A REPLICATION FUNCTION-ASSOCIATED WITH THE ACTIVATION DOMAIN OF THE EPSTEIN-BARR-VIRUS ZTA TRANSACTIVATOR

The Zta transactivator is crucial for both Epstein-Barr virus (EBV) ly tic gene expression and lytic DNA replication, We have used a cotransf ection-replication assay to examine the effect of mutations in the Zta activation domain (amino acids [aa] 1 to 167) on Zta replication acti vity. Deletion of Zta aa 25 to 86, which are critical for transcriptio nal activation of ori-Lyt, or aa 93 to 141 did not adversely affect re plication of an ori-Lyt-containing target plasmid. However, removal of aa 2 to 25 (Delta 2-25) abolished replication activity, Within this s ubdomain, deletion of aa 2 to 10 (Delta 2-10) or mutation of codons 18 and 19 (m18/19) or 22 and 26 (m22/26) did not affect replication comp etency, while deletion of codons 13 to 19 (Delta 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 e xpression from both BHLF1 promoter-chloramphenicol acetyltransferase c onstructions 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 fr om its transactivation activity and was supplied by the N-terminal reg ion encompassing aa 11 to 25. Replication by a subset of the impaired Zta mutants was partially rescued upon the addition of Rta to the repl ication assay. The contribution of Rta mapped to domain II of the Rta activation domain and was specific for this region, A chimeric Rta-EBN A-2 transactivation domain fusion, which retains the DNA-binding and t ransactivation properties associated with wild-type Rta, failed to res cue replication-deficient Zta, Our data suggest that Rta may act as an ancillary replication factor in EBV ori-Lyt DNA synthesis by stabiliz ing Zta-replisome interactions.