Isolation and Preliminary Characterization of Mitochondrial Spherules Involved in Flock House Virus RNA Replication

Abstract

The replication of genomic RNA by positive‐sense RNA viruses occurs in association with host membranes. Flock House virus (FHV) is an ideal system for studying these unique interactions since much of the molecular basis for the replication of its RNA is already understood. Protein A, the viral RNA‐dependent RNA polymerase protein, is targeted to the outer mitochondrial membrane (OMM) where it is responsible for viral RNA synthesis. OMM invaginations (or spherules) containing Protein A are formed and are known to be the site of viral RNA replication. Kopek and colleagues previously generated a three‐dimensional reconstruction of these spherules using EM tomography of thin‐sectioned FHV‐infected Drosophila cells. However, these structures have not been studied in the absence of chemical fixation and staining, nor have optimal RNA replication conditions been established for the wild‐type protein in the presence of host membranes.As part of a larger study directed at understanding the structural basis of spherule formation, we aimed to develop techniques for the robust production of replication‐induced OMM invaginations in isolated mitochondrial membranes for imaging by cryo‐electron microscopy (EM). We are now able to consistently isolate replication‐competent mitochondrial fragments and have optimized several reaction conditions enabling the imaging of spherules by cryo‐EM. Our results reveal a number of important initial insights, including the transient nature of the spherules as well as the possibility of visualizing replicated viral RNA.