A model on the role of co-opted ESCRT factors and lipids in formation of RNAi-insensitive VRCs during TBSV replication in yeast.

<p>(A) In wt yeast, the formation of tombusvirus-induced vesicle-like spherules (membrane invaginations) in the peroxisomal membranes is facilitated by the recruitment of the ESCRT proteins and enrichment of sterols and PE. The model predicts that the membranous VRC protects the viral dsRNA replication intermediate from DCR1, resulting in lack of siRNA generation and the increased stability of both newly-made TBSV (+)RNA and dsRNA replication intermediate, as shown. Note that ESCRT factors have been placed putatively in the VRCs. (B) In the absence of ESCRT-I or III proteins, tombusvirus replication proteins could only form membrane invaginations with large openings that make the viral dsRNA replication intermediate in VRCs exposed to DCR1, resulting in siRNA generation and the decreased stability of both newly-made TBSV (+)RNA and dsRNA replication intermediate. (C) In the absence of TBSV-induced sterol (shown here for ergosterol) enrichment within the replication compartment, the VRCs formed are less stable and the viral dsRNA replication intermediates in VRCs are exposed to DCR1, resulting in siRNA generation and the decreased stability of both newly-made TBSV (+)RNAs and dsRNA replication intermediate. (D) PE and phospholipid levels affect the speed of RNAi-insensitive VRC assembly. Left panel: when the VRC assembly is rapid due to high PE or phospholipid level in yeast, then components of RNAi machinery or ribonucleases have greatly reduced chance to enter the VRCs prior to the completion of VRC assembly. Right panel: when the VRC assembly is slow due to a limiting host factor (such as PE and phospholipid accessibility), then components of RNAi machinery (as indicated by the presence of DCR1 inside the spherule in the right panel) or ribonucleases might be able to enter the VRCs prior to the completion of VRC assembly. This deficiency in excluding ribonucleases from VRCs during slow VRC assembly process then leads to lower level of viral RNA accumulation.