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bioRxiv · 10.1101/2023.07.26.550743

Co-opted cytosolic host proteins form unique condensate substructures within the membranous tombusviral replication organelles

Lin, W.·Nagy, P. D.
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Abstract

Positive-strand RNA viruses co-opt intracellular organellar membranes for the biogenesis of viral replication organelles (VROs). The membranous VROs are the sites of viral replication. Tombusviruses co-opt numerous host proteins, many of them cytosolic, not membrane-bound, such as the glycolytic and fermentation enzymes. It is currently not known what type of molecular organization keeps these enzymes enriched and sequestered within the membranous VROs. Here, we show evidence that the tomato bushy stunt virus (TBSV) p33 and the closely-related carnation Italian ringspot virus (CIRV) p36 replication proteins sequester several co-opted cytosolic proteins in unique condensate substructures associated with the membranous VROs. We find that TBSV p33 and CIRV p36 replication proteins form droplets in vitro organized by their intrinsically disordered region (IDR). The replication proteins also organize the partitioning of several co-opted host proteins to p33/p36 droplets. We also show that the VRO-associated condensates containing co-opted glycolytic and fermentation enzymes are critical for local ATP production within the VRO to support the energy need of virus replication. A short segment with charged amino acids in p33/p36 IDR plays a critical role in droplet formation in vitro, in organizing the VRO-associated condensates and affects both ATP production in VROs and viral replication. We find that the co-opted ER membranes and actin filaments form meshworks within and around the condensate, likely contributing to the unique composition and structure of VROs. We propose that the p33/p36 replication proteins organize liquid-liquid phase separation of co-opted highly concentrated host proteins in condensate substructures within VROs to allow for the dynamic regulation of viral replication. Overall, we demonstrate that distinct substructures co-exist in tombusvirus VROs. These are the subverted membranes and condensate substructures, both of which are critical for VRO functions. The p33/p36 replication proteins induce and connect the two substructures within the VROs. Interfering with condensate formation in VROs might open up new antiviral approaches.

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Lin, W., Nagy, P. D.. 2023-07-26. Co-opted cytosolic host proteins form unique condensate substructures within the membranous tombusviral replication organelles. https://doi.org/10.1101/2023.07.26.550743

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