Abstract
Reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency requires the viral transactivator Rta to contact the host protein Jκ recombination signal-binding protein (RBP-Jκ or CSL). RBP-Jκ normally binds DNA sequence-specifically to determine the transcriptional targets of the Notch-signaling pathway, yet Notch alone cannot reactivate KSHV. We previously showed that Rta stimulates RBP-Jκ DNA binding to the viral genome. On a model viral promoter, this function requires Rta to bind to multiple copies of an Rta DNA motif (called "CANT" or Rta-c) proximal to an RBP-Jκ motif. Here, high-resolution ChIP/deep sequencing from infected primary effusion lymphoma cells revealed that RBP-Jκ binds nearly exclusively to different sets of viral genome sites during latency and reactivation. RBP-Jκ bound DNA frequently, but not exclusively, proximal to Rta bound to single, but not multiple, Rta-c motifs. To discover additional regulators of RBP-Jκ DNA binding, we used bioinformatics to identify cellular DNA-binding protein motifs adjacent to either latent or reactivation-specific RBP-Jκ-binding sites. Many of these cellular factors, including POU class homeobox (POU) proteins, have known Notch or herpesvirus phenotypes. Among a set of Rta- and RBP-Jκ-bound promoters, Rta transactivated only those that also contained POU motifs in conserved positions. On some promoters, POU factors appeared to inhibit RBP-Jκ DNA binding unless Rta bound to a proximal Rta-c motif. Moreover, POU2F1/Oct-1 expression was induced during KSHV reactivation, and POU2F1 knockdown diminished infectious virus production. Our results suggest that Rta and POU proteins broadly regulate DNA binding of RBP-Jκ during KSHV reactivation.
Highlights
Reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV) from latency requires the viral transactivator Rta to contact the host protein J recombination signal-binding protein (RBP-J or CSL)
We reasoned that valproic acid (VPA) would be the ideal stimulus to distinguish latent from reactivation-specific Rta and RBP-J– binding sites on the KSHV genome
We found that Rta and RBP-J bound to the Mta, Rta, and ORF50AS/K-bZIP promoters during reactivation, and the Rta and RBP-J peaks were close to each other (Fig. 2, asterisks, and Table S1A, peaks 49 –54, 57, 63–64, and B, peaks 38 – 44, 54, 56 –57)
Summary
A herpesvirus transactivator and cellular POU proteins extensively regulate DNA binding of the host Notch signaling protein RBP-J to the virus genome. Reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV) from latency requires the viral transactivator Rta to contact the host protein J recombination signal-binding protein (RBP-J or CSL). KSHV reactivation requires Rta to form a complex with the cellular protein recombination signal-binding protein (RBP)-J ( known as CSL and CBF-1) to bind and transactivate viral promoters (8 –10). Our laboratory has sought to define the molecular mechanisms required for forming transcriptionally productive Rta/ RBP-J/DNA complexes necessary for KSHV reactivation. To this end, we have focused on Rta transactivation of the promoter of the essential KSHV DE gene Mta. We showed a new mechanism of RBP-J– dependent transactivation in which Rta. stimulates RBP-J DNA binding in infected or uninfected cells [8]. Our new data set provides a basis for future analyses of regulated target promoter selectivity by RBP-J– dependent transactivators
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
