THE INTERPLAY BETWEEN CARNATION ITALIAN RINGSPOT VIRUS p36 REPLICASE PROTEIN AND MITOCHONDRIA ALTERS THE MITOCHONDRIAL FUNCTION IN YEAST CELLS

Positive-strand RNA [(+)RNA] viruses, which are agents of important diseases in humans, animals and plants, depend on the host endomembrane system for their replication. (+)RNA virus invariably replicate in association with specific host cell membranes, which are extensively rearranged to form partially enclosed vesicular enclaves, constituting the virus replication site. Carnation Italian ringspot virus (CIRV) genomic (+)RNA replication occurs on the mitochondrial outer membrane which is induced to proliferate and invaginates 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. In yeast cells, CIRV p36 is able to increase necrotic cell death and concomitantly decrease regulated cell death in response to acetic acid. p36-mitochondria interaction was analyzed in S. cerevisiae cells expressing CIRV p36 under the control of the inducible GAL1 promoter. p36-expressing cell growth on a non-fermentable carbon source was negatively affected, compared to that of control cells, indicating that CIRV p36 ectopic expression decreased respiratory yeast cell growth. Accordingly, the viral protein effect was more striking at temperatures favoring p36 expression. Confocal and electron microscopy analyses demonstrated that p36 expression in yeast cells altered the mitochondrial network. Measuring several parameters of mitochondrial function showed that heterologous expression of p36 decreased oxygen consumption in yeast cells due to respiratory chain complex impairment. Basal respiration rate and CCCP-stimulated maximal respiration rate were dramatically reduced in p36-expressing yeast cells as compared with control cells. Similar results were obtained in ADP-stimulated state 3 respiration measured in freshly isolated mitochondria, using succinate as a substrate. A significant reduction of the activity of complexes II + III and IV was observed in yeast. Immunoblot analysis of either whole cell lysates or cell membrane-enriched fractions from p36-expressing or control cells showed that mitochondrial proteins are not affected by p36 overexpression, since the level of marker proteins of mitochondrial matrix, inner and outer membrane was not changed by p36 expression. These data suggest that p36 alters mitochondrial function, without affecting mitochondrial biogenesis in yeast.