Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

Wu, Wenzhe; Wang, Zhaowei; Xia, Hongjie; Liu, Yongxiang; Qiu, Yang; Liu, Yujie; Hu, Yuanyang; Zhou, Xi

doi:10.1371/journal.pone.0086876

Cited by 13 publications

(16 citation statements)

References 65 publications

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“…2B). The Mn 2ϩ value is in agreement with previous biochemical characterizations (27,28), while the Mg 2ϩ concentration was similar to the amount previously used in cell extracts (26). Specific radioactivity (counts per minute per nanogram of RNA) of total RNA generated in the presence of 18 mM Mg 2ϩ or 1 mM Mn 2ϩ ( Fig.…”

Section: Resultssupporting

confidence: 89%

“…Optimization of FHV RNA replication system. We optimized a FHV RNA replication system described in two previous reports: the first outlined a cell-free replication system consisting of a lysate generated from FHV-infected Drosophila cells (26), and the second described the biochemical characterization of a protein A-maltose binding protein (MBP) fusion construct expressed in Escherichia coli (27). Our assay relied on isolation of crude mitochondria from FHV-infected Drosophila S2 cells by differential centrifugation.…”

Section: Resultsmentioning

confidence: 99%

“…Of interest during the optimization of this system was a discrepancy in the literature regarding the divalent cation required for optimal protein A activity. Two previous studies had reported reaction conditions for FHV replication: the first included 15 mM Mg 2ϩ in reactions using cell extracts (26,34) in contrast to the 1 mM Mn 2ϩ suggested by a more recent biochemical study performed using a purified protein A-MBP fusion protein (27). Our analysis suggests that, whereas protein A is able to generate the same RNA species in the presence of either metal ion, inclusion of magnesium in reaction mixtures results in the production of significantly more progeny RNA (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Short

Speir

Gopal

et al. 2016

J Virol

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Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ϳ50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single-and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Short

Speir

Gopal

et al. 2016

J Virol

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“…Localization to more than one site, possibly in alternate multimeric forms (Dye et al, 2005a), could be related to protein A’s many roles in spherule formation, negative- and positive-strand genomic RNA synthesis, sgRNA3 synthesis, and RNA capping (Ball, 1995; Eckerle et al, 2003; Johnson and Ball, 1999; Kopek et al, 2010; Lindenbach et al, 2002; Wu et al, 2014). Estimates of the number of spherules per cell also suggested a ratio of up to 100 protein A molecules per spherule, raising the possibility that protein A might form a lattice within spherules (Kopek et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

Cryo-electron tomography reveals novel features of a viral RNA replication compartment

Ertel

Benefield

Castaño-Díez

et al. 2017

eLife

133

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Positive-strand RNA viruses, the largest genetic class of viruses, include numerous important pathogens such as Zika virus. These viruses replicate their RNA genomes in novel, membrane-bounded mini-organelles, but the organization of viral proteins and RNAs in these compartments has been largely unknown. We used cryo-electron tomography to reveal many previously unrecognized features of Flock house nodavirus (FHV) RNA replication compartments. These spherular invaginations of outer mitochondrial membranes are packed with electron-dense RNA fibrils and their volumes are closely correlated with RNA replication template length. Each spherule’s necked aperture is crowned by a striking cupped ring structure containing multifunctional FHV RNA replication protein A. Subtomogram averaging of these crowns revealed twelve-fold symmetry, concentric flanking protrusions, and a central electron density. Many crowns were associated with long cytoplasmic fibrils, likely to be exported progeny RNA. These results provide new mechanistic insights into positive-strand RNA virus replication compartment structure, assembly, function and control.DOI: http://dx.doi.org/10.7554/eLife.25940.001

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“…The expression and purification of recombinant WhNV protein A and its derivatives were carried out as previously described Wu et al, 2014). Briefly, to obtain soluble recombinant protein, MBP-tagged FL protein A and its mutants as well as the negative control protein MBP were expressed in E. coli strain TB1 at 20 1C in the presence of 0.2 mM IPTG.…”

Section: Purification Of Protein a And Its Derivativesmentioning

confidence: 99%

The RNA binding of protein A from Wuhan nodavirus is mediated by mitochondrial membrane lipids

et al. 2014

Self Cite

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RNA replication of positive-strand (+)RNA viruses requires the lipids present in intracellular membranes, the sites of which viral replicases associate with. However, the direct effects of membrane lipids on viral replicases are still poorly understood. Wuhan nodavirus (WhNV) protein A, which associates with mitochondrial membranes, is the sole replicase required for RNA replication. Here, we report that WhNV protein A binds to RNA1 in a cooperative manner. Moreover, mitochondrial membrane lipids (MMLs) stimulated the RNA binding activity and cooperativity of protein A, and such stimulations exhibited strong selectivity for distinct phospholipids. Interestingly, MMLs stimulated the RNA-binding cooperativity only at higher protein A concentrations. Further investigation showed that MMLs stimulate the RNA binding of protein A by promoting its self-interaction. Finally, manipulating MML metabolism affected the protein A-induced RNA1 recruitment in cells. Together, our findings reveal the direct effects of membrane lipids on the RNA binding activity of a nodaviral replicase.

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Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

Cited by 13 publications

References 65 publications

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Cryo-electron tomography reveals novel features of a viral RNA replication compartment

The RNA binding of protein A from Wuhan nodavirus is mediated by mitochondrial membrane lipids

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