Flock House Virus B2 Research Articles

Subcellular Colocalization Assay of Host Factors with Viral Replication Complex in the dsRNA Reporter Nicotiana benthamiana.

As obligate pathogens, plant viruses co-opt several host factors for viral replication. Double-stranded RNA (dsRNA) plays important roles in plants, including eliciting innate immune responses and RNA interference. dsRNA also represents the genetic entities of a number of viruses and is a marker of infection by positive-sense single-stranded RNA viruses. Previous detection methods for RNA viruses basically relied on immunological methods, but later researchers discovered that the dsRNA-binding domain of the Flock house virus B2 protein is a perfect alternative to the J2 mAb for sensitive and rapid detection of long dsRNA in vitro and in vivo, and developed B2:GFP transgenic Nicotiana benthamiana line. This method describes in detail how to visualize host factors in the viral replication complex in time and space with the help of B2:GFP transgenic plants, exemplified by Turnip mosaic virus (TuMV), a representative virus member of the Potyviruses.

Inhibition of dicer activity in lepidopteran and dipteran cells by baculovirus-mediated expression of Flock House virus B2

Prior studies have suggested that insect DNA viruses are negatively affected by dicer-2-mediated RNA interference (RNAi). To examine this further, we utilized an in vitro assay to measure dicer activity in lepidopteran and dipteran cells, combined with baculoviruses expressing the RNAi suppressor B2 from Flock House virus or Aedes aegypti dicer-2 (Aedicer-2) using a constitutive heat shock promoter. Addition of cell lysates containing baculovirus-expressed B2 to lysates from dipteran (S2, Aag2) or lepidopteran (Sf9) cells inhibited endogenous dicer activity in a dose-dependent manner, while expression of Aedicer-2 restored siRNA production in Ae. albopictus C6/36 cells, which are dicer-2 defective. However, B2 expression from the constitutive heat shock promoter had no impact on baculovirus replication or virulence in cell lines or larvae that were either highly permissive (Trichoplusia ni) or less susceptible (Spodoptera frugiperda) to infection. We determined that this constitutive level of B2 expression had little to no ability to suppress dicer activity in cell lysates, but higher expression of B2, following heat shock treatment, inhibited dicer activity in all cells tested. Thus, we cannot rule out the possibility that optimized expression of B2 or other RNAi suppressors may increase baculovirus replication and expression of heterologous proteins by baculoviruses.

The cricket paralysis virus suppressor inhibits microRNA silencing mediated by the Drosophila Argonaute-2 protein.

Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway.

Crystallization and preliminary crystallographic analysis of a viral RNA-silencing suppressor encoded byWuhan nodavirus

Wuhan nodavirus (WhNV), which is a new member of the Nodaviridae family, encodes a viral protein, B2, that suppresses RNA silencing and host-cell RNA interference (RNAi)-mediated immunity. Although Flock House virus (FHV), another member of the Nodaviridae family, also produces a B2 protein with a similar function, the primary sequences of the B2 proteins from WhNV and FHV have no similarity. To gain a better understanding of the structural details and the mechanism of suppression of RNA silencing by WhNV B2 and to compare it with FHV B2, recombinant WhNV B2 protein has been overexpressed in Escherichia coli, purified and crystallized at 291 K using PEG 4000 as a precipitant. A 2.8 Å resolution data set has been collected from a single crystal at 100 K. This crystal belonged to space group P2₁2₁2₁, with unit-cell parameters a=27.3, b=45.6, c=133.9 Å, α=β=γ=90°. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient is 2.2 Å3 Da(-1).

Characterization of Virus-Encoded RNA Interference Suppressors in Caenorhabditis elegans

In fungi, plants, and invertebrates, antiviral RNA interference (RNAi) directed by virus-derived small interfering RNAs (siRNAs) represents a major antiviral defense that the invading viruses have to overcome in order to establish infection. As a counterdefense mechanism, viruses of these hosts produce diverse classes of proteins capable of suppressing the biogenesis and/or function of viral siRNAs. This RNA-directed viral immunity (RDVI) in the nematode Caenorhabditis elegans is known to exhibit some unique features. Currently, little is known about viral suppression of RNAi in C. elegans. Here, we show that ectopic expression of the B2 protein encoded by Flock House virus (FHV) suppresses RNAi induced by either long double-stranded RNA (dsRNA) or an FHV-based replicon and facilitates the natural infection of C. elegans by Orsay virus but is not active against RNA silencing mediated by microRNAs. We report the development of an assay for the identification of viral suppressor of RNAi (VSR) in C. elegans based on the suppression of a viral replicon-triggered RDVI by ectopic expression of candidate proteins. No VSR activity was detected for either of the two Orsay viral proteins proposed previously as VSRs. We detected, among the known heterologous VSRs, VSR activity for B2 of Nodamura virus but not for 2b of tomato aspermy virus, p29 of fungus-infecting hypovirus, or p19 of tomato bushy stunt virus. We further show that, unlike that in plants and insects, FHV B2 suppresses worm RDVI mainly by interfering with the function of virus-derived primary siRNAs.

Transgene‐mediated suppression of the RNA interference pathway in Aedes aegypti interferes with gene silencing and enhances Sindbis virus and dengue virus type 2 replication

RNA interference (RNAi) is the major innate antiviral pathway in Aedes aegypti that responds to replicating arboviruses such as dengue virus (DENV) and Sindbis virus (SINV). On the one hand, the mosquito's RNAi machinery is capable of completely eliminating DENV2 from Ae. aegypti. On the other, transient silencing of key genes of the RNAi pathway increases replication of SINV and DENV2, allowing the viruses to temporally overcome dose-dependent midgut infection and midgut escape barriers (MEB) more efficiently. Here we expressed Flock house virus B2 (FHV-B2) from the poly-ubiquitin (PUb) promoter in Ae. aegypti using the ΦC31 site-directed recombination system to investigate the impact of transgene-mediated RNAi pathway suppression on infections with SINV-TR339eGFP and DENV2-QR94, the latter of which has been shown to be confronted with a strong MEB in Ae. aegypti. FHV-B2 was constitutively expressed in midguts of sugar- and blood-fed mosquitoes of transgenic line PUbB2 P61. B2 over-expression suppressed RNA silencing of carboxypeptidase A-1 (AeCPA-1) in midgut tissue of PUbB2 P61 mosquitoes. Following oral challenge with SINV-TR339eGFP or DENV2-QR94, mean titres in midguts of PUbB2 P61 females were significantly higher at 7 days post-bloodmeal (pbm) than in those of nontransgenic control mosquitoes. At 14 days pbm, infection rates of carcasses were significantly increased in PubB2 P61 mosquitoes infected with SINV-TR339eGFP. Following infection with DENV2-QR94, midgut infection rates were significantly increased in the B2-expressing mosquitoes at 14 days pbm. However, B2 expression in PUbB2 P61 did not increase the DENV2-QR94 dissemination rate, indicating that the infection phenotype was not primarily controlled by RNAi.

Comparison of transgene expression in Aedes aegypti generated by mariner Mos1 transposition and ΦC31 site‐directed recombination

Transgenic mosquitoes generated by transposable elements (TEs) often poorly express transgenes owing to position effects. To avoid these effects, the ΦC31 site-directed recombination system was used to insert transgenes into a locus favourable for gene expression in Aedes aegypti. We describe phenotypes of mariner Mos1 TE and ΦC31 transgenic mosquitoes expressing the enhanced green fluorescent protein (EGFP) reporter in midguts of blood-fed females. Mosquitoes of nine TE-generated lines [estimated transformation frequency (TF): 9.3%] clearly expressed the eye-specific selection marker but only 2/9 lines robustly expressed the EGFP reporter. The piggyBac TE-generated ΦC31 docking strain, attP26, supported recombination with attB site containing donors at an estimated TF of 1.7-4.9%. Using a codon-optimized ΦC31 integrase mutant instead of the 'wild-type' enzyme did not affect TF. Site-directed recombination of line attP26 with an attB-containing donor expressing EGFP from the Ae. aegypti carboxypeptidase promoter produced one transgenic line with blood-fed females expressing the reporter in midgut tissue. Docking strain attP26 also supported robust expression of Flock House virus B2 from the Ae. aegypti polyubiquitin promoter. Our data confirm that eye-specific selection marker expression alone is not a reliable indicator for robust gene-of-interest expression in Ae. aegypti and that the ΦC31 system can ensure predictable transgene expression in this mosquito species.

Structure of the RNA-Binding Domain of Nodamura Virus Protein B2, a Suppressor of RNA Interference,

Protein B2 from Nodamura virus (NMV B2), a member of the Nodavirus family, acts as a suppressor of RNA interference (RNAi). The N-terminal domain of NMV B2, consisting of residues 1-79, recognizes double-stranded RNA (dsRNA). The 2.5 A crystal structure of the RNA-binding domain of NMV B2 shows a dimeric, helical bundle structure. The structure shows a conserved set of RNA-binding residues compared with flock house virus B2, despite limited sequence identity. The crystal packing places the RNA-binding residues along one face of symmetry-related molecules, suggesting a potential platform for recognition of dsRNA.

Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer

RNA silencing is a conserved pathway that functions as an antiviral mechanism. The majority of viruses encode silencing suppressors that interfere with siRNA- and miRNA-guided silencing pathways. The insect flock house virus B2 protein (FHVB2) functions as an RNAi silencing suppressor that inhibits siRNA biogenesis. Here, we describe the generation of a GFP silent sensor line (Sf21) and a GFP sensor line expressing FHVB2 to study RNAi suppression mechanisms. Overexpression of FHVB2 resulted in suppression of GFP-RNAi and resumption of GFP expression. Protein fractionation studies with FHVB2-transfected cells showed that FHVB2 associates with a high-molecular-weight complex of Dicer and dsRNA/siRNAs. Yeast two-hybrid and pulldown assays revealed an interaction between FHVB2 and Drosophila Dicer proteins that appeared to involve PAZ domains. To map the FHVB2 domains interacting with Dicer, we used a 17-residue C-terminal deletion mutant. RNAi suppression was reversed in cells transfected with the FHVB2 mutant as revealed by loss of GFP. Additional yeast two-hybrid and in vitro pulldown assays confirmed that the C-terminal region of FHVB2 was involved in the interaction with the PAZ domains of Dicers. These results thus reveal a novel interaction between FHVB2 and Dicer that leads to suppression of siRNA biogenesis.

Structural basis for RNA‐silencing suppression by Tomato aspermy virus protein 2b

The 2b proteins encoded by cucumovirus act as post-transcriptional gene silencing suppressors to counter host defence during infection. Here we report the crystal structure of Tomato aspermy virus 2b (TAV2b) protein bound to a 19 bp small interfering RNA (siRNA) duplex. TAV2b adopts an all alpha-helix structure and forms a homodimer to measure siRNA duplex in a length-preference mode. TAV2b has a pair of hook-like structures to recognize simultaneously two alpha-helical turns of A-form RNA duplex by fitting its alpha-helix backbone into two adjacent major grooves of siRNA duplex. The conserved pi-stackings between tryptophan and the 5'-terminal base of siRNA duplex from both ends enhance the recognition. TAV2b further oligomerizes to form a dimer of dimers through the conserved leucine-zipper-like motif at its amino-terminal alpha-helix. Biochemical experiments suggest that TAV2b might interfere with the post-transcriptional gene silencing pathway by directly binding to siRNA duplex.

Recent insights into the biology and biomedical applications of Flock House virus.

Flock House virus (FHV) is a nonenveloped, icosahedral insect virus whose genome consists of two molecules of single-stranded, positive-sense RNA. FHV is a highly tractable system for studies on a variety of basic aspects of RNA virology. In this review, recent studies on the replication of FHV genomic and subgenomic RNA are discussed, including a landmark study on the ultrastructure and molecular organization of FHV replication complexes. In addition, we show how research on FHV B2, a potent suppressor of RNA silencing, resulted in significant insights into antiviral immunity in insects. We also explain how the specific packaging of the bipartite genome of this virus is not only controlled by specific RNA-protein interactions but also by coupling between RNA replication and genome recognition. Finally, applications for FHV as an epitopepresenting system are described with particular reference to its recent use for the development of a novel anthrax antitoxin and vaccine.

Identification of critical residues in nervous necrosis virus B2 for dsRNA-binding and RNAi-inhibiting activity through by bioinformatic analysis and mutagenesis

It is known that the non-structural B2 protein of nervous necrosis virus (NNV) plays an important role in viral replication and can inhibit the RNA interference system of the host cell. Moreover, the mechanism of NNV B2 protein to inhibit RNAi is by sequestration and protection of double strand (ds) RNA. In the flock house virus (FHV), a model alphanodavirus, the structural and mutational analysis of B2 identified that the positively charged Arg54 of the α2 helix mediated the dsRNA-binding activity. According to the betanodavirus B2 protein alignment and modeling results, the amino acid sequences and the predicted structure of betanodavirus B2 are different from alphanodaviruses. It was suggested that the four Arg residues of α3 helix between amino residues 52–60 of B2 may be involved in dsRNA-binding activity. Thus, this study replaced these four Arg residues with Gln at position 52 (R52Q), 53 (R53Q), 59 (R59Q), and 60 (R60Q) by site-directed mutagenesis method. The dsRNA-binding assays of these B2 mutants demonstrated that mB2(R53Q) and mB2(R60Q) mutants are dsRNA-binding defective. Moreover, we have found mB2(R53Q) and mB2(R60Q) could not antagonize RNAi by using HeLa cell as an RNAi inhibition model. These results suggested that Arg53 and Arg60 of betanodavirus B2 protein may be similar to Arg54 of alphanodavirus FHV B2 protein and are critical for dsRNA binding and RNAi-inhibiting. This study may serve as an example where bioinformatic analysis of related viral genomes may lead to meaningful structural and functional clues for certain viral proteins.

The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of double‐stranded RNA recognition

We report the structure of the flock house virus B2 protein, a potent suppressor of RNA interference (RNAi) in animals and plants. The B2 protein is a homodimer in solution and contains three alpha-helices per monomer. Chemical shift perturbation shows that an antiparallel arrangement of helices (alpha2/alpha2') forms an elongated binding interface with double-stranded RNA (dsRNA). This implies a novel mode of dsRNA recognition and provides insights into the mechanism of RNAi suppression by B2.

Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans

The worm Caenorhabditis elegans is a model system for studying many aspects of biology, including host responses to bacterial pathogens, but it is not known to support replication of any virus. Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs and may act cooperatively. However, it is not known whether the single Dicer of worms and mammals is able to initiate the small RNA-guided RNA interference (RNAi) antiviral immunity as occurs in plants and insects. Here we show complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. We show that FHV replication in C. elegans triggers potent antiviral silencing that requires RDE-1, an Argonaute protein essential for RNAi mediated by small interfering RNAs (siRNAs) but not by microRNAs. This immunity system is capable of rapid virus clearance in the absence of FHV B2 protein, which acts as a broad-spectrum RNAi inhibitor upstream of rde-1 by targeting the siRNA precursor. This work establishes a C. elegans model for genetic studies of animal virus-host interactions and indicates that mammals might use a siRNA pathway as an antiviral response.