RNA binding proteome profiling reveals RNA-dependent activation of oncogenic host signaling during KSHV infection

Abstract Viruses encode RNA-binding proteins (RBPs) to manipulate host processes and promote infection. However, due to limited genomic space, many viral proteins are multifunctional and may engage RNA through non-canonical domains, making their identification difficult using traditional sequence-based approaches. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of many AIDS-associated cancers. Here, leveraging an unbiased proteomic approach we identified all RBPs in KSHV-infected primary effusion lymphoma (PEL) cells, uncovering 7 previously unrecognized KSHV-encoded RBPs. Among these is viral Interferon Regulatory Factor 1 (vIRF1), which we found is both necessary and sufficient to activate oncogenic RAS/MAPK signaling through an RNA-dependent interaction with host G3BP proteins. Importantly, a KSHV vIRF1 mutant lacking the ability to bind RNA exhibits a B cell-specific replication defect, highlighting the essential role of RNA interactions in viral gene regulation and pathogenesis. These findings reveal a novel strategy by which KSHV hijacks RNA-mediated signaling to support its lifecycle.