Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway

Abstract

The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional repressor proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional repressor protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the corepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.

Latency-Associated Nuclear Antigen of Kaposi Sarcoma–Associated Herpesvirus Promotes Angiogenesis through Targeting Notch Signaling Effector Hey1

“…We demonstrated that KSHV infection unregulated the level of expression of endogenous Hey1 in B and 293T cells and LANA takes major responsibility for it. Our results showed that Hey1 was degraded by the ubiquitinproteasome system, as indicated by other research (47,48). Ectopic expression of LANA significantly inhibited the degradation of Hey1, in a dose-dependent manner.…”

Murine Gammaherpesvirus 68 Evades Host Cytokine Production via Replication Transactivator-Induced RelA Degradation

“…To remove genomic DNA, total RNA was digested with RNase-free DNase I (New England Biolabs) at 37°C for 1 h. DNase I digestion was quenched by heat inactivation at 70°C for 20 min, and total RNA was repurified with TRIzol reagent. cDNA was prepared with 1.5 g total RNA, reverse transcriptase (Invitrogen), and 0.5 g/l primer oligo(dT) [12][13][14][15][16][17][18][19] . RNA was then removed by incubation with RNase H (Epicentre).…”

Oxidative Stress Induces Reactivation of Kaposi's Sarcoma-Associated Herpesvirus and Death of Primary Effusion Lymphoma Cells

“…Several cellular factors, such as XBP-1, Ras, Ets-1, PARP-1, hKFC, CBP, the SWI/SNF chromatin remodeling complex, the TRAP/Mediator complex, RBP-J, human Notch intracellular domain, and HMGB1, have been shown to promote KSHV reactivation and/or lytic gene expression (11,31,32,44,71,72,75,76), suggesting a close link between many cellular processes and KSHV reactivation. Other cellular factors, such as Oct-2, KAP-1, and Hey1, were found to inhibit KSHV reactivation and/or lytic gene expression (12,19,25). However, the regulation of KSHV reactivation by cellular signals is still not fully understood.…”