Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi

Contains fulltext : 136405.pdf (publisher's version ) (Open Access)The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary 'arms race' may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors

Application of bacteriophages EP75 and EP335 efficiently reduces viable cell counts of Escherichia coli O157 on beef and vegetables

Shiga toxin producing Escherichia coli (STEC) are common etiological agents of food borne illnesses and outbreaks, most often caused by consuming contaminated beef products, followed by raw vegetables and dairy products. Patients infected with E. coli O157 are more likely hospitalized than patients infected with non-O157 STEC, making E. coli O157 an important target for microbiological interventions. We show that a cocktail of bacteriophages EP75 and EP335 effectively reduces E. coli O157 on beef, romaine lettuce, spinach, and zucchini. Treatment of contaminated beef with either 2 × 107 or 1 × 108 PFU/cm2 of bacteriophage cocktail EP75/EP335 resulted in reductions of 0.8–1.1 log10 CFU/cm2 and 0.9–1.3 log10 CFU/cm2, respectively (P < 0.0001). Similarly, bacteriophage treatments of contaminated romaine lettuce, zucchini, or spinach showed significant (P < 0.05) E. coli O157 reductions of 0.7–1.9 log10 CFU/cm2 (2 × 107 PFU/cm2), and 1.4–2.4 log10 CFU/cm2 (1 × 108 PFU/cm2). An E. coli O157 reduction of 0.9 log10 and 2.0 log10 was observed already 30 min after phage application of 1 × 108 PFU/cm2 on beef and romaine lettuce, respectively. These data show that bacteriophages EP75 and EP335 can be effectively used as a processing aid on beef and vegetables, and thereby can aid industry to reduce the risk of E. coli O157 food poisoning

Structural and functional characterization of the receptor binding proteins of Escherichia coli O157 phages EP75 and EP335

ISSN:2001-037

VP1 suppressor activity is species-specific.

<p>(<b>A</b>) Western blot analysis of S2 cells expressing V5 epitope-tagged VP1 from <i>D. melanogaster</i> Nora virus (DmelNV) and <i>D. immigrans</i> Nora-like virus (DimmNV). S2 cells were transfected with plasmids encoding full-length VP1 (FL) and C-terminal (ΔC) or N-terminal (ΔN) deletions thereof. Expression of the VP1 constructs was analyzed by western blot using an anti-V5 (α-V5) antibody. Detection of tubulin with anti-tubulin (α-tub) antibody was used as a loading control. Molecular mass (in kDa) is indicated on the left. For DmelNV VP1^ΔN284, bands of lower mobility were observed in addition to the expected 26 kDa protein, the nature of which remains unknown. Note that these additional bands are not consistently observed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256.s002" target="_blank">Figure S2A</a>, lane 5, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256-vanMierlo2" target="_blank">[14]</a>). (<b>B</b>) RNAi sensor assay in S2 cells. Firefly luciferase (Fluc) and <i>Renilla</i> luciferase (Rluc) reporter plasmids were transfected into S2 cells, together with plasmids encoding the indicated VP1 constructs. Two days after transfection, S2 cells were soaked in either control (Ctrl) dsRNA or Fluc dsRNA, and luciferase activities were measured the next day. Fluc counts were normalized to Rluc counts, and presented as fold silencing relative to the corresponding control dsRNA treatment. (<b>C</b>) Hairpin-based RNAi sensor assay in S2 cells. S2 cells were transfected with plasmids coding for Fluc, Rluc, and an Rluc-hairpin RNA together with a control vector (Vector) or plasmids encoding the N-terminal deletion mutants of DmelNV VP1^ΔN284 or DimmNV VP1^ΔN295. Rluc counts were normalized to Fluc counts, and presented as fold silencing over non-hairpin control transfections. Bars in Panels B and C represent means and standard deviations of three independent biological replicates. One-way ANOVA followed by Dunnett's <i>post hoc</i> test was used to evaluate whether VP1 constructs significantly suppressed RNAi relative to the vector control (light gray bar). ** <i>P</i><0.01; *** <i>P</i><0.001; ns, not significant.</p

Species-specific inhibition of AGO2 slicer activity.

<p>(<b>A</b>) <i>In vitro</i> RNA cleavage (slicer) assays in lysates from <i>D. melanogaster</i> embryos (left panel) or <i>D. immigrans</i> embryos (right panel). Radioactively cap-labelled target RNA was incubated in embryo lysate together with a non-specific control siRNA (lanes 1 and 6) or a target specific siRNA (lanes 2–5, 7–10). Target cleavage was determined either in the absence of recombinant protein (lanes 2 and 7) or in the presence of 0.3 µM of MBP (lanes 3 and 8), MBP-DmelNV VP1 (lanes 4 and 9), or DimmNV VP1 (lanes 5 and 10). (<b>B</b>) Quantification of target cleavage in <i>D. melanogaster</i> and <i>D. immigrans</i> embryo lysate in the presence of MBP, DmelNV VP1, or DimmNV VP1 protein. The fraction of cleaved RNA was determined by dividing the intensity of the cleavage product by the total intensity of cleavage product and non-cleaved target. Data are normalized to MBP. Bars represent means and standard deviations of two independent experiments.</p

VP1 suppressor activity is species-specific.

<p>(<b>A</b>) Western blot analysis of S2 cells expressing V5 epitope-tagged VP1 from <i>D. melanogaster</i> Nora virus (DmelNV) and <i>D. immigrans</i> Nora-like virus (DimmNV). S2 cells were transfected with plasmids encoding full-length VP1 (FL) and C-terminal (ΔC) or N-terminal (ΔN) deletions thereof. Expression of the VP1 constructs was analyzed by western blot using an anti-V5 (α-V5) antibody. Detection of tubulin with anti-tubulin (α-tub) antibody was used as a loading control. Molecular mass (in kDa) is indicated on the left. For DmelNV VP1^ΔN284, bands of lower mobility were observed in addition to the expected 26 kDa protein, the nature of which remains unknown. Note that these additional bands are not consistently observed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256.s002" target="_blank">Figure S2A</a>, lane 5, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256-vanMierlo2" target="_blank">[14]</a>). (<b>B</b>) RNAi sensor assay in S2 cells. Firefly luciferase (Fluc) and <i>Renilla</i> luciferase (Rluc) reporter plasmids were transfected into S2 cells, together with plasmids encoding the indicated VP1 constructs. Two days after transfection, S2 cells were soaked in either control (Ctrl) dsRNA or Fluc dsRNA, and luciferase activities were measured the next day. Fluc counts were normalized to Rluc counts, and presented as fold silencing relative to the corresponding control dsRNA treatment. (<b>C</b>) Hairpin-based RNAi sensor assay in S2 cells. S2 cells were transfected with plasmids coding for Fluc, Rluc, and an Rluc-hairpin RNA together with a control vector (Vector) or plasmids encoding the N-terminal deletion mutants of DmelNV VP1^ΔN284 or DimmNV VP1^ΔN295. Rluc counts were normalized to Fluc counts, and presented as fold silencing over non-hairpin control transfections. Bars in Panels B and C represent means and standard deviations of three independent biological replicates. One-way ANOVA followed by Dunnett's <i>post hoc</i> test was used to evaluate whether VP1 constructs significantly suppressed RNAi relative to the vector control (light gray bar). ** <i>P</i><0.01; *** <i>P</i><0.001; ns, not significant.</p

Species-specific interaction between VP1 and AGO2.

<p>(<b>A</b>) V5 Immunoprecipitation (V5-IP) of lysates from S2 cells transfected with FLAG-tagged Dmel AGO2 expression plasmid and either V5-tagged DmelNV VP1, DimmNV VP1, or V5-control plasmids (−). Input, supernatant after immunoprecipitation (Sup), and the immunoprecipitate (V5-IP) were analyzed by western blot (WB) using anti-V5 (α-V5) or anti-FLAG (α-FLAG) antibodies. (<b>B</b>) V5 immunoprecipitation of S2 cells transfected with plasmids encoding V5-tagged DmelNV VP1, DimmNV VP1, or V5-control vector (−). Input, sup, and IP fractions were analyzed by western blot using antibodies for endogenous AGO2 (α-Dmel AGO2) and V5 (α-V5). (<b>C</b>) V5 immunoprecipitation on lysates from S2 cells co-transfected with plasmids encoding FLAG-tagged Dimm AGO2 and either V5-tagged DmelNV VP1, DimmNV VP1, or V5-control vector (−). VP1 and Dimm AGO2 proteins were detected on western blot using anti-V5 (α-V5) and anti-FLAG (α-FLAG) antibodies, respectively. Asterisks (*) indicate a non-specific background band; triangles indicate AGO2. For these experiments the corresponding DmelNV VP1^ΔN284 and DimmNV VP1^ΔN295 constructs were used (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004256#ppat.1004256.s001" target="_blank">Figure S1</a>).</p

Nora virus VP1 suppresses RNAi <i>in vitro</i>.

<p>(<b>A</b>) Schematic representation of the Nora virus genome with its four predicted ORFs in three different reading frames. There is a 7-nt overlap between ORF1 and ORF2 and a 26-nt overlap between ORF2 with ORF3. An intergenic region of 85 nt separates ORF3 and ORF4. (<b>B</b>) Western blot analysis of V5-epitope tagged Nora virus expression constructs. Two days after transfection of the indicated plasmids into S2 cells, expression of the constructs was analyzed by Western blot using the V5 antibody (αV5). Asterisks (*) indicate additional bands that do not correspond to the expected size of the full-length protein product. (<b>C</b>) RNAi reporter assay in <i>Drosophila</i> S2 cells. Copper-inducible plasmids encoding Fluc and Rluc were transfected into S2 cells together with a construct expressing Nora virus ORF1, 3, and 4, encoding viral protein 1 (VP1), VP3, and VP4, respectively. Two days after transfection, dsRNA targeting Fluc or GFP (Ctrl) was added to the medium. Seven hours later, expression of FLuc and Rluc was induced and luciferase activity was measured the next day. FLuc counts were normalized to Rluc counts and presented as fold silencing relative to the control GFP dsRNA. Plasmids encoding DCV 1A and the K73A mutant (DCV 1A mut) were used as controls. (<b>D</b>) siRNA-based RNAi reporter assay. The experiment was performed as described in panel C, but 21-nt Fluc siRNAs were cotransfected with the reporter plasmids to silence gene expression. An siRNA targeting the human MDA5 gene was used as a non-silencing control (Ctrl). Bars in panel C represent averages and standard deviations of five independent samples; bars in panel D represent averages and standard deviations of three independent samples. Panel C and D are representative for two and three independent experiments, respectively.</p