Co‐opted cytosolic proteins form condensate substructures within membranous replication organelles of a positive‐strand RNA virus

Summary Positive‐strand RNA viruses co‐opt organellar membranes for biogenesis of viral replication organelles (VROs). Tombusviruses also co‐opt pro‐viral cytosolic proteins to VROs. It is currently not known what type of molecular organization keeps co‐opted proteins sequestered within membranous VROs. In this study, we employed tomato bushy stunt virus (TBSV) and carnation Italian ringspot virus (CIRV) – Nicotiana benthamiana pathosystems to identify biomolecular condensate formation in VROs. We show that TBSV p33 and the CIRV p36 replication proteins sequester glycolytic and fermentation enzymes in unique condensate substructures associated with membranous VROs. We find that p33 and p36 form droplets in vitro driven by intrinsically disordered region. The replication protein organizes partitioning of co‐opted host proteins into droplets. VRO‐associated condensates are critical for local adenosine triphosphate production to support energy for virus replication. We find that co‐opted endoplasmic reticulum membranes and actin filaments form meshworks within and around VRO condensates, contributing to unique composition and structure. We propose that p33/p36 organize liquid–liquid phase separation of co‐opted concentrated host proteins in condensate substructures within membranous VROs. Overall, we demonstrate that subverted membranes and condensate substructures co‐exist and are critical for VRO functions. The replication proteins induce and connect the two substructures within VROs.