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Kaposi’s sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. RTA activates promoters by several mechanisms. RTA can bind to sequences in viral promoters and activate transcription. In addition, RTA interacts with the cellular recombination signal sequence-binding protein-J kappa (RBP- Jκ), a transcriptional repressor, converts the repressor into an activator and activates viral promoters via RBP- Jκ. Because RBP- Jκ is required for RTA to activate lytic replication, it is important to understand how RTA cooperates with RBP- Jκ protein to activate KSHV lytic replication program. Previously, we identified an RTA mutant, RTA-K152E, which has a defect in its direct DNA-binding activity. In this report, the effect of the mutant RTA on KSHV activation via RBP- Jκ protein is examined. We demonstrate that RTA-K152E interacts with RBP- Jκ physically and the mutant RTA and RBP-Jκ complex binds to target DNA properly in vivo and in vitro. However, the complex of RTA-K152E and RBP- Jκ does not activate transcription. Furthermore, the RTA mutant (RTA-K12E) inhibits cellular Notch-mediated RBP- Jκ activation. These data collectively suggest that the complex between KSHV RTA and cellular RBP- Jκ and the subsequent DNA binding by the complex are not sufficient for the activation of RBP- Jκ protein. Other factor(s) whether additional cofactor(s) in the complex or the intrinsic conformation of RTA, are predicted to be required for the activation of RBP- Jκ protein by RTA.