Kaposi's sarcoma-associated herpesvirus (KSHV) is an important pathogen in Kaposi's sarcoma and abnormal lymphoproliferation. KSHV open reading frame 50 (ORF50), a homolog of the Epstein-Barr virus immediate-early gene product RTA, activates early and late gene transcription in the KSHV lytic cycle, and its expression is closely correlated with KSHV-related diseases. ORF50 interacts with the cellular proteins CBP and histone deacetylase and represses p53-induced apoptosis through a CBP-related mechanism. We show here that KSHV ORF50 also interacts with STAT3. ORF50 stimulated transcription of STAT-driven reporter genes, and interleukin-6 and v-Src further activated this stimulating effect of ORF50. Physical association of STAT3 and ORF50 required the carboxyl-terminal transactivation domain of ORF50 and multiple regions within STAT3. ORF50 recruited STAT3 to the nucleus and induced the dimerization of STAT3 monomers in the absence of STAT3 phosphorylation. We show here that KSHV ORF50 activates STAT3-mediated transcription through direct interaction without mediating tyrosine phosphorylation.Kaposi's sarcoma-associated herpesvirus (KSHV), 1 also known as human herpesvirus 8, is an important pathogen in Kaposi's sarcoma and multicentric Castleman's disease (1, 2). In a KSHV infection, the viral DNA is found principally in B lymphocytes, and KSHV is in fact associated with abnormal lymphoproliferation such as primary effusion lymphoma (3). In a Kaposi's sarcoma tumor, KSHV DNA is found in spindle cells, and viral lytic replication is important for Kaposi's sarcoma tumor development (4). KSHV is a member of the ␥-herpesvirus family and has homology to the other herpesviruses, herpesvirus saimiri, and Epstein-Barr virus (EBV).An important step in the KSHV life is the switch from latency to lytic replication. Upon chemical induction, KSHV produces RNA transcripts from its immediate-early genes. These transcripts encode viral transcriptional activator proteins such as open reading frame 50 (ORF50) and K8, which are necessary to induce lytic replication (5). ORF50 is a homolog of the EBV immediate-early gene product RTA. It has been reported that ORF50 activates the lytic cycle of KSHV and is expressed earlier than K8, a homolog of the EBV ZTA protein, which induces the lytic cycle of EBV (6, 7). ORF50 activates expression of the early and late genes in the KSHV lytic cycle (8). Demethylation of the ORF50 promoter closely correlates with expression of the ORF50 gene and induction of the lytic phase (9). Biopsies from patients with KSHV-related diseases such as Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma show demethylation of the ORF50 promoter, whereas samples from a latently infected KSHV carrier show a methylated ORF50 promoter. These data suggest that the expression of ORF50 is very important for the development of KSHV-associated diseases. ORF50 binds to the cellular proteins CBP and histone deacetylase, and these binding events activate and repress ORF50-activated viral transc...