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 (Δ2- 25) abolished replication activity. Within this subdomain, deletion of aa 2 to 10 (Δ2-10) 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 (Δ13-19) 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.
ASJC Scopus subject areas
- Insect Science