The Nodamura virus RNA-dependent RNA polymerase localizes to mitochondria in transformed yeast cells

The goal of our laboratory is to decipher the mechanisms of viral RNA replication. We study this process in Nodamura virus (NoV), which replicates its positive-strand RNA genome to high levels in a wide range of host cells, including yeast. NoV provides an excellent model for studying replication of more complex, positive-strand RNA viruses pathogenic to humans. The NoV genome is bipartite. The RNA1 genome segment encodes the viral RNA-dependent RNA polymerase (RdRp), which catalyzes viral RNA replication via negative strand intermediates. Many RNA viruses replicate their genomes in membrane-bound replication complexes. The related Flock House virus replicates on mitochondrial membranes in infected Drosophila cells and transformed yeast. These organelles must also play an essential role for NoV, since infected mouse muscle tissue exhibits mitochondrial clustering and membrane rearrangement. We hypothesize that NoV RNA replication complexes localize to mitochondria in transformed yeast cells. To facilitate detection of the NoV RdRp in yeast cells, we added a C-terminal hemagglutinin (HA) epitope tag and placed its expression under control of an inducible yeast promoter. We used immunofluorescence confocal microscopy with mouse anti-HA primary and FITC-labeled anti-mouse secondary antibodies to determine the subcellular localization of NoV RdRp in transformed S. cerevisiae at 30°C. The RdRp co-localized with the mitochondrial dye Mitotracker Red and with monoamine oxidase, an outer mitochondrial membrane protein. Co-localization was lost if the RdRp was mutated to remove a predicted membrane-associated region essential for RNA replication. We conclude that the NoV RdRp localizes to mitochondria in transformed yeast cells.