Subversion of selective autophagy for the biogenesis of tombusvirus replication organelles inhibits autophagy

Elaborate viral replication organelles (VROs) are formed to support positive-strand RNA virus replication in infected cells. VRO formation requires subversion of intracellular membranes by viral replication proteins. Here, we showed that the key ATG8f autophagy protein and NBR1 selective autophagy receptor were co-opted by Tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus. Knockdown of ATG8f or NBR1 in plants led to reduced tombusvirus replication, suggesting pro-viral function for selective autophagy. BiFC and proximity-labeling experiments showed that the TBSV p33 replication protein interacted with ATG8f and NBR1 to recruit them to VROs. In addition, we observed that several core autophagy proteins, such as ATG1a, ATG4, ATG5, ATG101 and the plant-specific SH3P2 autophagy adaptor proteins were also re-localized to TBSV VROs, suggesting that TBSV hijacks the autophagy machinery in plant cells. We demonstrated that subversion of autophagy components facilitated the recruitment of VPS34 PI3 kinase and enrichment of phospholipids, such as phosphatidylethanolamine and PI3P phosphoinositide in the VRO membranes. Hijacking of autophagy components into TBSV VROs led to inhibition of autophagic flux. We also found that a fraction of the subverted ATG8f and NBR1 was sequestered in biomolecular condensates associated with VROs. We propose that the VRO-associated condensates trap those autophagy proteins, taking them away from the autophagy pathway. Overall, tombusviruses hijack selective autophagy to provide phospholipid-rich membranes for replication and to regulate the antiviral autophagic flux.