB (A-CREB) (58), on basal activity (D) and RTA induction (E) of the PCSK1−113 reporter. F, luciferase assay comparing levels of induction of PCSK1 reporter constructs shown in A by RTA and CREB/Bt₂cAMP (db-cAMP). G, luciferase assay showing RTA inhibition of CREB activity on the PCSK1−113 reporter. Error bars, S.D.

FIGURE 5.

Inhibition of CREB-driven promoter by RTA. A, diagram of the DUSP1 promoter showing location of full and half CREB-response elements and deletion mutant. B and C, luciferase assays showing inhibition of the DUSP1 reporters by wild type but not mutant RTA in tetracycline-induced cell lines (B) and transfected 293 cells (C). Error bars, S.D.

FIGURE 6.

Partial reversal of RTA inhibition by specific coactivators. A, luciferase activity of pCRE-Luc reporter in the presence of wild type RTA and either wild type or Y134F mutant CREB. B, luciferase activity of pCRE-Luc reporter in the presence of CREB/dibutyryl-cAMP, RTA and coactivators CBP, Med12, and Med23. C, schematic diagram outlining potential mechanism for opposing effects of RTA on promoters driven by primary response factors, such as CREB. Error bars, S.D.