We show that brome mosaic virus (BMV) RNA replication protein 1a, 2a polymerase, and a cis-acting replication signal recapitulate the functions of Gag, Pol, and RNA packaging signals in conventional retrovirus and foamy virus cores. Prior to RNA replication, 1a forms spherules budding into the endoplasmic reticulum membrane, sequestering viral positive-strand RNA templates in a nuclease-resistant, detergent-susceptible state. When expressed, 2a polymerase colocalizes in these spherules, which become the sites of viral RNA synthesis and retain negative-strand templates for positive-strand RNA synthesis. These results explain many features of replication by numerous positive strand RNA viruses and reveal that these viruses, reverse transcribing viruses, and dsRNA viruses share fundamental similarities in replication and may have common evolutionary origins.

The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40-to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus-and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.Positive-strand RNA viruses are responsible for a wide range of diseases in humans, animals, and plants. Clinically relevant members of this group cause significant morbidity and mortality and include viruses from the Picornaviridae, Caliciviridae, Togaviridae, and Flaviviridae families. Although these pathogens represent a prominent component of the growing list of emerging and potentially devastating viral diseases (40), current therapies for positive-strand RNA virus infections are limited to a few marginally effective drugs (36). The design and investigation of novel and broadly effective therapies require the identification and characterization of fundamental mechanisms in positive-strand RNA virus replication and pathogenesis, such as replication complex formation.Flock house virus (FHV) and the closely related black beetle virus (BBV) are the best-studied alphanodaviruses in the Nodaviridae family (2). FHV was originally isolated from the grass grub Costelytra zealandica (12, 57) and contains one of the smallest known genomes of any animal RNA virus. The 4.5-kb FHV genome is bipartite, with two capped but nonpolyadenylated RNAs copackaged into a 29-nm nonenveloped virion with an icosahedral (Tϭ3) capsid (56, 57). The larger 3.1-kb RNA species (RNA1) encodes protein A (2, 11), a 112-kDa protein with s...