Repression of EEF1D by KSHV RTA promotes viral lytic reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes lifelong latency in the host but can reactivate in the lytic cycle under specific pathophysiological conditions. The replication and transcription activator (RTA) serves as the master regulator of this latent-to-lytic switch. Although RTA is well known as a potent viral transcriptional activator and E3 ubiquitin ligase, its role in modulating host gene expression remains incompletely understood. In this study, we identified eukaryotic translation elongation factor 1δ (EEF1D) as a previously unrecognized host inhibitory factor that inhibits KSHV reactivation. Functional analyses revealed that ectopic expression of EEF1D suppressed viral lytic replication, whereas EEF1D depletion enhanced viral reactivation. KSHV RTA counteracts this inhibition through two complementary mechanisms: interaction with EEF1D protein to promote its ubiquitin-proteasome-mediated degradation and, more prominently, repression of EEF1D transcription through promoter silencing. Dual-luciferase reporter assays further revealed that this transcriptional repression is conserved among primate γ-herpesviruses, including KSHV, rhesus rhadinovirus, and Epstein-Barr virus, but is absent in murine γ-herpesvirus 68. Mechanistically, RTA induces DNMT3A-dependent hypermethylation of the EEF1D promoter, a process facilitated by the transcription factor, PATZ1. Collectively, these findings reveal a previously unrecognized repressive function of RTA on the host gene EEF1D, highlighting an additional layer of viral strategy to promote lytic reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) establishes lifelong latency in host cells but can periodically reactivate, a process that is essential for viral spread and disease development. The viral replication and transcription activator (RTA) serves as the master switch of this transition; however, the host factors that inhibit reactivation and the mechanisms by which RTA overcomes them remain incompletely defined. In this study, we identified the host protein eukaryotic translation elongation factor 1δ (EEF1D) as a previously unrecognized inhibitor of KSHV lytic replication. However, RTA inhibited EEF1D expression at both the protein and transcriptional levels. These findings expand the functional repertoire of RTA by revealing its ability to repress host gene transcription, providing new insights into viral persistence and pathogenesis.