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: 9.34). 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.

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    • "Moreover, membranes may also provide key lipids or protein cofactors for activation of the viral replicase. Accordingly, special membrane invaginations, called spherules, consisting of lipid membranes bended inward that contain viral replication proteins and recruited host proteins, have been documented for several (þ )RNA viruses (Barajas et al., 2009, 2014; de Castro et al., 2013; Kopek et al., 2007; McCartney et al., 2005; Schwartz et al., 2002). These viral-induced spherules are the sites of viral replication. "
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    • "numerous vesicles of about 50 nm in diameter, which might connect to an internal lumen or to the cytoplasm through a neck as shown for other viruses belonging to different families. For example, a study of the vesicles induced by Flock house virus (FHV) in mitochondria revealed the presence of the virus replication protein within similar vesicles, called there " spherules, " and the connection to the outside through a channel of sufficient diameter to allow the transit of nucleotides or newly synthesized RNA or both (Kopek et al. 2007). "
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    • "The larger segment, RNA1, encodes the RNA-dependent RNA polymerase (RdRp), which establishes replication complexes on the surface of mitochondria (Miller and Ahlquist, 2002; Miller et al., 2001). More specifically, RNA synthesis occurs in so-called spherules, which represent invaginations of the outer membrane of the organelle (Kopek et al., 2007). The smaller genome segment, RNA2, encodes capsid protein alpha, which co-packages one molecule of RNA1 and RNA2 into progeny particles that have T ¼3 icosahedral symmetry (Fisher and Johnson, 1993; Friesen and Rueckert, 1981; Krishna and Schneemann, 1999). "
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