A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator

Abstract

The Zta transactivator is crucial for both Epstein-Barr virus (EBV) lytic gene expression and lytic DNA replication. We have used a cotransfection-replication assay to examine the effect of mutations in the Zta activation domain (amino acids [aa] 1 to 167) on Zta replication activity. Deletion of Zta aa 25 to 86, which are critical for transcriptional activation of ori-Lyt, or aa 93 to 141 did not adversely affect replication of an ori-Lyt-containing target plasmid. However, removal of aa 2 to 25 (⌬2-25) abolished replication activity. Within this subdomain, deletion of aa 2 to 10 (⌬2-10) or mutation of codons 18 and 19 (m18/19) or 22 and 26 (m22/26) did not affect replication competency, while deletion of codons 13 to 19 (⌬13-19) or mutation at codons 12 and 13 (m12/13) impaired Zta replication function. Each of the replication-negative Zta variants was capable of transactivating expression from both BHLF1 promoter-chloramphenicol acetyltransferase constructions and the BMRF1 promoter on endogenous EBV genomes in Raji cells with efficiency comparable to that of the wild-type polypeptide. Thus, a replication contribution of Zta was functionally separable from its transactivation activity and was supplied by the N-terminal region encompassing aa 11 to 25. Replication by a subset of the impaired Zta mutants was partially rescued upon the addition of Rta to the replication assay. The contribution of Rta mapped to domain II of the Rta activation domain and was specific for this region. A chimeric Rta-EBNA-2 transactivation domain fusion, which retains the DNA-binding and transactivation properties associated with wild-type Rta, failed to rescue replication-deficient Zta. Our data suggest that Rta may act as an ancillary replication factor in EBV ori-Lyt DNA synthesis by stabilizing Zta-replisome interactions.

Improving nuclear envelope dynamics by EBV BFRF1 facilitates intranuclear component clearance through autophagy

“…Akata cells with EBV were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (HyClone Laboratories) (32). For EBV induction, NA cells were transfected with plasmid pRTS15 expressing the Rta transactivator (33). Akata cells (1 3 10 6 cells/ml) were induced with 0.8% (v/v) goat anti-human IgG antibody (Cappel, Malvern, PA, USA).…”

Cellular transcription factors recruit viral replication proteins to activate the Epstein–Barr virus origin of lytic DNA replication, oriLyt

“…Numerous cellular and viral promoters contain binding In contrast, viral lytic proteins cannot be detected in the latent phase, but stimuli, such as TPA, lead to the activation of the lytic activator BZLF1, which in turn results in the induction of viral lytic genes including viral DNA replication proteins (reviewed in Speck et al, 1997). BZLF1 is also capable of activating oriLyt directly by binding to DNA motifs, socalled BZLF1-responsive DNA elements (ZRE), located in the upstream component (Lieberman et al, 1990;Schepers et al, 1993aSchepers et al, , 1996Sarisky et al, 1996;Askovic and Baumann, 1997). Proteinprotein interactions have been demonstrated to exist between the DNA-binding domain of BZLF1 and the DNA polymerase accessory factor (BMRF1) (Zhang et al, 1996), and between the transactivation domain of BZLF1 and both the viral helicase (BBLF4) and the primase subcomplex (composed of BSLF1 and BBLF2/3) (Gao et al, 1998).…”

Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLyt-dependent DNA replication and formation of replication compartments in cotransfection assays

“…HCMV(AD169)-infected cell DNA was purified as described previously (47) to serve as a DNA template source from which to clone the viral replication genes. The EBV parent clones containing the replication genes from which second-generation expression vectors were constructed were described previously (67). Synthetic oligonucleotide primers (JHU Core Facility) were used to amplify by PCR the coding regions of viral replication genes extending from the ATG translational start site to the translational stop codon as indicated in Table 1.…”

“…When this assay approach (10,93) was applied for replication of EBV oriLyt in Vero cells, it resulted in the identification of six core replication proteins, as well as three IE transactivators, Zta, Rta, and Mta (30,31). Subsequently, both Rta and Mta were found to be dispensable when the core components were placed into heterologous SV40 enhancerdriven expression vectors (31,67). Although attempts to replace individual EBV replication genes by their HSV-1 counterparts proved futile, a group substitution approach aided in resolving which EBV replication gene(s) provided the essential origin-binding function.…”