Alteration of a single serine in the basic domain of the Epstein-Barr virus ZEBRA protein separates its functions of transcriptional activation and disruption of latency

Francis, Amy; Gradoville, Lyndle; Miller, George

doi:10.1128/jvi.71.4.3054-3061.1997

Cited by 56 publications

(21 citation statements)

References 49 publications

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“…Amplification of this plasmid is a direct consequence of DNA replication during the lytic phase. Substitution of serine 186 by alanine in BZLF1 completely abolished p526 amplification (data not shown), as has been concluded recently (23).…”

Section: Fig 5 Phosphopeptide Mapping Of Bzlf1 (A)supporting

confidence: 84%

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Activation of the Epstein-Barr Virus Transcription Factor BZLF1 by 12- O -Tetradecanoylphorbol-13-Acetate-Induced Phosphorylation

Baumann¹,

Mischak²,

Dammeier³

et al. 1998

J Virol

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BZLF1 is a member of the extended AP-1 family of transcription factors which binds to specific BZLF1 sequence motifs within early Epstein-Barr virus (EBV) promoters and to closely related AP-1 motifs. BZLF1’s activity is regulated at the transcriptional level as well as through protein interactions and posttranslational modifications. Phorbol esters or immunoglobulin cross-linking both reactivate EBV from latently infected B cells via transactivation of BZLF1. We report here that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is capable of inducing BZLF1’s activity even further. The induction occurs at the posttranscriptional level and depends on a single serine residue located in the DNA binding domain of BZLF1. This serine residue (S186) is phosphorylated by protein kinase C in vitro and in vivo after stimulation with TPA. Phosphorylation of S186 per se interferes with the DNA binding affinity of BZLF1 in vitro but is mandatory for TPA-induced increase in DNA binding of BZLF1, as shown in gel retardation assays and reconstruction experiments with cellular extracts. In transcriptional reporter assays, S186 is essential for the activation of BZLF1 by TPA. Presumably, a yet-to-be-identified cellular factor restores the DNA binding affinity and enhances the transcriptional activity of S186-phosphorylated BZLF1, which is required to induce the lytic phase of EBV’s life cycle.

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Section: Fig 5 Phosphopeptide Mapping Of Bzlf1 (A)supporting

confidence: 84%

“…BZLF1 is also found phosphorylated in vivo, but the relevance of this finding is not clear (13). In addition, a consensus phosphorylation motif for protein kinase C (PKC) has been proposed within the DNA binding domain of BZLF1 at serine amino acid residue 186, which was found to be critical for the full biological activity of BZLF1 (23).…”

mentioning

confidence: 99%

Activation of the Epstein-Barr Virus Transcription Factor BZLF1 by 12- O -Tetradecanoylphorbol-13-Acetate-Induced Phosphorylation

Baumann¹,

Mischak²,

Dammeier³

et al. 1998

J Virol

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“…Expression vectors. Expression vectors for the wild-type EBV BZLF1 gene and the Z(S186A) mutant containing EBV genomic DNA driven by the cytomegalovirus (CMV) immediate early (IE) promoter in pHD1013 have been previously described (29,31). Expression vectors for wild-type human c-Jun, wild-type human c-Fos, c-Jun(A266S), and c-Fos(A151S) cloned with a FLAG tag in pCMV/Flag2 (Sigma-Aldrich) or untagged in pCMV6-XL5 vector (OriGene) were previously described (32).…”

Section: Methodsmentioning

confidence: 99%

Mutant Cellular AP-1 Proteins Promote Expression of a Subset of Epstein-Barr Virus Late Genes in the Absence of Lytic Viral DNA Replication

Lyons

Heiden

et al. 2018

J Virol

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Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator. Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.

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“…Zta stabilizes the formation of a DNA-bound complex containing the basal transcription factors TFIID and TFIIA (14,39), and it is most active on promoters containing noncanonical TATA boxes that have reduced affinity for TFIID (38,42). Both DNA binding and activation of endogenous viral promoters are modified by phosphorylation of Zta (25,34). In addition to regulating EBV lytic promoters, Zta modifies cellular gene expression (8,9,23) and has been found to interact with a variety of cellular proteins, including the retinoic acid receptor, NF-B/p65, and p53 (29,54,64,73).…”

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confidence: 99%

The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

et al. 1998

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The Epstein-Barr virus transactivator Zta triggers lytic gene expression and is essential for replication of the lytic origin, oriLyt. Previous analysis indicated that the Zta activation domain contributed a replication-specific function. We now show that the Zta activation domain interacts with components of the EBV helicase-primase complex. The three helicase-primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc epitope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (primase subcomplex) were separately transfected, the proteins localized to the cytoplasm. Interaction between Zta and the components of the helicase-primase complex was tested by examining the ability of Zta to alter the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; similarly, cotransfection of Zta with the primase subcomplex led to nuclear translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This relocalization provides evidence for an interaction between Zta and the helicase and Zta and the primase subcomplex. An affinity assay using glutathioneS-transferase–Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino acids 1 to 133). In the nuclear relocalization assay, the amino-terminal 25 amino acids of Zta were required for efficient interaction with the primase subcomplex but not for interaction with BBLF4. Evidence for interaction between oriLyt bound Zta and the helicase-primase complex was obtained in a superactivation assay using an oriLyt-chloramphenicol acetyltransferase (CAT) reporter. Zta activated expression from a CAT reporter containing the complete oriLyt region and regulated by the oriLyt BHLF1 promoter. Cotransfection of the helicase-primase proteins, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression. This assay also provided evidence for an interaction between the single-stranded DNA binding protein, BALF2, and the Zta-tethered helicase-primase complex. The helicase-primase interaction is consistent with a role for Zta in stabilizing the formation of an origin-bound replication complex.

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Alteration of a single serine in the basic domain of the Epstein-Barr virus ZEBRA protein separates its functions of transcriptional activation and disruption of latency

Cited by 56 publications

References 49 publications

Activation of the Epstein-Barr Virus Transcription Factor BZLF1 by 12- O -Tetradecanoylphorbol-13-Acetate-Induced Phosphorylation

Activation of the Epstein-Barr Virus Transcription Factor BZLF1 by 12- O -Tetradecanoylphorbol-13-Acetate-Induced Phosphorylation

Mutant Cellular AP-1 Proteins Promote Expression of a Subset of Epstein-Barr Virus Late Genes in the Absence of Lytic Viral DNA Replication

The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

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