Dual Roles for an Arginine-Rich Motif in Specific Genome Recognition and Localization of Viral Coat Protein to RNA Replication Sites in Flock House Virus-Infected Cells

Venter, Philip A.; Marshall, Dawn; Schneemann, Anette

doi:10.1128/jvi.01780-08

Cited by 36 publications

(45 citation statements)

References 63 publications

(64 reference statements)

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“…In addition, in both Drosophila and BHK21 cells, this distribution is independent of the coat protein, excluding the possibility that coat protein is required for RNA transport or that the RNA is transported inside assembled particles. The apparent movement of progeny RNA from the replication site to the site of translation and assembly in the periphery of the cell presents a departure from our previous model, in which we proposed that coat protein travels from the site of translation to the site of replication in order to retrieve the RNAs for assembly (32). In light of the current data, the latter scenario appears to be less likely.…”

Section: Discussioncontrasting

confidence: 53%

“…Transfected cells were incubated at 28°C for 15 h before being fixed in 0.3% glutaraldehyde and 3% paraformaldehyde (in PBS, 1 mM EGTA, 1 mM MgCl 2 , pH 6.8) for 10 min at room temperature. All indirect immunofluorescence and MitoTracker staining was done as previously reported (32). Monoclonal mouse anti-B2 antibodies were used at a 1:6,000 dilution.…”

Section: Methodsmentioning

confidence: 99%

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Cytoplasmic Granule Formation and Translational Inhibition of Nodaviral RNAs in the Absence of the Double-Stranded RNA Binding Protein B2

Petrillo

Venter

Short

et al. 2013

J Virol

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bFlock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3= end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.T he nodaviruses are a family of positive-strand RNA viruses that naturally infect insects and fish. They have a bipartite genome that contains approximately 4,500 bases and encodes three proteins. The most thoroughly studied nodavirus is the insect virus Flock House virus (FHV), which has served as an important model system to investigate the structural and molecular basis of RNA replication, virus assembly, and inhibition of antiviral host responses (1). The larger of the two genomic RNAs, RNA1 (3.1 kb), encodes the RNAdependent RNA polymerase (RdRp; 112 kDa), which is located on the outer membrane of mitochondria in infected cells (2, 3). Viral RNA synthesis induces so-called spherules (4), i.e., membrane invaginations, which are thought to sequester the replication complexes and double-stranded RNA (dsRNA) intermediates to protect them from RNA silencing, a major antiviral pathway activated in insects upon infection with RNA viruses (5). Further protection from RNA silencing is afforded by FHV protein B2 (11.6 kDa), a dsRNA binding protein that is translated from RNA3 (387 nucleotides), a subgenomic RNA derived from the 3= end of RNA1 (6-8). The capsid protein, protein alpha (43 kDa), is translated from RNA2 (1.4 kb), the second genomic RNA segment.Although a seemingly simple virus, FHV uses a sophisticated regulatory system to control its gene expression. This regulation occurs at several levels and is currently incompletely understo...

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Section: Discussioncontrasting

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Cytoplasmic Granule Formation and Translational Inhibition of Nodaviral RNAs in the Absence of the Double-Stranded RNA Binding Protein B2

Petrillo

Venter

Short

et al. 2013

J Virol

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“…Consistent with the electrostatic interaction driving assembly, previous work also showed that deletions of the basic residues in the CP results in reduced packaging of viral RNA. 51,52 Although L-Glu carries a negative charge at neutral pH, it did not serve as substrate for successful CLP assembly. In previous studies using 12mer and 6mer oligonucleotides, CLPs were also not formed.…”

Section: ■ Discussionmentioning

confidence: 99%

The Packaging of Different Cargo into Enveloped Viral Nanoparticles

Fan

Tsvetkova²,

Khuong³

et al. 2012

Mol. Pharmaceutics

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Viral nanoparticles used for biomedical applications must be able to discriminate between tumor or virus-infected host cells and healthy host cells. In addition, viral nanoparticles must have the flexibility to incorporate a wide range of cargo, from inorganic metals to mRNAs to small molecules. Alphaviruses are a family of enveloped viruses for which some species are intrinsically capable of systemic tumor targeting. Alphavirus virus-like particles, or viral nanoparticles, can be generated from in vitro self-assembled core-like particles using nonviral nucleic acid. In this work, we expand on the types of cargo that can be incorporated into alphavirus core-like particles and the molecular requirements for packaging this cargo. We demonstrate that different core-like particle templates can be further enveloped to form viral nanoparticles that are capable of cell entry. We propose that alphaviruses can be selectively modified to create viral nanoparticles for biomedical applications and basic research.

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“…For example, deletion of C-terminal residues 382 to 407 resulted in packaging large amounts of random cellular RNA and various amounts of viral RNA (35), while deletion of N-terminal residues 2 to 31 affected the packaging efficiency of F2 but not F1 (26). Analysis that is more recent showed that the packaging of F1 is determined by an N-terminal arginine-rich motif (ARM) region located between positions 32 and 50 (43). Based on these observations in conjunction with confocal microscopic analyses, it was proposed that an ARM is required for the trafficking of CP to mitochondria where FHV RNA replication occurs actively (43).…”

mentioning

confidence: 99%

“…Analysis that is more recent showed that the packaging of F1 is determined by an N-terminal arginine-rich motif (ARM) region located between positions 32 and 50 (43). Based on these observations in conjunction with confocal microscopic analyses, it was proposed that an ARM is required for the trafficking of CP to mitochondria where FHV RNA replication occurs actively (43). Despite these advances, the underlying molecular mechanism regulating genomepackaging specificity is not well understood.…”

mentioning

confidence: 99%

A Physical Interaction between Viral Replicase and Capsid Protein Is Required for Genome-Packaging Specificity in an RNA Virus

Seo

Kwon

Rao

2012

J Virol

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Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans ( Poliovirus ), insects ( Flock house virus [FHV]), and plants ( Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV- Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.

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Dual Roles for an Arginine-Rich Motif in Specific Genome Recognition and Localization of Viral Coat Protein to RNA Replication Sites in Flock House Virus-Infected Cells

Cited by 36 publications

References 63 publications

Cytoplasmic Granule Formation and Translational Inhibition of Nodaviral RNAs in the Absence of the Double-Stranded RNA Binding Protein B2

Cytoplasmic Granule Formation and Translational Inhibition of Nodaviral RNAs in the Absence of the Double-Stranded RNA Binding Protein B2

The Packaging of Different Cargo into Enveloped Viral Nanoparticles

A Physical Interaction between Viral Replicase and Capsid Protein Is Required for Genome-Packaging Specificity in an RNA Virus

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