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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 does not inhibit Slicer activity of pre-assembled RISC. (A) In vitro RNA cleavage (Slicer) assay in Drosophila embryo lysates to analyze the effect of 340R on RISC assembly and subsequent AGO2 catalytic activity. Embryo lysates were pre-incubated for 30 min with recombinant proteins (lanes 3–8) or protein storage buffer (lanes 1 and 2), followed by the addition of Fluc-specific siRNAs (siFluc, lanes 2–8) or non-specific control siRNAs (siCtrl, lane 1). After another 30-min incubation, a radioactive cap-labeled Fluc target RNA was added to the reaction mixture. Target cleavage was analyzed on a denaturing gel after a further 2-h incubation. (B) Slicer assay to monitor the effect of WT 340R on Slicer activity of a pre-assembled RISC. Recombinant proteins (lanes 3–8) were added after RISC assembly for 30 min with siFluc (lanes 2–4) or siCtrl (lane 1). After a further 30-min incubation, target RNA was added and the reaction was allowed to proceed for 2 h before analysis. Nora virus VP1 was analyzed at a concentration of 0.3 μM, all other proteins at 1.5 μM.
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Figure 5: 340R does not inhibit Slicer activity of pre-assembled RISC. (A) In vitro RNA cleavage (Slicer) assay in Drosophila embryo lysates to analyze the effect of 340R on RISC assembly and subsequent AGO2 catalytic activity. Embryo lysates were pre-incubated for 30 min with recombinant proteins (lanes 3–8) or protein storage buffer (lanes 1 and 2), followed by the addition of Fluc-specific siRNAs (siFluc, lanes 2–8) or non-specific control siRNAs (siCtrl, lane 1). After another 30-min incubation, a radioactive cap-labeled Fluc target RNA was added to the reaction mixture. Target cleavage was analyzed on a denaturing gel after a further 2-h incubation. (B) Slicer assay to monitor the effect of WT 340R on Slicer activity of a pre-assembled RISC. Recombinant proteins (lanes 3–8) were added after RISC assembly for 30 min with siFluc (lanes 2–4) or siCtrl (lane 1). After a further 30-min incubation, target RNA was added and the reaction was allowed to proceed for 2 h before analysis. Nora virus VP1 was analyzed at a concentration of 0.3 μM, all other proteins at 1.5 μM.

Mentions: Having shown that 340R interferes with the initiation steps of the RNAi pathway, we wondered whether 340R also inhibits the effector phase of the RNAi response. We thus monitored slicing of a radioactively 5′ cap-labeled target RNA (44) in the presence or absence of 340R. Drosophila embryo lysates were incubated with a 492-nt Fluc target RNA sequence and a Fluc-specific siRNA that triggers target RNA cleavage into a 164-nt 5′ cleavage product (Figure 5A, lane 2). This specific cleavage product was not detected after incubation with a non-specific control siRNA (Figure 5A, lane 1). In a first approach, we analyzed whether 340R interferes with RISC assembly and subsequent target RNA cleavage. To this end, we incubated recombinant proteins with embryo lysate before the addition of siRNAs (27). Using this approach, we observed that WT 340R efficiently inhibited target RNA cleavage (Figure 5A, lane 4). In contrast, the K89A and dsRBD100 mutants did not suppress slicing (Figure 5A, lanes 5 and 6), similar to MBP alone (lane 3). To differentiate between the effect of 340R on RISC assembly and target slicing, we next tested whether WT 340R affects slicing by interfering with a pre-assembled RISC. To allow mature RISC formation, we pre-incubated embryo extracts with siRNAs before the addition of 340R. Neither MBP alone (Figure 5B, lane 3) nor WT or mutant 340R (lanes 4–6) inhibited target RNA cleavage under these conditions. In contrast, the positive control Nora virus VP1 efficiently inhibited AGO2-mediated target cleavage in both experimental approaches (Figure 5A and B, lane 7) (27). These results demonstrate that 340R does not inhibit the activity of a pre-assembled mature RISC. Because 340R binds siRNAs (Figure 3C and D), we propose that 340R interferes with the RISC assembly process by preventing siRNA loading into AGO2.


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 does not inhibit Slicer activity of pre-assembled RISC. (A) In vitro RNA cleavage (Slicer) assay in Drosophila embryo lysates to analyze the effect of 340R on RISC assembly and subsequent AGO2 catalytic activity. Embryo lysates were pre-incubated for 30 min with recombinant proteins (lanes 3–8) or protein storage buffer (lanes 1 and 2), followed by the addition of Fluc-specific siRNAs (siFluc, lanes 2–8) or non-specific control siRNAs (siCtrl, lane 1). After another 30-min incubation, a radioactive cap-labeled Fluc target RNA was added to the reaction mixture. Target cleavage was analyzed on a denaturing gel after a further 2-h incubation. (B) Slicer assay to monitor the effect of WT 340R on Slicer activity of a pre-assembled RISC. Recombinant proteins (lanes 3–8) were added after RISC assembly for 30 min with siFluc (lanes 2–4) or siCtrl (lane 1). After a further 30-min incubation, target RNA was added and the reaction was allowed to proceed for 2 h before analysis. Nora virus VP1 was analyzed at a concentration of 0.3 μM, all other proteins at 1.5 μM.
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Figure 5: 340R does not inhibit Slicer activity of pre-assembled RISC. (A) In vitro RNA cleavage (Slicer) assay in Drosophila embryo lysates to analyze the effect of 340R on RISC assembly and subsequent AGO2 catalytic activity. Embryo lysates were pre-incubated for 30 min with recombinant proteins (lanes 3–8) or protein storage buffer (lanes 1 and 2), followed by the addition of Fluc-specific siRNAs (siFluc, lanes 2–8) or non-specific control siRNAs (siCtrl, lane 1). After another 30-min incubation, a radioactive cap-labeled Fluc target RNA was added to the reaction mixture. Target cleavage was analyzed on a denaturing gel after a further 2-h incubation. (B) Slicer assay to monitor the effect of WT 340R on Slicer activity of a pre-assembled RISC. Recombinant proteins (lanes 3–8) were added after RISC assembly for 30 min with siFluc (lanes 2–4) or siCtrl (lane 1). After a further 30-min incubation, target RNA was added and the reaction was allowed to proceed for 2 h before analysis. Nora virus VP1 was analyzed at a concentration of 0.3 μM, all other proteins at 1.5 μM.
Mentions: Having shown that 340R interferes with the initiation steps of the RNAi pathway, we wondered whether 340R also inhibits the effector phase of the RNAi response. We thus monitored slicing of a radioactively 5′ cap-labeled target RNA (44) in the presence or absence of 340R. Drosophila embryo lysates were incubated with a 492-nt Fluc target RNA sequence and a Fluc-specific siRNA that triggers target RNA cleavage into a 164-nt 5′ cleavage product (Figure 5A, lane 2). This specific cleavage product was not detected after incubation with a non-specific control siRNA (Figure 5A, lane 1). In a first approach, we analyzed whether 340R interferes with RISC assembly and subsequent target RNA cleavage. To this end, we incubated recombinant proteins with embryo lysate before the addition of siRNAs (27). Using this approach, we observed that WT 340R efficiently inhibited target RNA cleavage (Figure 5A, lane 4). In contrast, the K89A and dsRBD100 mutants did not suppress slicing (Figure 5A, lanes 5 and 6), similar to MBP alone (lane 3). To differentiate between the effect of 340R on RISC assembly and target slicing, we next tested whether WT 340R affects slicing by interfering with a pre-assembled RISC. To allow mature RISC formation, we pre-incubated embryo extracts with siRNAs before the addition of 340R. Neither MBP alone (Figure 5B, lane 3) nor WT or mutant 340R (lanes 4–6) inhibited target RNA cleavage under these conditions. In contrast, the positive control Nora virus VP1 efficiently inhibited AGO2-mediated target cleavage in both experimental approaches (Figure 5A and B, lane 7) (27). These results demonstrate that 340R does not inhibit the activity of a pre-assembled mature RISC. Because 340R binds siRNAs (Figure 3C and D), we propose that 340R interferes with the RISC assembly process by preventing siRNA loading into AGO2.

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