The Kaposi's sarcoma-associated herpesvirus open reading frame 50 (ORF50) protein (called Rta), is necessary and sufficient for reactivation of the virus from latency. We previously demonstrated that a truncated mutant of ORF50 lacking its C-terminal transcriptional activation domain, called ORF50⌬STAD, formed mixed multimers with wild-type (WT) ORF50 and functioned as a dominant negative inhibitor of reactivation. For this report, we investigated the requirements for multimerization of ORF50/Rta in transactivation and viral reactivation. We analyzed multimerization of WT, mutant, and chimeric ORF50 proteins, using Blue Native polyacrylamide gel electrophoresis and size exclusion chromatography. WT and mutant ORF50 proteins form tetramers and higherorder multimers, but not monomers, in solution. The proline-rich, N-terminal leucine heptapeptide repeat (LR) of ORF50 (amino acids [aa] 244 to 275) is necessary but not sufficient for oligomer formation and functions in concert with the central portion of ORF50/Rta (aa 245 to 414). The dominant negative mutant ORF50⌬STAD requires the LR to form mixed multimers with WT ORF50 and inhibit its function. In the context of the WT ORF50/Rta protein, mutagenesis of the LR, or replacement of the LR by heterologous multimerization domains from the GCN4 or p53 proteins, demonstrates that tetramers of Rta are sufficient for transactivation and viral reactivation. Mutants of Rta that are unable to form tetramers but retain the ability to form higher-order multimers are reduced in function or are nonfunctional. We concluded that the proline content, but not the leucine content, of the LR is critical for determining the oligomeric state of Rta.Epidemiologic, serologic, and histopathologic studies have established Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8) as the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) (13,18,29,41,60,75). Reactivation of KSHV from latency is a crucial step in KS development. The viral load in the peripheral blood is directly proportional to the risk of KS progression and the stage of KS (1,5,9,26,69,94), and treatments that reduce the KSHV load are accompanied by KS regression (6,11,12,39,40,45,56,59,66,70,76,95). Most of the candidate pathogenic genes of KSHV (encoding proteins with cell growth deregulatory and immunomodulatory functions) are expressed in the delayed early class of the lytic-gene expression program (7,14,19,23,24,27,28,38,43,48,65,72,82). It is likely that reactivation of KSHV contributes to cancer initiation both by facilitating dissemination of the virus and by permitting expression of lytic-cycle genes with direct roles in pathophysiology. Therefore, a complete understanding of KSHV pathogenesis demands elucidation of the mechanisms that regulate viral reactivation and progression through the lytic cycle.We and others have demonstrated that the KSHV protein Rta (for "replication and transcriptional activator," expressed from open reading frame 50 [ORF50]) is both necess...