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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.

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340R binds long dsRNA and duplex siRNAs. (A) EMSA of 126-nt blunt dsRNA with WT 340R and the K89A and dsRBD100 mutants. Buffer only (-, lane 1) and decreasing concentrations of MBP were included as negative controls (lanes 2–4; 10, 3.2 and 0.4 μM). An MBP-DCV 1A fusion protein was included as positive control (lane 5; 0.1 μM). WT and K89A 340R were tested in 2-fold serial dilutions starting at 0.4 μM (WT, lanes 6–10) and 3.2 μM (K89A, lanes 11–15). dsRBD100 340R protein was tested in 10-fold dilutions starting at 10 μM (lanes 16–18). (B) Quantification of the fraction bound probe at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line). Data represent means and standard deviations of three independent experiments. (C) EMSA of 21-nt siRNAs containing 2-nt 3′ overhangs with MBP (lane 2; 8 μM) and 2-fold serial dilutions of WT 340R (lanes 3–8; starting at 2 μM), and the K89A and dsRBD100 mutants (lanes 9–13 and 14–18, respectively; starting at 8 μM). A representative experiment of three independent experiments is shown in panels A and C. (D) Quantification of the fraction bound siRNA at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line). Data represent means and standard deviations of three independent experiments. (E) EMSA of 19-nt blunt dsRNA with decreasing amounts of recombinant proteins. Protein concentrations are as described in panel (C). (F) Quantification of the fraction bound 19-nt blunt dsRNA probe at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line).
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Figure 3: 340R binds long dsRNA and duplex siRNAs. (A) EMSA of 126-nt blunt dsRNA with WT 340R and the K89A and dsRBD100 mutants. Buffer only (-, lane 1) and decreasing concentrations of MBP were included as negative controls (lanes 2–4; 10, 3.2 and 0.4 μM). An MBP-DCV 1A fusion protein was included as positive control (lane 5; 0.1 μM). WT and K89A 340R were tested in 2-fold serial dilutions starting at 0.4 μM (WT, lanes 6–10) and 3.2 μM (K89A, lanes 11–15). dsRBD100 340R protein was tested in 10-fold dilutions starting at 10 μM (lanes 16–18). (B) Quantification of the fraction bound probe at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line). Data represent means and standard deviations of three independent experiments. (C) EMSA of 21-nt siRNAs containing 2-nt 3′ overhangs with MBP (lane 2; 8 μM) and 2-fold serial dilutions of WT 340R (lanes 3–8; starting at 2 μM), and the K89A and dsRBD100 mutants (lanes 9–13 and 14–18, respectively; starting at 8 μM). A representative experiment of three independent experiments is shown in panels A and C. (D) Quantification of the fraction bound siRNA at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line). Data represent means and standard deviations of three independent experiments. (E) EMSA of 19-nt blunt dsRNA with decreasing amounts of recombinant proteins. Protein concentrations are as described in panel (C). (F) Quantification of the fraction bound 19-nt blunt dsRNA probe at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line).

Mentions: To directly analyze dsRNA binding by 340R, we performed EMSAs using different dsRNA substrates. Full-length WT 340R and the VSR-defective mutants K89A and dsRBD100 were fused to MBP and affinity-purified from E. coli. We first tested whether these recombinant proteins can bind radioactively labeled, 126-bp long dsRNA. As positive control, we included recombinant MBP-DCV 1A. As expected, no shift in mobility of long dsRNA on native polyacrylamide gels was observed with increasing concentrations of MBP (Figure 3A, compare lanes 2–4 with lane 1). By contrast, addition of DCV 1A resulted in protein-dsRNA complex formation (Figure 3A, lane 5), which is in line with previous observations (11,27). Similarly, WT 340R bound dsRNA in a dose-dependent manner (Figure 3A, lanes 6–10). Interestingly, the K89A mutant could still bind long dsRNA, although 8-fold higher protein concentrations were required for a complete dsRNA shift (Figure 3A, lanes 11–15). Indeed, WT 340R had a ∼12-fold higher affinity for long dsRNA than the K89A mutant (dissociation constants of 138.8 ± 34.0 nM and 1626 ± 412.2 nM, respectively, Figure 3B). No dsRNA-binding activity was detected for dsRBD100, even when a 25-fold higher protein concentration was tested (Figure 3A, lanes 16–18). These results are in line with the results from the RNAi reporter assay, in which we observed slight VSR activity for the K89A mutant and a lack of VSR activity for dsRBD100 (Figure 2D).


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)

340R binds long dsRNA and duplex siRNAs. (A) EMSA of 126-nt blunt dsRNA with WT 340R and the K89A and dsRBD100 mutants. Buffer only (-, lane 1) and decreasing concentrations of MBP were included as negative controls (lanes 2–4; 10, 3.2 and 0.4 μM). An MBP-DCV 1A fusion protein was included as positive control (lane 5; 0.1 μM). WT and K89A 340R were tested in 2-fold serial dilutions starting at 0.4 μM (WT, lanes 6–10) and 3.2 μM (K89A, lanes 11–15). dsRBD100 340R protein was tested in 10-fold dilutions starting at 10 μM (lanes 16–18). (B) Quantification of the fraction bound probe at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line). Data represent means and standard deviations of three independent experiments. (C) EMSA of 21-nt siRNAs containing 2-nt 3′ overhangs with MBP (lane 2; 8 μM) and 2-fold serial dilutions of WT 340R (lanes 3–8; starting at 2 μM), and the K89A and dsRBD100 mutants (lanes 9–13 and 14–18, respectively; starting at 8 μM). A representative experiment of three independent experiments is shown in panels A and C. (D) Quantification of the fraction bound siRNA at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line). Data represent means and standard deviations of three independent experiments. (E) EMSA of 19-nt blunt dsRNA with decreasing amounts of recombinant proteins. Protein concentrations are as described in panel (C). (F) Quantification of the fraction bound 19-nt blunt dsRNA probe at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line).
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Figure 3: 340R binds long dsRNA and duplex siRNAs. (A) EMSA of 126-nt blunt dsRNA with WT 340R and the K89A and dsRBD100 mutants. Buffer only (-, lane 1) and decreasing concentrations of MBP were included as negative controls (lanes 2–4; 10, 3.2 and 0.4 μM). An MBP-DCV 1A fusion protein was included as positive control (lane 5; 0.1 μM). WT and K89A 340R were tested in 2-fold serial dilutions starting at 0.4 μM (WT, lanes 6–10) and 3.2 μM (K89A, lanes 11–15). dsRBD100 340R protein was tested in 10-fold dilutions starting at 10 μM (lanes 16–18). (B) Quantification of the fraction bound probe at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line). Data represent means and standard deviations of three independent experiments. (C) EMSA of 21-nt siRNAs containing 2-nt 3′ overhangs with MBP (lane 2; 8 μM) and 2-fold serial dilutions of WT 340R (lanes 3–8; starting at 2 μM), and the K89A and dsRBD100 mutants (lanes 9–13 and 14–18, respectively; starting at 8 μM). A representative experiment of three independent experiments is shown in panels A and C. (D) Quantification of the fraction bound siRNA at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line). Data represent means and standard deviations of three independent experiments. (E) EMSA of 19-nt blunt dsRNA with decreasing amounts of recombinant proteins. Protein concentrations are as described in panel (C). (F) Quantification of the fraction bound 19-nt blunt dsRNA probe at different protein concentrations for WT 340R (black line) and the K89A mutant (gray line).
Mentions: To directly analyze dsRNA binding by 340R, we performed EMSAs using different dsRNA substrates. Full-length WT 340R and the VSR-defective mutants K89A and dsRBD100 were fused to MBP and affinity-purified from E. coli. We first tested whether these recombinant proteins can bind radioactively labeled, 126-bp long dsRNA. As positive control, we included recombinant MBP-DCV 1A. As expected, no shift in mobility of long dsRNA on native polyacrylamide gels was observed with increasing concentrations of MBP (Figure 3A, compare lanes 2–4 with lane 1). By contrast, addition of DCV 1A resulted in protein-dsRNA complex formation (Figure 3A, lane 5), which is in line with previous observations (11,27). Similarly, WT 340R bound dsRNA in a dose-dependent manner (Figure 3A, lanes 6–10). Interestingly, the K89A mutant could still bind long dsRNA, although 8-fold higher protein concentrations were required for a complete dsRNA shift (Figure 3A, lanes 11–15). Indeed, WT 340R had a ∼12-fold higher affinity for long dsRNA than the K89A mutant (dissociation constants of 138.8 ± 34.0 nM and 1626 ± 412.2 nM, respectively, Figure 3B). No dsRNA-binding activity was detected for dsRBD100, even when a 25-fold higher protein concentration was tested (Figure 3A, lanes 16–18). These results are in line with the results from the RNAi reporter assay, in which we observed slight VSR activity for the K89A mutant and a lack of VSR activity for dsRBD100 (Figure 2D).

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.

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Related in: MedlinePlus