Subversion of phosphorylated SR proteins by enterovirus A71 in IRES-dependent translation revealed by RNA-interactome analysis

During infection by positive-sense single-stranded RNA viruses, understanding the mechanisms governing the fate of viral RNA, whether directed towards translation, replication, or virion assembly, remains a significant challenge. In this study, we conducted RNA-interactome analysis using metabolic labeling coupled with quantitative proteomics to investigate the protein composition of temporal ribonucleoprotein complexes (RNPs) during enterovirus A71 (EV-A71) infection. Comparative analysis of RNPs during the early and late infection stages, representing the eclipse and maturation phases, revealed dynamic RNP remodeling over time. This remodeling process involved the exchange of nuclear RNA binding proteins with cytoplasmic membrane-associated proteins. Notably, EV-A71 infection induced the phosphorylation and cytoplasmic re-localization of nuclear serine and arginine-rich (SR) proteins, as determined using pan-SR protein antibodies, with these proteins found to co-localize with viral RNAs. Knockdown of specific SR proteins, including SRSF4, SRSF5, and SRSF6, significantly reduced viral growth, highlighting their critical role in the infection process. Intriguingly, these phosphorylated SR proteins cofractionated with the translation machinery rather than the replication organelles, a phenomenon predominantly observed during the early infection phase and abolished in the late phase. Importantly, inhibition of SR protein phosphorylation using the kinase inhibitors SRPKIN-1 and TG003 significantly impaired IRES-dependent translation and EV-A71 replication. These findings underscore the pivotal role of SR protein phosphoregulation during the eclipse phase of EV-A71 infection in facilitating the formation of translation-competent complexes. Furthermore, they highlight the potential of targeting SR protein phosphorylation as a promising strategy for antiviral development.