Three-Dimensional Analysis of a Viral RNA Replication Complex Reveals a Virus-Induced Mini-Organelle

Abstract

Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)–infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside ∼50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of ∼10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of ∼100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions. Whereas cells store and replicate their genomes as DNA, most viruses have RNA genomes that replicate by using virus-specific pathways in the host cell. The largest class of RNA viruses, the positive-strand RNA viruses, replicate their genomes on intracellular membranes. However, little is understood about how and why these viruses use membranes in RNA replication. The well-studied flock house virus (FHV) replicates its RNA on mitochondrial membranes. We found that the single FHV RNA replication factor and newly synthesized FHV RNA localized predominantly in numerous infection-specific membrane vesicles inside the outer mitochondrial membrane. We used electron microscope tomography to image these membranes in three dimensions and found that the interior of each vesicle was connected to the cytoplasm by a single necked channel large enough to import ribonucleotide substrates and to export product RNA. The results suggest that FHV uses these vesicles as replication compartments, which may also protect replicating RNA from competing processes and host defenses. These findings complement results from other viruses to support possible parallels between genome replication by positive-strand RNA viruses and two distinct virus classes, double-stranded RNA and reverse-transcribing viruses.