KSHV-encoded vIRF3 Cooperates with Cellular IRF4 to Drive Super-Enhancer Activity through Complex DNA Elements

The Kaposi’s sarcoma-associated herpesvirus (KSHV) oncoprotein vIRF3 is essential for the survival of primary effusion lymphoma (PEL) cells. vIRF3 cooperates with cellular IRF4 to activate super-enhancers (SEs) driving oncogenes including MYC and IRF4 itself. However, the vIRF3/IRF4-responsive DNA sequences underlying this cooperation are unknown. Investigating the IRF4-SE, we mapped its vIRF3/IRF4-responsiveness to a complex ∼83 bp region, which retained cooperative activation by vIRF3 and IRF4 and was activated by vIRF3 but not IRF4 alone. vIRF3-mediated activation depended on an AP1 site, while the cooperation of vIRF3 with IRF4 required the DNA binding ability of IRF4 and IRF-related motifs that do not participate in canonical AP1-IRF (AICE) composite sites. These motifs are necessary but insufficient to confer responsiveness outside their native sequence context, suggesting that vIRF3/IRF4-mediated IRF4-SE activation requires an extended composite element. DNA pulldowns confirmed the importance of the identified motifs for association of vIRF3 and IRF4 with the IRF4-SE. A PEL MYC-SE similarly depended on an extended responsive element containing a critical AP1 site, within a functional AICE motif. Together, our results show that vIRF3 activates oncogenic SEs by co-opting complex genetic elements that may accommodate previously unknown IRF4 binding configurations, improving our understanding of vIRF3 and IRF4-dependent oncogenesis.