AbstractRNA is a central molecule for RNA viruses, acting as mRNA and genome. However, the interactions that viral (v)RNA establishes with the host cell is only starting to be elucidated. Here, we determine with unprecedented depth the composition of the ribonucleoproteins (RNPs) of the prototypical arthropod-borne Sindbis virus (SINV) using viral RNA interactome capture. We show that SINV RNAs engage with hundreds of cellular proteins and pathways, including a group of nuclear RNA-binding proteins (RBPs) with unknown roles in infection. Combining subcellular fractionation and proteomics with several orthogonal approaches, we demonstrate that these nuclear RBPs are selectively redistributed to the cytoplasm after infection, where they associate with the viral replication organelles. These nuclear RBPs potently supress viral gene expression, with activities spanning viral species and families. Our study provides a comprehensive and systematic analysis of SINV RNP composition, revealing a network of nuclear RBPs with moonlighting antiviral function.Research highlightsSINV RNAs interact with over four hundred cellular RBPsSINV induces selective cytoplasmic translocation of a subset of nuclear RBPsThese nuclear RBPs display potent antiviral effectsThe SF3B complex binds to SINV RNA and supresses infection in a splicing-independent manner
