The γ-herpesviral TATA box binding protein directly interacts with RNA Polymerase II to direct late gene transcription

{beta}- and {gamma}-herpesviruses include the oncogenic human viruses Kaposis sarcoma-associated virus (KSHV) and Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV), which is a significant cause of congenital disease. Near the end of their replication cycle, these viruses transcribe their late genes in a manner distinct from host transcription. Late gene transcription requires six virally-encoded proteins, one of which is a functional mimic of host TATA-box-binding protein (TBP) that is also involved in recruitment of RNA polymerase II (Pol II) via unknown mechanisms. Here, we applied biochemical protein interaction studies together with electron microscopy-based imaging of a reconstituted human preinitiation complex to define the mechanism underlying Pol II recruitment. These data revealed that the herpesviral TBP, encoded by ORF24 in KSHV, makes a direct protein-protein contact with the C-terminal domain of host RNA polymerase II (Pol II), which is a unique feature that functionally distinguishes viral from cellular TBP. The interaction is mediated by the N-terminal domain (NTD) of ORF24 through a conserved motif that is shared in its {beta}- and {gamma}-herpesvirus homologs. Thus, these herpesviruses employ an unprecedented strategy in eukaryotic transcription, wherein promoter recognition and polymerase recruitment are facilitated by a single transcriptional activator with functionally distinct domains. SIGNIFICANCE STATEMENTThe {beta}- and {gamma}-herpesviruses mediate their late gene transcription through a set of viral transcriptional activators (vTAs). One of these vTAs, ORF24 in Kaposis sarcoma-associated herpesvirus (KSHV), is a mimic of host TATA-box-binding protein (TBP). We demonstrate that the N-terminal domain of ORF24 and its homologs from other {beta}- and {gamma}-herpesviruses directly bind the unstructured C-terminal domain (CTD) of RNA Pol II. This functionally distinguishes the viral TBP mimic from cellular TBP, which does not bind Pol II. Thus, herpesviruses encode a transcription factor that has the dual ability to directly interact with promoter DNA and the polymerase, a property which is unique in eukaryotic transcription and is conceptually akin to prokaryotic transcription factors.