Carnation Italian ringspot virus p36 is targeted to mitochondria in human cells

BACKGROUND AND AIMS. Positive-strand RNA [(+)RNA] viruses include the agents of important human, animal and plant diseases. Regardless of the host, (+)RNA virus replication invariably occurs in association with host cell membranes, which are induced to proliferate and are rearranged to form partially enclosed vesicular enclaves, where the virus replication complex is assembled, and replication takes place. Carnation Italian ringspot virus (CIRV) is a member of the species Tombusvirus dianthi (genus Tombusvirus, family Tombusviridae). In infected plant cells, CIRV (+)RNA genome replication occurs on the mitochondrial outer membrane, which invaginates to produce numerous vesicles between the inner and outer mitochondrial membranes. CIRV replicase-associated p36 protein is required for targeting and anchoring the virus replication complex to the mitochondrial outer membrane in plant and when expressed in Saccharomyces cerevisiae. In yeast cells, CIRV p36 alters the mitochondrial structure and function. The aim of the study was to investigate whether CIRV p36 mitochondrial targeting signal is functional when the viral protein is expressed in human cells. METHODS. The sequence of CIRV p36, native or fused to the c-Myc epitope at the N-terminus, was cloned in plasmid pEGFP-N1 under the control of the constitutive CMV promoter, upstream and downstream the EGFP sequence, to obtain plasmids pEGFP-p36, pEGFP-mycp36, p36-EGFP, mycp36-EGFP. The four plasmids were separately transfected into Human Embryonic Kidney (HEK) 293 and HeLa cells. Cells transfected with empty pEGFP-N1 plasmid served as control. p36 heterologous expression was visualized by confocal microscopy of EGFP fluorescence in cells stained with the mitochondria-specific dye MitoTrackerTM. The mitochondrial morphology was analyzed by electron microscopy. RESULTS. CIRV p36 heterologous expression was obtained in HEK 293 cells, both when fused in frame at the C- or N-terminus of EGFP. Transfected HEK 293 cells had size and shape identical to untransformed cells, with several of them in the process of division. Confocal microscopy analysis showed that in cells transfected with pEGFP-N1 the green fluorescence was distributed throughout the cytoplasm, whereas in cells expressing EGFP fused to either terminus of the p36 protein (pEGFP-p36 or p36-EGFP), green fluorescence was restricted to defined regions of the cytoplasm, in proximity of the cell membrane. These areas had a punctuate appearance or were organized into larger patches. The same areas stained red when cells were treated with MitoTrackerTM showing that they might be constituted by mitochondria. Similarly, immortalized HeLa cells transfected with the same constructs showed mitochondria localization in aggregates close to the cell membrane. CONCLUSIONS. The plant virus replication-associated p36 protein was shown to localize to mitochondria in human cells, driving mitochondria concentration to defined areas in the cytoplasm, thus paralleling the results observed in plant and yeast cells. This confirms the conservation of replication mechanisms among (+)RNA viruses and supports simple plant viruses as valuable tools for studying (+)RNA virus replication and identifying molecular targets to develop new antiviral strategies.