Article

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

Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
PLoS Biology (Impact Factor: 11.77). 10/2007; 5(9):e220. DOI: 10.1371/journal.pbio.0050220
Source: PubMed

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 approximately 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 approximately 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 approximately 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.

Full-text

Available from: Benjamin G Kopek, Jun 10, 2015
0 Followers
 · 
123 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Viral factories are novel structures built by viruses in infected cells. During their construction organelles are recruited and build a large scaffold for viral replication and morphogenesis. We have studied how a bunyavirus uses the Golgi to build the factory. With the help of confocal and 3D ultrastructural imaging together with molecular mapping in situ and in vitro we have characterized a tubular structure that harbours the viral replication complexes in a globular domain. Numerous ribonucleoproteins were released from purified tubes disrupted in vitro. Actin and myosin I were identified by peptide mass fingerprinting in isolated tubes while actin and the viral NSm non-structural protein were detected in the tubes' internal proteinaceous scaffold by immunogold labelling. Studies with NSm deletion mutants and drugs affecting actin showed that both NSm and actin are key factors for tube and virus assembly in Golgi. Three-dimensional reconstructions based on oriented serial sections of infected cells showed that tubes anchor cell organelles to Golgi stacks and make contacts with intracellular viruses. We propose that this new structure, unique among enveloped viruses, assembles in association with the most stable component of Golgi stacks, the actin-containing matrix scaffold, connecting viral replication and morphogenesis inside viral factories.
    Cellular Microbiology 07/2008; 10(10):2012-28. DOI:10.1111/j.1462-5822.2008.01184.x · 4.82 Impact Factor
  • Source
    11/2014; 1(1):237-259. DOI:10.1146/annurev-virology-031413-085532
  • Future Virology 12/2014; 9(12):1089-1104. DOI:10.2217/fvl.14.95 · 1.00 Impact Factor