Host microRNA-31-5p regulates KHDRBS3 expression and modulates viral gene expression during KSHV lytic reactivation

Oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) employs a biphasic life cycle consisting of latency and lytic replication to achieve lifelong infection. Despite its essential role in KSHV persistence and tumorigenicity, much remains unknown about how KSHV lytic reactivation is regulated. Leveraging high-throughput transcriptomics, we identify microRNA-31-5p (miRNA-31-5p) as a key regulator of KSHV lytic reactivation, capable of modulating expression of KSHV lytic genes and progression through the lytic cascade. Mechanistically, miR-31-5p controls the KSHV lytic switch by regulating expression of the RNA-binding protein KHDRBS3. miR-31-5p-mediated KHDRBS3 repression results in decreased nascent transcription of crucial viral lytic genes including the main KSHV transcription factor RTA. We characterize KHDRBS3 as a major host factor that is critical for KSHV lytic replication and uncover its key role in KSHV lytic gene transcription. Our results highlight a pivotal role for the miR-31-5p/KHDRBS3 axis in modulating KSHV reactivation and provide insights into gene-expression regulation of lytic KSHV.