The expression of a plant virus replicase protein affects the nature of acetic acid-induced programmed cell death in Saccharomyces cerevisiae

The replication of positive-strand RNA [(+)RNA] viruses is always associated with specific cell membranes. The association of viral replicase proteins with mitochondria has been studied with Carnation Italian ringspot virus (CIRV), a (+)RNA plant virus replicating in membranous structures originating from vesiculation of the mitochondrial outer membrane. The replication-associated protein p36 is responsible for targeting and anchoring CIRV replication complex to the mitochondrial membrane. Yet, the mechanisms by which (+)RNA viruses control host cell fate are still unknown.Programmed cell death (PCD) is a primordial response of virus-infected cells, whose mechanism is conserved from yeast to mammals.Taking advantage of the successful CIRV replication in the yeast Saccharomyces cerevisiae,we investigated whether and how the heterologous expression of the mitochondria-targeted CIRV p36 alone or in combination with the replicase protein p95 and DI RNA could affect yeast cell viability or stress-induced PCD. S. cerevisiae YPH499 cells were transformed with plasmids containing the CIRV p36 sequence, fused or not to GFP, cloned under the control of the inducible GAL1 promoter. Acetic acid-induced PCD (AA-PCD) was triggered and cell viability measured as in Giannattasio et al. (2005).The type of yeast cell death was analyzed by measuring plasma membrane integrity and PS externalization by propidium iodide and FITC-annexin V staining, respectively, and fluorescence microscopy. Heterologous expression of p36/p95 did not affect yeast cell viability up to 72 h whereas p36 alone slowed down cell growth after 24 h expression compared to control cells, with virtually no loss in plasma membrane integrity. Interestingly,after AA-PCD induction, p36-expressing cells lost viability at the same rate as the controls but showed a change in the type of cell death judged by apoptotic and necrotic marker analysis. S. cerevisiae is a powerful model organism for virus-host interaction studies. Due to the conservation of replication mechanisms among (+)RNA viruses, the data obtained with plain model viruses in this simple eukaryotic host can potentially be transferred to pathogens of higher eukaryotes. In addition, this system can be extended to investigate cellular pathways such as PCD.