The Kaposi's sarcoma-associated herpesvirus TBP mimic uses a non-canonical DNA binding mode to promote viral late gene transcription.

Kaposi's sarcoma-associated herpesvirus (KSHV) orchestrates late gene transcription through viral transcriptional activators that hijack host RNA polymerase II machinery, maintaining selectivity for viral promoters. Among these, the KSHV protein ORF24 serves as a TATA-binding protein (TBP) mimic essential for recognizing viral late promoters, although the molecular mechanisms underlying its function remain poorly characterized. Here, we used AlphaFold3 to predict the structure of ORF24 in complex with DNA and validated key features in both transfected cells and during KSHV lytic replication. Structural modeling revealed that ORF24 employs a non-canonical DNA binding mode where the C-terminal domain (CTD) makes critical DNA contacts beyond the canonical TBP fold. Targeted mutagenesis confirmed that ORF24 requires conserved TBP-like phenylalanines alongside a polar-rich binding interface distinct from cellular TBP. During infection, both the TBP-like domain and CTD are essential for ORF24 occupancy at viral late promoters. Most surprisingly, we discovered that ORF24 pre-assembles with RNA polymerase II and the viral protein ORF34 to achieve stable promoter binding. This cooperative assembly mechanism represents a fundamental departure from stepwise eukaryotic transcription initiation, resembling a prokaryotic strategy within the eukaryotic nucleus.