In transfected mammalian cells, the Nodamura virus RdRp localizes to the outer mitochondrial membrane and causes apparent clustering of the mitochondria

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 bipartite positive-strand RNA genome to high levels in a wide range of host cells. NoV serves as a model for the study of genome replication of other positive-strand RNA viruses pathogenic to humans. NoV RNA1 encodes the viral RNA-dependent RNA polymerase (RdRp) that catalyzes viral RNA replication. The related Flock House virus replicates its genome on mitochondrial membranes. For NoV, infected mouse tissue exhibits mitochondrial clustering and membrane rearrangement. We hypothesize that this represents formation of viral RNA replication complexes. To determine the subcellular localization of NoV RdRp in mammalian cells, we expressed it with a C-terminal hemagglutinin (HA) tag and detected it using immunofluorescence with HA-specific antibodies and confocal microscopy. The RdRp localized to mitochondria and caused mitochondrial clustering similar to that described for NoV-infected muscle tissue. This was dependent on the presence of a predicted membrane-associated region. To determine whether the RdRp binds to mitochondrial membranes, we used selective membrane permeabilization and immunofluorescence confocal microscopy. When only the plasma membrane was permeabilized, the RdRp co-localized with monoamine oxidase (MAO), an outer mitochondrial membrane protein. When both the plasma and mitochondrial membranes were permeabilized, the RdRp and MAO both co-localized with cytochrome c oxidase subunit III, an inner mitochondrial membrane protein. These results suggest that the NoV RdRp localizes to the outer mitochondrial membrane in transfected mammalian cells.