Human herpesvirus-8 (HHV-8), also known as Kaposi sarcoma-associated herpesvirus (KSHV), is etiologically linked to primary effusion lymphoma (PEL). At least 10 KSHV-encoded proteins with potential roles in KSHV-associated neoplasia have been identified. However, with few exceptions, these putative oncogenes were analyzed in heterologous systems only using overexpression of single genes. Thus, the pathogenetic relevance of most of these putative oncogenes remains essentially unclear. We used RNA interference (RNAi) to knock down the expression of several KSHV genes in cultured PEL cells carrying the KSHV genome. The viral interferon-regulatory factor-3 (vIRF-3) was found to be required for proliferation and survival of cultured PEL cells. Knock-down of vIRF-3 expression by various RNAi approaches unequivocally resulted in reduced proliferation and increased activity of caspase-3 and/or caspase-7. Thus, vIRF-3 can be seen as a bona fide oncogene of KSHV-associated lymphoma. Surprisingly, although the related Epstein-Barr virus (EBV) is usually sufficient to immortalize human B lymphocytes, silencing of vIRF-3 reduced the viability of both EBV(-) and EBV(+) PEL cells. This suggests that KSHV is the driving force in the pathogenesis of PEL.