Carnation Italian ringspot virus p36 protein expression in Saccharomyces cerevisiae induces changes in mitochondrial function

"Positive-strand RNA [(+)RNA] viruses include pathogens responsible for a number of diseases in humans, animals and plants. (+)RNA virus replication invariably occurs in association with specific host cell membranes, which are extensively rearranged to form partially enclosed vesicular enclaves, to scaffold and protect genome replication from the host plant defense reaction. The association of (+)RNA virus replication with the limiting membrane of peroxisomes and mitochondria has been studied with members of the genus Tombusvirus (family Tombusviridae). The tombusvirus carnation Italian ringspot virus (CIRV) genomic (+)RNA replication occurs on the mitochondrial outer membrane to produce numerous vesicles between the inner and outer membrane. CIRV p36 protein is required for targeting and anchoring the virus replication complex to the mitochondrial outer membrane in plant and when ectopically expressed in Saccharomyces cerevisiae. Interaction of CIRV p36 with yeast mitochondria is associated with increase in necrotic cell death and concomitant decrease in regulated cell death in response to acetic acid. Thus, in order to investigate p36-mitochondria interaction, we analyzed yeast cells expressing CIRV p36 under the control of the inducible GAL1 promoter, and measured several parameters of mitochondrial function. Concomitantly we also expressed the p33 replicase protein of another tombusvirus (cymbidium ringspot virus, CymRSV), which is known to be targeted to the endoplasmic reticulum in yeast cells. Endogenous and CCCP-stimulated respiration was dramatically reduced in p36-expressing yeast cells as compared with control cells. Similar results were obtained with isolated mitochondria using succinate as a substrate. A significant reduction of the activity of complexes II + III and IV was observed in yeast spheroplasts. No significant changes in mitochondrial respiration were observed in yeast cells expressing p33. Immunoblot analysis of either whole cell lysates or cell membrane-enriched fractions from p36- and p33-expressing or control cells showed that the level of marker proteins of mitochondrial matrix, inner and outer membrane was not changed by p36/p33 expression. These data suggest that p36 specifically alters mitochondrial function, without affecting mitochondrial biogenesis in yeast."