The immediate early viral protein replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for activating the lytic cycle of KSHV. RTA induces the KSHV lytic cycle by several mechanisms, acting as a viral transcription factor that directly induces viral and host genes and acting as a viral E3 ubiquitin ligase by degrading host proteins that block viral lytic replication. Recently, we have characterized the global gene expression changes in primary effusion lymphoma (PEL) upon lytic reactivation of KSHV, which also led to the identification of rapidly downregulated genes such as ID2, an inhibitor of basic helix-loop-helix transcription factors. Here, we demonstrate that ID2 overexpression in PEL ablates KSHV lytic reactivation, indicating that ID2 inhibits the KSHV lytic cycle. Furthermore, we show that while ID2 is highly expressed during latency, its protein level is rapidly reduced by 4 h postinduction during lytic reactivation. Our results indicate that RTA binds to ID2 and induces its degradation during the KSHV lytic cycle by N-terminal ubiquitination through the ubiquitin-proteasome pathway. Importantly, we found that not only KSHV RTA but also its Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) homologs interact with ID2, and they can induce the degradation of all four members of the ID protein family, suggesting an evolutionarily conserved interplay between gammaherpesvirus RTAs and ID proteins. Taken together, we propose that ID2 acts as a repressor of the KSHV lytic cycle, which is counteracted by its RTA-mediated degradation. We also predict that ID proteins may act as restriction factors of the lytic phase of the other gammaherpesviruses as well. IMPORTANCE In addition to its transcription regulatory role, RTA is also known to have an E3 ubiquitin ligase activity, which RTA utilizes for inducing protein degradation. However, it is still largely unknown what host factors are downregulated during KSHV lytic reactivation by RTA-mediated protein degradation and what the biological significance of the degradation of these host factors is. In this study, we discovered that RTA employs N-terminal ubiquitination to induce degradation of ID2, a potent transcription repressor of host genes, via the ubiquitin-proteasome pathway to promote KSHV lytic reactivation in PEL cells. Furthermore, we found that not only KSHV RTA but also RTA of EBV and MHV68 gammaherpesviruses can induce the degradation of all four human ID proteins, indicating that the interplay between gammaherpesvirus RTAs and ID proteins is evolutionarily conserved.
Keywords: ID proteins; ID2; Kaposi's sarcoma-associated herpesvirus; PEL; RTA; lytic cycle; reactivation; ubiquitination.