UC Berkeley

In the beta- and gammaherpesviruses, a specialized complex of viral transcriptional activators (vTAs) coordinate to direct expression of virus-encoded late genes, which are critical for the production of infectious virions. The vTAs in Kaposi’s sarcoma-associated herpesvirus (KSHV) are ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. While the general organization of the vTA complex has been mapped, the individual roles of these proteins, and how they coordinate to activate late gene promoters, remains largely unknown. Thus, we set out to determine the roles of ORF18, which is a highly interconnected vTA component and of ORF24, which is a putative structural and functional TATA-binding protein (TBP) mimic.

We first performed a comprehensive mutational analysis of the conserved residues in ORF18. The mutants were largely selective for disrupting the interaction with ORF30 but not the other three ORF18 binding partners: ORF31, ORF34, and ORF66. Furthermore, disrupting the ORF18-ORF30 interaction weakened the vTA complex as a whole, and an ORF18 point mutant that failed to bind ORF30 was unable to complement an ORF18 null virus. Thus, contacts between individual vTAs are critical, as even small disruptions in this complex result in profound defects in KSHV late gene expression. These findings underscore how individual interactions between the late gene transcription components are critical for both the stability and function of the complex.

Next, we examined the mechanism by which ORF24 binds RNA polymerase II (Pol II). Previous work in our lab found that ORF24 is a modular protein that binds to viral late gene promoters through its central TBP-like domain. Residues in the N-terminus of ORF24 bind and recruit human Pol II to activate the expression of late genes. We found that ORF24 interacts directly with the heptapeptide repeats of the carboxy terminal domain of Pol II, suggesting that ORF24 may be involved in bringing Pol II to sites of active viral late gene transcription. Collectively, our data highlight how KSHV (and likely other gamma and betaherpesviruses) direct robust transcription of late genes via a unique and streamlined mechanism of pre-initiation complex assembly.