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.
Highlights
Positive-strand RNA [(þ)RNA] viruses contain messengersense, single-stranded RNA in their virions; they represent over a third of known virus genera; and they include many important human, animal, and plant pathogens [1]
Protein A, the only flock house virus (FHV) protein needed for RNA replication, is directed by an Nterminal targeting and transmembrane sequence to outer mitochondrial membranes, where it colocalizes by immunofluorescence with the sites of viral RNA synthesis [8,38]
The results show that FHV spherules are compartments or mini-organelles for viral RNA synthesis, which form by invagination of the outer mitochondrial membrane and communicate with the cytoplasm through ;10-nm diameter necks
Summary
Positive-strand RNA [(þ)RNA] viruses contain messengersense, single-stranded RNA in their virions; they represent over a third of known virus genera; and they include many important human, animal, and plant pathogens [1]. For many (þ)RNA viruses—including alphaviruses [5], other members of the alphavirus-like superfamily [15], rubiviruses [7,20], flaviviruses [21], tombusviruses [22], and others [4,23,24,25] —RNA replication occurs in association with ;50–70-nm diameter membranous vesicles or spherules that form in the lumen of specific secretory compartments or organelles The similarity of these structures suggests that RNA replication by such otherwise distinct viruses involves important conserved features related to membranes.
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