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


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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Available from: Benjamin G Kopek, Oct 03, 2015
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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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    ABSTRACT: Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation and enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells.
    Virology 12/2014; 471:72-80. DOI:10.1016/j.virol.2014.10.005 · 3.32 Impact Factor
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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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    ABSTRACT: Nodaviruses are icosahedral viruses with a bipartite, positive-sense RNA genome. The two RNAs are packaged into a single virion by a poorly understood mechanism. We chose two distantly related nodaviruses, Flock House virus and Nodamura virus, to explore formation of viral reassortants as a means to further understand genome recognition and encapsidation. In mixed infections, the viruses were incompatible at the level of RNA replication and their coat proteins segregated into separate populations of progeny particles. RNA packaging, on the other hand, was indiscriminate as all four viral RNAs were detectable in each progeny population. Consistent with the trans-encapsidation phenotype, fluorescence in situ hybridization of viral RNA revealed that the genomes of the two viruses co-localized throughout the cytoplasm. Our results imply that nodaviral RNAs lack rigorously defined packaging signals and that co-encapsidation of the viral RNAs does not require a pair of cognate RNA1 and RNA2.
    Virology 04/2014; s 454–455(1):280–290. DOI:10.1016/j.virol.2014.03.003 · 3.32 Impact Factor
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    • "Previous studies have revealed that FHV protein A contains the conserved glycine-aspartate-aspartate (GDD) box that is present in all nodaviruses and strictly required for RNA replication by all known RdRPs of (+)-RNA viruses [22]. It has also been reported that FHV protein A replicates viral RNA in concert with the mitochondrial outer membrane and other viral or cellular factors [23], [24], [25], [26], [27], and mediates the formation of viral RNA replication complexes and small spherules by inducing membrane rearrangement [24], [26]. "
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    ABSTRACT: Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3'-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3' initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.
    PLoS ONE 01/2014; 9(1):e86876. DOI:10.1371/journal.pone.0086876 · 3.23 Impact Factor
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