The Epstein-Barr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16

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.

Section: Ap-1(a/s) Activates Expression Of a Subset Of Viral Lytic Gementioning

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

“…BRLF1 is translated from a bicistronic mRNA which also encodes another IE gene product, BZLF1 (12,20). These two EBV gene products function in activating expression from a number of viral (3,5,8,13,15,18,28) and perhaps cellular (7) promoters, thereby initiating the lytic cascade. However, HVS R RNA is expressed as an early transcript in HVS-infected cells (23,25).…”

mentioning

confidence: 99%

The herpesvirus saimiri ORF50 gene, encoding a transcriptional activator homologous to the Epstein-Barr virus R protein, is transcribed from two distinct promoters of different temporal phases

Whitehouse

Carr

Griffiths

et al. 1997

The mRNA species encoding the herpesvirus saimiri (HVS) homolog of the Epstein-Barr virus R transcriptional activator (termed ORF50) have been identified and used to determine transcriptional start sites within the gene. The first transcript is spliced and starts from a promoter within ORF49 containing a single intron; the second is produced from a promoter within the second exon and is in the same reading frame. The spliced transcript is detected at early times during productive virus replication in OMK cells, whereas the nonspliced transcript is detected later. The spliced transcript is fivefold-more potent in activating the delayed-early ORF6 promoter; the function of the nonspliced transcript is unclear. Thus, the role of this protein in activating herpesvirus saimiri from the latent state may differ significantly from that of the Epstein-Barr virus R protein.Herpesvirus saimiri (HVS) establishes asymptomatic infections involving a subset of T lymphocytes in its natural host, the squirrel monkey (Saimiri sciureus), but causes fatal T-cell lymphomas and lymphoproliferative diseases in other species of New World primates (6). HVS has been classified as a gamma-2 herpesvirus and shares significant homology with Epstein-Barr virus (EBV) (1,2,9,10,16,24). The genomes of HVS and EBV are generally colinear, in that homologous genes are found in approximately equivalent locations and in the same relative orientations in the two viruses (22). As in many other herpesviruses, gene expression during lytic infection occurs in three main temporal phases: immediate-early (IE), delayed-early (DE), and late (17). The major IE transcript in HVS is encoded by the HindIII-G IE gene (ORF14) (2,(25)(26)(27). Analysis of the sequence shows that it does not exhibit homology with any EBV-encoded proteins (25), but it does contain local homology with a putative superantigen (29); however, its function remains to be established. The 52-kDa product of the HVS IE gene (ORF57) is homologous to the EBV transactivator encoded by BMLF1 (also known as Mta, M-IE, or IE-2) and to the 27-kDa or IE 63-kDa protein encoded by UL54 of herpes simplex virus 1 and RF4 of varicellazoster virus (4,14,21,22,24).The EcoRI-D or HVS R protein transactivator (ORF50) is homologous to the BRLF1 gene product (also known as R or Rta) (23). BRLF1 is translated from a bicistronic mRNA which also encodes another IE gene product, BZLF1 (12, 20). These two EBV gene products function in activating expression from a number of viral (3,5,8,13,15,18,28) and perhaps cellular (7) promoters, thereby initiating the lytic cascade. However, HVS R RNA is expressed as an early transcript in HVS-infected cells (23, 25). The HVS R gene is proposed to be spliced with more than one exon, although the initiation codon has not been mapped. Despite the likelihood of a spliced transcript, there is evidence that a DNA sequence within the identified exon is able to produce a functionally active protein capable of activating a DE ORF6 gene promoter, a component of the major DNA binding prot...

“…Activation by Zta in the absence of DNA binding has also been observed with certain targets (28). The Rta transcriptional activator encoded by BRLF1 (38) also binds DNA as a dimer (34,54) and contains a powerful negatively charged, VP16-like activation domain (39). Rta can contact both TBP and TFIIB (53).…”

mentioning

confidence: 99%

Replication of Epstein-Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays

Fixman

Hayward

1995

183

104

Using a transient replication assay in which cosmid DNAs were cotransfected into Vero cells, we had previously demonstrated that oriLyt replication required six Epstein-Barr virus (EBV)-encoded replication genes. No oriLyt origin-binding protein was identified in this study, but oriLyt replication in the cotransfection assay was also dependent on the three lytic cycle transactivators Zta, Rta, and Mta and an activity encoded by the EBV SalI F fragment. We have now used expression plasmids for the six known replication proteins to further examine the question of the requirement for an oriLyt origin-binding protein. The activity in SalI-F was shown to be encoded by BKRF3. The predicted product of this open reading frame is an enzyme, uracyl DNA glycosylase, not an origin-binding protein, and is dispensable for replication in assays using expression plasmids. BBLF2, which is positionally related to the gene for the herpes simplex virus (HSV) UL9 originbinding protein, was confirmed to be expressed as a spliced transcript with BBLF3 and not as an independent product. Examination of the requirement for the EBV transactivators revealed that Rta, while contributing to replication efficiency, was dispensable. Mta could be substituted by HSV IE63, and in complementation experiments with HSV replication genes, Mta was no longer required for replication of EBV oriLyt, suggesting that the contribution of Mta to replication may be indirect. Zta continued to be required for detectable oriLyt replication both with the EBV replication proteins and in the complementation assays with HSV replication proteins. We conclude that EBV does not encode an equivalent of HSV UL9 and that Zta is the sole virally encoded protein serving an essential origin-binding function.