Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

Abstract

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...

Disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor

“…Although requiring investigation, this would not be without precedent among positive-strand RNA viruses. Nodavirus RNA replication occurs on outer mitochondrial membranes in insect cells (41). It could explain mitochondrial abnormalities observed in early ultrastructural studies of acute hepatitis A in chimpanzees; a finding not present in acute hepatitis B (42).…”

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

“…Whether these viruses pack into microcrystals is dependent on the specimen preparation technique used for EM (Lanman et al, 2007, submitted this issue). In addition, FHV replication takes over the cellular machinery, in particular the mitochondria and re-fashion it into a distinct morphology (Miller et al, 2001). Therefore, late stage FHV infected DL1 cells represent a good test specimen for our hybrid aldehyde fixation/HPF methods.…”

Subcellular localization and live-cell imaging of the Helicoverpa armigera stunt virus replicase in mammalian and Spodoptera frugiperda cells

“…When expressed alone in cells, foot-and-mouth disease virus non-structural proteins 2B and 2C are found in association with ER and Golgi membranes (Moffat et al, 2005). Flock House virus, which is related structurally to the tetraviruses, replicates its RNA in association with the outer mitochondrial membrane in infected Drosophila cells (Miller et al, 2001). This localization is independent of infection and viral RNA replication and can be redirected to the ER, resulting in a replicase that is functional both in vivo and in vitro (Miller & Ahlquist, 2002;Miller et al, 2003).…”