Genome-Wide Identification of Direct RTA Targets Reveals Key Host Factors for Kaposi’s Sarcoma-Associated Herpesvirus Lytic Reactivation

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus, which maintains the persistent infection of the host by intermittently reactivating from latently infected cells to produce viral progenies. While it is established that the replication and transcription activator (RTA) viral transcription factor is required for the induction of lytic viral genes for KSHV lytic reactivation, it is still unknown to what extent RTA alters the host transcriptome to promote KSHV lytic cycle and viral pathogenesis. To address this question, we performed a comprehensive time course transcriptome analysis during KSHV reactivation in B-cell lymphoma cells and determined RTA-binding sites on both the viral and host genomes, which resulted in the identification of the core RTA-induced host genes (core RIGs). We found that the majority of RTA-binding sites at core RIGs contained the canonical RBP-Jκ-binding DNA motif. Subsequently, we demonstrated the vital role of the Notch signaling transcription factor RBP-Jκ for RTA-driven rapid host gene induction, which is consistent with RBP-Jκ being essential for KSHV lytic reactivation. Importantly, many of the core RIGs encode plasma membrane proteins and key regulators of signaling pathways and cell death; however, their contribution to the lytic cycle is largely unknown. We show that the cell cycle and chromatin regulator geminin and the plasma membrane protein gamma-glutamyltransferase 6, two of the core RIGs, are required for efficient KSHV reactivation and virus production. Our results indicate that host genes that RTA rapidly and directly induces can be pivotal for driving the KSHV lytic cycle. IMPORTANCE The lytic cycle of KSHV is involved not only in the dissemination of the virus but also viral oncogenesis, in which the effect of RTA on the host transcriptome is still unclear. Using genomics approaches, we identified a core set of host genes which are rapidly and directly induced by RTA in the early phase of KSHV lytic reactivation. We found that RTA does not need viral cofactors but requires its host cofactor RBP-Jκ for inducing many of its core RIGs. Importantly, we show a critical role for two of the core RIGs in efficient lytic reactivation and replication, highlighting their significance in the KSHV lytic cycle. We propose that the unbiased identification of RTA-induced host genes can uncover potential therapeutic targets for inhibiting KSHV replication and viral pathogenesis.

A herpesvirus transactivator and cellular POU proteins extensively regulate DNA binding of the host Notch signaling protein RBP-Jκ to the virus genome

“…Several known targets for Rta and RBP-J were identified in our data, including the Mta, K-bZIP, and PAN promoters (Table S1). Whereas previous papers also suggested a large number of potential Rta and RBP-J targets on the viral genome (19,(57)(58)(59), the breadth of the genome bound by Rta and RBP-J in our work exceeds those papers and may be due to technical differences.…”

Epigenetic factor siRNA screen during primary KSHV infection identifies novel host restriction factors for the lytic cycle of KSHV

“…We speculate that the reason we did not see more virus production is that the shRNA knockdown of host epigenetic factors might be incomplete. Alternatively, the shRNA inhibition of epigenetic factors interfere with the induction of RTA's host target genes that are critical for virus production, which can limit the magnitude of lytic activation (S4 Fig) [19]. Nevertheless, these results suggest that the epigenetic factor shRNA knockdown may also increase lytic viral DNA replication, which can in part contribute to the increased lytic viral gene expression and virus production in a subpopulation of infected cells.…”

“…293TBAC16 wild-type and RTA-KO cell lines were cultured in DMEM with 10% FBS, P/S, and 300 μg/ml hygromycin. TRExBCBL1-3xFLAG-RTA cell line was cultured in RPMI medium containing 10% Tet System Approved FBS (TaKaRa), P/S, and 20 μg/ ml hygromycin [19].…”

“…Of the lytic genes, the transcriptional regulation of ORF50, which encodes the viral replication and transcription activator (RTA), has been studied in more detail because of its essential role in the lytic cycle [17]. RTA is an immediate-early gene whose expression is required and sufficient to induce many of the lytic genes and a set of host genes, which collectively are necessary for the induction of viral DNA replication and virus production [17,18,19]. RTA promoter has bivalent chromatin enriched in both the activating H3K4me3 and the repressive H3K27me3 histone marks, which can inhibit RTA expression during latency, while simultaneously priming the RTA promoter for rapid activation during the lytic cycle [6,7].…”

Epigenetic control in Kaposi sarcoma-associated herpesvirus infection and associated disease

“…RTA binds to GMNN, a protein involved in cell cycle and chromatin remodeling [138] PEL cell lines (TRExBCBL1-3xFLAG-RTA); in vitro infection (iSLK); ectopic expression (HEK293T, BJAB, iSLK) at most sites, it is interesting that one study observed LANAbinding peaks which partially overlapped with Stat1-binding sites in the promoters of gamma interferon (IFNγ) regulated genes. Indeed, IFNγ treatment demonstrated that LANA could counteract activation of a subset of these genes, suggesting that some of the chromatin changes associated with KSHV infection may conditionally alter gene expression only after activation of specific pathways [140].…”