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

Abstract

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.

An Epigenetic Journey: Epstein-Barr Virus Transcribes Chromatinized and Subsequently Unchromatinized Templates during Its Lytic Cycle

“…Expression of BLRF2, another leaky-late gene, could be detected even in the absence of viral lytic replication, but protein levels of BLRF2 increased in the presence of viral DNA replication, indicating that BLRF2 is expressed by both replication-dependent and replication-independent mechanisms. In support of these findings, a recent study by Lyons et al (33) showed that mutant versions of the cellular protein AP-1, which can assume some functions of BZLF1, can activate the transcription of some EBV genes that were previously classified as late genes even in the absence of viral DNA lytic replication. Using CAGE-Seq data, Djavadian et al (24) have newly classified these viral genes activated by the mutant AP-1 proteins as leaky-late genes or, in some cases, as early genes.…”

A Noncanonical Basic Motif of Epstein-Barr Virus ZEBRA Protein Facilitates Recognition of Methylated DNA, High-Affinity DNA Binding, and Lytic Activation

“…Furthermore, high-affinity binding of ZEBRA to nonmethylated ZEBRA response elements in the EBV origin of lytic replication is needed for inducing DNA amplification (29). As little as a 2-fold decrease in affinity for ZREs in oriLyt is associated with a defect in DNA replication (30). Mutation of the RRTRK motif to AATAA was associated with profound defects in DNA binding, DNA replication, and late gene expression ( Fig.…”

“…HKB5/B5 is a hybrid cell line derived from fusion of HH514-16 Burkitt lymphoma cells with human embryonic kidney cell line 293 (kindly provided by M.-S. Cho, Bayer Corporation, Berkeley, Calif.) maintained on RPMI 1640 supplemented with 10% FBS, 50 U/ml penicillin-streptomycin, and 1 g/ml amphotericin B. 293T cells, human embryonic kidney cells which were transformed with sheared human adenovirus DNA, and simian virus 40 T antigen were maintained on DMEM with 10% FBS (30,31).…”

“…Viral transcript levels were assessed from 100 ng total RNA using an iTaq universal SYBR green one-step kit (Bio-Rad) in a total volume of 25 l. The level of 18S RNA was measured to normalize the total amount of RNA. Each sample was analyzed in triplicate (30,35). See Table S1 for sequences of primers.…”

Identification of ARKL1 as a Negative Regulator of Epstein-Barr Virus Reactivation

“…Consistent with this hypothesis, we showed that ARKL1 overexpression suppressed Jun activity in a Jun reporter assay. Some EBV lytic proteins in addition to BZLF1 can be directly regulated by AP-1 transcription factors, such as Jun (28,65,66). Therefore, the effect of ARKL1 on the EBV lytic cycle may not be limited to regulation of BZLF1 expression, although that alone would have a major impact on lytic infection.…”