Positive-strand RNA viruses are important pathogens of humans and plants. These viruses built viral replication organelles (VROs) with the help of co-opted host proteins and intracellular membranes to support robust virus replication in infected cells. Tomato bushy stunt virus (TBSV), a model (+)RNA virus, assembles membranous VROs, which are associated with vir-condensate substructures driven by TBSV p33 replication-associated protein. In this work, we provide evidence that the peroxisome-associated TBSV and the mitochondria-associated carnation Italian ringspot virus hijack the host small ubiquitin-like modifier (SUMO) machinery in yeast model host and plants. Based on knockdown of components of the SUMO pathway, we show that SUMO machinery acts as a cellular proviral dependency factor during TBSV replication. The sumoylation machinery was found to be partially retargeted from the nucleus into vir-condensate associated with membranous VROs through direct interactions with TBSV p33. We developed a yeast-based sumoylation assay that demonstrated p33 sumoylation. Absence of sumoylation or mutations in SIM SUMO-interacting motif in p33 replication protein reduced the ability of p33 to form droplets in vitro via phase separation. We demonstrate that p33 sumoylation and its intrinsically disordered region play noncomplementary roles in droplet formation. Mutations in p33 sumoylation sites and p33-SIM sequence resulted in reduced-sized VROs, which showed diminished protection of TBSV p33 and the viral RNA from degradation and also reduced viral RNA recombination. Altogether, the co-opted host sumoylation machinery promotes viral replication and RNA recombination. This finding could provide opportunities for antiviral interventions via targeting protein posttranslational modifications.

• PMID: 40512796
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Journal Article

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Lin W, Nagy PD

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