The EV-A71 RNA-binding protein Matrin 3 facilitates viral replication by stabilizing viral RNA via ZDHHC20-mediated...

Enterovirus 71 (EV-A71) is a major causative agent of severe hand, foot, and mouth disease (HFMD) in young children. However, the molecular interplay between EV-A71 and host factors, particularly how these interactions govern viral replication, remains incompletely understood. To systematically identify host factors involved in EV-A71 infection, we performed comprehensive identification of RNA-binding proteins (RBPs) by mass spectrometry (ChIRP-MS) during viral infection and identified 374 host proteins associated with the EV-A71 genomic RNA. Integrative analysis of existing datasets further revealed a broader association between Matrin 3 (MATR3) and enteroviruses, which we functionally validated through gain- and loss-of-function assays, demonstrating that MATR3 acts as a proviral factor essential for efficient EV-A71 replication. Mechanistically, we show that MATR3 specifically binds to the 3' untranslated region (3'UTR) of EV-A71 RNA via its second RNA recognition motif (RRM2). This interaction promotes RNA-dependent condensate formation of MATR3, thereby enhancing viral RNA stability. Furthermore, MATR3 recruits the host palmitoyltransferase ZDHHC20 to the viral ribonucleoprotein complex, facilitating MATR3 palmitoylation. This posttranslational modification promotes the assembly of a stable ribonucleoprotein complex that cooperatively stabilizes the viral genome and supports efficient replication. Together, this study delineates a mechanistic pathway by which EV-A71 co-opts MATR3 through condensate formation and palmitoylation to stabilize its genomic RNA and promote viral replication, and provides a comprehensive profiling dataset of host proteins associated with EV-A71 RNA.