KSHV-encoded vIRF3 cooperates with cellular IRF4 to drive super-enhancer activity through complex DNA elements.

The Kaposi's sarcoma-associated herpesvirus oncoprotein vIRF3 is essential for primary effusion lymphoma (PEL) cell survival and cooperates with cellular interferon regulatory factor 4 (IRF4) to activate super-enhancers (SEs) controlling MYC and IRF4. However, the DNA sequence requirements for this cooperation are unknown. Using reporter assays, we mapped vIRF3/IRF4 responsiveness within the IRF4-SE to an ∼83-bp element activated by vIRF3 alone and cooperatively by vIRF3 and IRF4. vIRF3-dependent activation required an AP-1 motif, whereas cooperation with IRF4 required IRF4 DNA binding and IRF-related motifs outside canonical AP-1-IRF composite elements (AICEs). These motifs were necessary but insufficient outside their native sequence context, indicating a requirement for extended flanking sequences. DNA pulldowns revealed distinct motif requirements for vIRF3 and IRF4 association with the IRF4-SE. A distal MYC-SE required a similarly extended region containing a functional AICE2 motif, with vIRF3 activity depending primarily on its AP-1 half-site. Motif requirements were preserved in integrated reporter assays. BATF promoted vIRF3/IRF4-dependent activation preferentially in integrated reporters and was required to maintain vIRF3 and IRF4 occupancy at the endogenous MYC-SE. These findings define context-dependent DNA and cofactor requirements through which vIRF3 co-opts IRF4-regulated oncogenic enhancers in PEL.