KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus whose lytic replication cycle is induced and driven by the viral transcription factor RTA (replication and transcription activator). Identifying what cellular and viral proteins RTA is interacting with during lytic reactivation can help to better understand how RTA promotes the lytic cycle. Using a proteomic approach, we identified the cellular E3 ubiquitin ligase complex RNF20/40 as a novel interacting partner of RTA. One of the major roles of this protein complex is the monoubiquitylation of histone H2B at lysine 120 on cellular chromatin, which is a hallmark of transcriptionally active genes. While the role of RNF20/40 has been studied on different host genomes, its potential to regulate viral genes remains largely unexplored. Here, we show that the shRNA inhibition of RNF20 expression impairs RTA-induced host and viral lytic gene expression, viral DNA replication, and virus production. In addition, we found that the association of RNF20 with the KSHV episome increases during lytic reactivation, and the binding of RTA to viral promoters is required for the synergistic promoter activation by RTA and RNF20/40. Interestingly, the inhibition of RNF20 expression during lytic reactivation of KSHV did not affect H2BK120ub level but it did reduce RNA polymerase II occupancy on viral promoters. Overall, our data suggest that RTA is hijacking the cellular E3 ligase complex RNF20/40 to increase the level of transcriptionally active RNA polymerase II on viral gene promoters thereby facilitating lytic gene expression to advance the viral lytic cycle.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing human herpesvirus that establishes a persistent infection in humans. The lytic viral cycle plays a crucial part in lifelong infection as it is involved in the viral dissemination. The master regulator of the KSHV lytic replication cycle is the viral replication and transcription activator (RTA) protein, which is necessary and sufficient to push the virus from latency into the lytic phase. Thus, the identification of host factors utilized by RTA for controlling the lytic cycle can help to find novel targets that could be used for the development of antiviral therapies against KSHV. Using a proteomics approach, we have identified a novel interaction between RTA and the cellular E3 ubiquitin ligase complex RNF20/40, which we have shown to be necessary for promoting RTA-induced KSHV lytic cycle.