# The Kaposi’s sarcoma-associated herpesvirus TBP mimic uses a noncanonical DNA-binding mode to promote viral late gene transcription

## TL;DR

This paper reveals how a Kaposi’s sarcoma-associated herpesvirus protein mimics a host DNA-binding protein to control viral gene expression using a unique mechanism.

## Contribution

The study identifies a noncanonical DNA-binding mode of the KSHV protein ORF24 and its cooperative assembly with RNAPII and ORF34 for late gene transcription.

## Key findings

- ORF24 uses a noncanonical DNA-binding mode involving its C-terminal domain for viral late promoter recognition.
- ORF24 pre-assembles with RNAPII and ORF34 to achieve stable promoter binding during KSHV lytic replication.
- The TBP-like domain and CTD of ORF24 are essential for its function at viral late promoters.

## Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) orchestrates late gene transcription through viral transcriptional activators that hijack host RNA polymerase II (RNAPII) 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 noncanonical 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 RNAPII 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.

Graphical Abstract