An integrative approach identifies direct targets of the late viral transcription complex and an expanded promoter recognition motif in Kaposi’s sarcoma-associated herpesvirus

Abstract

The structural proteins of DNA viruses are generally encoded by late genes, whose expression relies on recruitment of the host transcriptional machinery only after the onset of viral genome replication. β and γ-herpesviruses encode a unique six-member viral pre-initiation complex (vPIC) for this purpose, although how the vPIC directs specific activation of late genes remains largely unknown. The specificity underlying late transcription is particularly notable given that late gene promoters are unusually small, with a modified TATA-box being the only recognizable element. Here, we explored the basis for this specificity using an integrative approach to evaluate vPIC-dependent gene expression combined with promoter occupancy during Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. This approach distinguished the direct and indirect targets of the vPIC, ultimately revealing a novel promoter motif critical for KSHV vPIC binding. Additionally, we found that the KSHV vPIC component ORF24 is required for efficient viral DNA replication and identified a ORF24 binding element in the origin of replication that is necessary for late gene promoter activation. Together, these results identify an elusive element that contributes to vPIC specificity and suggest novel links between KSHV DNA replication and late transcription.

Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

“…RT-qPCR-based measurements of viral RNA from the ORF68 (early) and K8.1 (late) loci confirmed that the selective absence of late proteins in the ORF66.stop infections was due to a transcriptional defect ( Figure 1F ). The moderate decrease in both the transcript and protein of early gene ORF68 is consistent with the observation that most viral transcripts are downregulated in the absence of the vPIC component ORF24 (1). We also observed less ORF6 (the ssDNA binding protein involved in viral DNA replication) in the ORF66.stop samples, which could explain the reduced levels of viral DNA replication observed in the ORF66.stop cell line ( Figure 1D-E ).…”

“…It is well established that late gene transcription is licensed by the initiation of viral genome replication (15-17). Although it has been previously reported that other vTA mutants do not exhibit a defect in viral genome replication (6, 8-10), we recently reported that mutations in ORF24 result in a ∼6-fold defect in viral genome replication (1). Similarly, we find that the ORF66.stop virus has a modest ∼3-fold defect in viral genome replication that is rescued in the ORF66.MR virus ( Figure 1D ).…”

“…ORF24 is the only vTA known to directly contact promoter DNA, although other vTAs likely co-localize at late gene promoters via protein-protein interactions within the complex (1, 6). However, given that ORF66 possesses Cx n C motifs, which are frequently found in nucleic acid binding proteins (19), we considered that it might independently bind promoter DNA.…”

“…In this regard, we generated KSHV containing C-terminally HA tagged ORF66 in an otherwise WT BAC16 background (66HA) or in a BAC16 lacking ORF24 (66HA/24S) to evaluate whether ORF66 associates with the late gene promoter and, if so, whether its association requires ORF24. We also generated KSHV with N-terminally HA tagged ORF24 (HA24; (1)) and lacking either ORF66 (HA24/66S) or ORF30 (H24/30S) to determine whether promoter binding by ORF24 is influenced by other vTAs. Unlike ORF66, the ORF30 vTA is a small protein with no predicted nucleic acid binding properties and its only connection with the complex occurs through ORF18 (see Figure 2A ) (11).…”

“…Immediate early and early genes possess promoters similar to host promoters, with canonical TATA boxes. In contrast, late genes have minimal promoters, characterized by the presence of a non-canonical TAT T box, which in KSHV is followed by an RVNYS motif (1-3).…”