Conserved Cx _n C Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

Abstract

Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposi’s sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus, deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is dependent upon a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we found that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV viral preinitiation complex and suggests that it functions as a complex to recognize late gene promoters. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. The release of infectious virions requires the production of capsid proteins and other late genes, whose production is transcriptionally controlled by a complex of six virally encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function five of its six components play in transcription. Here, we demonstrate that ORF66 is an essential component of this complex in KSHV and that its inclusion in the complex depends upon its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examined the assembly of the viral preinitiation complex at late gene promoters and found that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral preinitiation complex.

Murine Cytomegalovirus Protein pM49 Interacts with pM95 and Is Critical for Viral Late Gene Expression

“…However, studies are mostly restricted to the functional level due to low accumulation of vTFs and difficulties in their mechanism of study. Recent work in KSHV demonstrated that ORF66, the homolog of pM49, was essential for viral late gene expression via interaction between ORF34, the homolog of pM95 (31,32). Those studies, together with our results, provide a shred of evidence that the regulatory mechanism of viral late genes by vTFs is probably very conserved between betaherpesviruses and gammaherpesviruses.…”

“…pM49, pM95, pM79 and pM91 interacted directly or indirectly to form the core within the vTF complex; pM87 was thought to function as a hub to bridge pM92 and the core through the pM87-pM95 and pM87-pM92 interaction. Based on the research from Glaunsinger's group (31) and our references, we propose a hypothesis for the mechanism of the MCMV vTF transcription regulation system; we speculate that the members of the core complex, pM49, pM95, pM91, and pM79, may have the same role in viral late gene regulation. Although viral late gene expression regulatory mechanism remains largely unknown, it has been reported that the pM79 analog pUL79 serves as an elongation factor, but with no effects on RNA polymerase II (RNAPII) recruitment (28).…”

“…Since pM49 and pM79 belong to the same vTF complex component, we speculate that pM49, along with pM91 and pM95, may enhance transcription elongation as well. Besides the discovery of the role for pUL79, ORF66 and ORF30 were reported to be required for ORF24 occupancy in the KSHV genome (31). By analogy with KSHV vTFs, it is possible that pM49, pM91, and other core complex members are also involved in the regulation of pM87 localization to the MCMV genome.…”

The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

“…We assessed the ability of the NTD chimeras to functionally complement ORF24 using an established transfection-based late gene transcription assay [13][14][15]. The six vTAs (ORFs 18, 30, 31, 34, 66 and either WT ORF24, its homologs, or chimeras) were co-transfected into HEK293T cells, along with a firefly luciferase reporter controlled by the K8.1 late gene promoter or, as a control, the early ORF57 promoter.…”

“…First, β/γ late gene transcription is regulated in part by a core promoter sequence 12-15 base pairs in length that has a TATT motif followed by a RVNYS motif in lieu of the canonical TATA box found in early viral promoters and in some cellular promoters [8][9][10][11]. Additionally, late gene expression requires at least six viral proteins, called viral transcriptional activators (vTAs), which form a complex at late gene promoters [12][13][14][15][16][17]. Little is known about the functional role these vTAs play in late gene transcription.…”