Limits...
A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response.

Bronkhorst AW, van Cleef KW, Venselaar H, van Rij RP - Nucleic Acids Res. (2014)

Bottom Line: Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R.Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs).We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

Show MeSH

Related in: MedlinePlus

Viral protein 340R requires a functional dsRBD to suppress RNAi. (A) Alignment of the dsRBD of 340R (aa 30–100) to the dsRBDs of proteins from different model organisms and to the dsRBD of DCV 1A. Red boxes indicate residues that interact with dsRNA in structural analyses of the second dsRBD of Xenopus laevis RNA-binding protein A (Xl RBPA2) (56). Residues predicted to be involved in RNA binding or conserved amino acids were selected for site-directed mutagenesis. Position of secondary structures are indicated above the alignment. (B) Homology model of 340R in complex with dsRNA. The protein is shown in cartoon-view with the WT residue K89 shown in red (left panel) or with the WT residue shown in green and the mutant residue (Alanine) shown in red (right panel). The RNA is shown in ball-and-stick view, without atomic details (left) and with all atoms (right). The side-chain of residue K89 is positioned toward the phosphate backbone of the RNA and likely binds dsRNA through an electrostatic interaction of the positively charged Lysine with the negatively charged phosphates. The substitution of this Lysine into the small and hydrophobic Alanine is likely to abolish the interaction. See Supplementary Figure S2 for other residues selected for site-directed mutagenesis. (C) Western blot analysis of V5 epitope-tagged WT and mutant 340R from transfected S2 cells. Proteins were detected using anti-V5 antibodies (α-V5) or, as a loading control, using anti-α-tubulin (α-Tub) antibodies. Molecular mass (in kDa) is indicated on the left of the image. The predicted molecular weight for dsRBD100 is 14.7 kDa. FL, full-length. The asterisk (*) indicates a 340R-specific processing or degradation product. (D) dsRNA-induced RNAi reporter assay. The experiment was performed as described in the legend to Figure 1B, using expression plasmids for WT and mutant 340R. (E) siRNA-induced RNAi reporter assay. Sequential transfection was performed as described in the legend to Figure 1E, using expression plasmids for WT and mutant 340R and CrPV 1A. Difference in RNAi efficiency compared to controls (dark gray bars) was analyzed by one-way ANOVA followed by a post hoc Dunnett's test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231766&req=5

Figure 2: Viral protein 340R requires a functional dsRBD to suppress RNAi. (A) Alignment of the dsRBD of 340R (aa 30–100) to the dsRBDs of proteins from different model organisms and to the dsRBD of DCV 1A. Red boxes indicate residues that interact with dsRNA in structural analyses of the second dsRBD of Xenopus laevis RNA-binding protein A (Xl RBPA2) (56). Residues predicted to be involved in RNA binding or conserved amino acids were selected for site-directed mutagenesis. Position of secondary structures are indicated above the alignment. (B) Homology model of 340R in complex with dsRNA. The protein is shown in cartoon-view with the WT residue K89 shown in red (left panel) or with the WT residue shown in green and the mutant residue (Alanine) shown in red (right panel). The RNA is shown in ball-and-stick view, without atomic details (left) and with all atoms (right). The side-chain of residue K89 is positioned toward the phosphate backbone of the RNA and likely binds dsRNA through an electrostatic interaction of the positively charged Lysine with the negatively charged phosphates. The substitution of this Lysine into the small and hydrophobic Alanine is likely to abolish the interaction. See Supplementary Figure S2 for other residues selected for site-directed mutagenesis. (C) Western blot analysis of V5 epitope-tagged WT and mutant 340R from transfected S2 cells. Proteins were detected using anti-V5 antibodies (α-V5) or, as a loading control, using anti-α-tubulin (α-Tub) antibodies. Molecular mass (in kDa) is indicated on the left of the image. The predicted molecular weight for dsRBD100 is 14.7 kDa. FL, full-length. The asterisk (*) indicates a 340R-specific processing or degradation product. (D) dsRNA-induced RNAi reporter assay. The experiment was performed as described in the legend to Figure 1B, using expression plasmids for WT and mutant 340R. (E) siRNA-induced RNAi reporter assay. Sequential transfection was performed as described in the legend to Figure 1E, using expression plasmids for WT and mutant 340R and CrPV 1A. Difference in RNAi efficiency compared to controls (dark gray bars) was analyzed by one-way ANOVA followed by a post hoc Dunnett's test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.

Mentions: The IIV-6 340R gene encodes a 23-kDa protein that contains a 70-aa dsRBD flanked by a 30-aa N-terminal sequence and a 73-aa C-terminal sequence. Alignment of the the dsRBD of 340R to the dsRBDs of DCV 1A and cellular proteins from different model organisms shows that conserved amino acids are present throughout the motif (Figure 2A). Homology modeling suggests that the dsRBD of 340R adopts the expected αβββα topology, in which two α helices are packed along a three-stranded antiparallel β sheet, and that the dsRBD is preceded by an N-terminal helical structure (Figure 2B). Based on these analyses, we selected for site-directed mutagenesis four highly conserved residues (L35Y, F63A and AA92LL) and two residues within a region expected to interact with dsRNA (K86A and K89A) (Figure 2A and B and Supplementary Figure S2). In addition, we generated a C-terminally truncated version of 340R, consisting of the N-terminal 100-aa that contains the complete dsRBD (dsRBD100).


A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response.

Bronkhorst AW, van Cleef KW, Venselaar H, van Rij RP - Nucleic Acids Res. (2014)

Viral protein 340R requires a functional dsRBD to suppress RNAi. (A) Alignment of the dsRBD of 340R (aa 30–100) to the dsRBDs of proteins from different model organisms and to the dsRBD of DCV 1A. Red boxes indicate residues that interact with dsRNA in structural analyses of the second dsRBD of Xenopus laevis RNA-binding protein A (Xl RBPA2) (56). Residues predicted to be involved in RNA binding or conserved amino acids were selected for site-directed mutagenesis. Position of secondary structures are indicated above the alignment. (B) Homology model of 340R in complex with dsRNA. The protein is shown in cartoon-view with the WT residue K89 shown in red (left panel) or with the WT residue shown in green and the mutant residue (Alanine) shown in red (right panel). The RNA is shown in ball-and-stick view, without atomic details (left) and with all atoms (right). The side-chain of residue K89 is positioned toward the phosphate backbone of the RNA and likely binds dsRNA through an electrostatic interaction of the positively charged Lysine with the negatively charged phosphates. The substitution of this Lysine into the small and hydrophobic Alanine is likely to abolish the interaction. See Supplementary Figure S2 for other residues selected for site-directed mutagenesis. (C) Western blot analysis of V5 epitope-tagged WT and mutant 340R from transfected S2 cells. Proteins were detected using anti-V5 antibodies (α-V5) or, as a loading control, using anti-α-tubulin (α-Tub) antibodies. Molecular mass (in kDa) is indicated on the left of the image. The predicted molecular weight for dsRBD100 is 14.7 kDa. FL, full-length. The asterisk (*) indicates a 340R-specific processing or degradation product. (D) dsRNA-induced RNAi reporter assay. The experiment was performed as described in the legend to Figure 1B, using expression plasmids for WT and mutant 340R. (E) siRNA-induced RNAi reporter assay. Sequential transfection was performed as described in the legend to Figure 1E, using expression plasmids for WT and mutant 340R and CrPV 1A. Difference in RNAi efficiency compared to controls (dark gray bars) was analyzed by one-way ANOVA followed by a post hoc Dunnett's test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231766&req=5

Figure 2: Viral protein 340R requires a functional dsRBD to suppress RNAi. (A) Alignment of the dsRBD of 340R (aa 30–100) to the dsRBDs of proteins from different model organisms and to the dsRBD of DCV 1A. Red boxes indicate residues that interact with dsRNA in structural analyses of the second dsRBD of Xenopus laevis RNA-binding protein A (Xl RBPA2) (56). Residues predicted to be involved in RNA binding or conserved amino acids were selected for site-directed mutagenesis. Position of secondary structures are indicated above the alignment. (B) Homology model of 340R in complex with dsRNA. The protein is shown in cartoon-view with the WT residue K89 shown in red (left panel) or with the WT residue shown in green and the mutant residue (Alanine) shown in red (right panel). The RNA is shown in ball-and-stick view, without atomic details (left) and with all atoms (right). The side-chain of residue K89 is positioned toward the phosphate backbone of the RNA and likely binds dsRNA through an electrostatic interaction of the positively charged Lysine with the negatively charged phosphates. The substitution of this Lysine into the small and hydrophobic Alanine is likely to abolish the interaction. See Supplementary Figure S2 for other residues selected for site-directed mutagenesis. (C) Western blot analysis of V5 epitope-tagged WT and mutant 340R from transfected S2 cells. Proteins were detected using anti-V5 antibodies (α-V5) or, as a loading control, using anti-α-tubulin (α-Tub) antibodies. Molecular mass (in kDa) is indicated on the left of the image. The predicted molecular weight for dsRBD100 is 14.7 kDa. FL, full-length. The asterisk (*) indicates a 340R-specific processing or degradation product. (D) dsRNA-induced RNAi reporter assay. The experiment was performed as described in the legend to Figure 1B, using expression plasmids for WT and mutant 340R. (E) siRNA-induced RNAi reporter assay. Sequential transfection was performed as described in the legend to Figure 1E, using expression plasmids for WT and mutant 340R and CrPV 1A. Difference in RNAi efficiency compared to controls (dark gray bars) was analyzed by one-way ANOVA followed by a post hoc Dunnett's test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.
Mentions: The IIV-6 340R gene encodes a 23-kDa protein that contains a 70-aa dsRBD flanked by a 30-aa N-terminal sequence and a 73-aa C-terminal sequence. Alignment of the the dsRBD of 340R to the dsRBDs of DCV 1A and cellular proteins from different model organisms shows that conserved amino acids are present throughout the motif (Figure 2A). Homology modeling suggests that the dsRBD of 340R adopts the expected αβββα topology, in which two α helices are packed along a three-stranded antiparallel β sheet, and that the dsRBD is preceded by an N-terminal helical structure (Figure 2B). Based on these analyses, we selected for site-directed mutagenesis four highly conserved residues (L35Y, F63A and AA92LL) and two residues within a region expected to interact with dsRNA (K86A and K89A) (Figure 2A and B and Supplementary Figure S2). In addition, we generated a C-terminally truncated version of 340R, consisting of the N-terminal 100-aa that contains the complete dsRBD (dsRBD100).

Bottom Line: Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R.Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs).We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

Show MeSH
Related in: MedlinePlus