Heterologous expression of p36 replicase of Carnation Italian ringspot virus in Saccharomyces cerevisiae

Positive-strand RNA [(+)RNA] viruses, the largest class of viruses, include many important pathogens of humans, animals and plants, sharing common replication mechanisms. A highly conserved feature of (+)RNA virus replication is the association of the viral replication complex with specific intracellular membranes, which are induced to proliferate and are extensively rearranged to form vesicles (or spherules). These partially closed vesicular enclaves constitute the confined environment in which virus and host factors concentrate to allow for a productive viral RNA synthesis, under conditions protected from host defense reactions. Virus-encoded proteins are responsible for the intracellular localization of the replication complex and for the formation of spherules. The association of viral replicase proteins with the outer membrane of mitochondria has been studied in details with Carnation Italian ringspot virus (CIRV, genus Tombusvirus, family Tombusviridae), a virus with a (+)RNA genome 4.8 kb in size, containing five ORFs. In infected plants, CIRV replication takes place in membranous structures originating from vesiculation of the mitochondrial outer membrane. The signals targeting and anchoring CIRV replication complex to the mitochondrial membrane are contained in the 36-kDa product of ORF1 (p36). Most traits of CIRV replication can be reconstituted in Saccharomyces cerevisiae cells, thus representing a good model for virus-host interaction studies. Heterologous expression of p36 protein fused or not to GFP localizes to mitochondria in yeast cells and causes organelle and membrane proliferation. To gain insights into the interaction between p36 and mitochondria, the effects were studied of p36 heterologous expression on yeast cell viability as well as on programmed cell death induced by acetic acid. It was shown that p36 affects cell viability and seems to exert an inhibitory effect on the nature of acetic acid-induced cell death. Due to the conservation of replication mechanisms between (+)RNA viruses, data obtained with simple model viruses, like CIRV, in a simple eukaryotic host, could be extended to pathogens of higher eukaryotes.