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Mosquito and Drosophila entomobirnaviruses suppress dsRNA- and siRNA-induced RNAi.

van Cleef KW, van Mierlo JT, Miesen P, Overheul GJ, Fros JJ, Schuster S, Marklewitz M, Pijlman GP, Junglen S, van Rij RP - Nucleic Acids Res. (2014)

Bottom Line: We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs).VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs.Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3.

View Article: PubMed Central - PubMed

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

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CYV and DXV VP3 inhibit Dicer, but not Slicer activity. (A) Dicer assays in extracts from Drosophila S2 (first panel), Aedes U4.4 (second panel), Culex Hsu (third panel) and Culex CT (fourth panel) cells. Radioactively-labeled 126-nt dsRNA was incubated in the extracts in the presence of MBP-tagged CYV or DXV VP3. Glutathione S-transferase (GST)-tagged DCV 1A served as positive control and MBP and GST alone as negative controls. Processing of the dsRNA into 21-nt siRNAs was monitored by denaturing polyacrylamide gel electrophoresis followed by autoradiography. Protein concentrations were as follows: 10-fold dilutions starting at 1 μM for CYV VP3 (lanes 6–8, 11–13, 16–18 and 21–23) and DXV VP3 (lanes 3–5); 1 μM for MBP (lanes 9, 14, 19 and 24); 0.5 μM for DCV 1A (lane 1) and GST (lane 2). Reactions with buffer instead of recombinant protein (No protein; lanes 10, 15, 20 and 25) were used as controls for Dcr-2 activity. (B) Dicer assays in extracts from infected S2 (left panel) and CT (right panel) cells. Radioactively-labeled 126-nt dsRNA was incubated in mixtures of mock- and CYV-infected cell extracts. The production of siRNAs was monitored as in (A). Ratios of mock- to CYV-infected cell extracts were as follows: 4:0 (lanes 5 and 10); 3:1 (lanes 4 and 9); 2:2 (lanes 3 and 8); 1:3 (lanes 2 and 7); 0:4 (lanes 1 and 6). (C) Slicer assay in Drosophila embryo lysates. Embryo lysates were first incubated with non-specific control (siCtrl; lane 1) or firefly luciferase (siFLuc; lanes 2–12) siRNAs. MBP-tagged CYV or DXV VP3 was added after 30 min. MBP-tagged Nora virus VP1 (Nora VP1) served as positive control and MBP alone as negative control. After another 30 min, the radioactive 5′ cap-labeled FLuc target RNA was added. Cleavage of the target RNA was monitored by denaturing polyacrylamide gel electrophoresis followed by autoradiography. Protein concentrations were as follows: 2-fold dilutions starting at 1.2 μM for CYV VP3 (lanes 5–7) and DXV VP3 (lanes 8–10); 1.2 μM (lane 3) or 0.3 μM (lanes 4 and 12) for MBP; 0.3 μM for Nora VP1 (lane 11). Reactions with buffer instead of recombinant protein (lanes 1 and 2) were used as controls for Slicer activity.
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Figure 6: CYV and DXV VP3 inhibit Dicer, but not Slicer activity. (A) Dicer assays in extracts from Drosophila S2 (first panel), Aedes U4.4 (second panel), Culex Hsu (third panel) and Culex CT (fourth panel) cells. Radioactively-labeled 126-nt dsRNA was incubated in the extracts in the presence of MBP-tagged CYV or DXV VP3. Glutathione S-transferase (GST)-tagged DCV 1A served as positive control and MBP and GST alone as negative controls. Processing of the dsRNA into 21-nt siRNAs was monitored by denaturing polyacrylamide gel electrophoresis followed by autoradiography. Protein concentrations were as follows: 10-fold dilutions starting at 1 μM for CYV VP3 (lanes 6–8, 11–13, 16–18 and 21–23) and DXV VP3 (lanes 3–5); 1 μM for MBP (lanes 9, 14, 19 and 24); 0.5 μM for DCV 1A (lane 1) and GST (lane 2). Reactions with buffer instead of recombinant protein (No protein; lanes 10, 15, 20 and 25) were used as controls for Dcr-2 activity. (B) Dicer assays in extracts from infected S2 (left panel) and CT (right panel) cells. Radioactively-labeled 126-nt dsRNA was incubated in mixtures of mock- and CYV-infected cell extracts. The production of siRNAs was monitored as in (A). Ratios of mock- to CYV-infected cell extracts were as follows: 4:0 (lanes 5 and 10); 3:1 (lanes 4 and 9); 2:2 (lanes 3 and 8); 1:3 (lanes 2 and 7); 0:4 (lanes 1 and 6). (C) Slicer assay in Drosophila embryo lysates. Embryo lysates were first incubated with non-specific control (siCtrl; lane 1) or firefly luciferase (siFLuc; lanes 2–12) siRNAs. MBP-tagged CYV or DXV VP3 was added after 30 min. MBP-tagged Nora virus VP1 (Nora VP1) served as positive control and MBP alone as negative control. After another 30 min, the radioactive 5′ cap-labeled FLuc target RNA was added. Cleavage of the target RNA was monitored by denaturing polyacrylamide gel electrophoresis followed by autoradiography. Protein concentrations were as follows: 2-fold dilutions starting at 1.2 μM for CYV VP3 (lanes 5–7) and DXV VP3 (lanes 8–10); 1.2 μM (lane 3) or 0.3 μM (lanes 4 and 12) for MBP; 0.3 μM for Nora VP1 (lane 11). Reactions with buffer instead of recombinant protein (lanes 1 and 2) were used as controls for Slicer activity.

Mentions: VSRs of different viruses may target different aspects of the RNAi machinery, such as Dcr-2-mediated cleavage of dsRNA and slicing of target RNAs by AGO2. To characterize the VSR activity of CYV and DXV VP3 in more detail, we performed a series of biochemical assays using maltose-binding protein (MBP)-tagged recombinant proteins purified from Escherichia coli. We first tested whether the recombinant VP3 proteins interfere with dicing of dsRNA, the initiation phase of the RNAi pathway. We incubated radioactively-labeled 126-nt dsRNA in D. melanogaster S2, A. albopictus U4.4, C. quinquefasciatus Hsu and C. tarsalis CT cell extracts and monitored its processing into 21-nt siRNAs on denaturing polyacrylamide gels. The dsRNA was efficiently processed into siRNAs in extracts from all cell types (Figure 6A, lanes 10, 15, 20 and 25). Processing of the dsRNA was, however, inhibited in a dose-dependent manner by the VP3 proteins of both CYV (Figure 6A, lanes 6–8, 11–13, 16–18 and 21–23) and DXV (Figure 6A, lanes 3–5), and in the presence of DCV 1A (Figure 6A, lane 1), a VSR that is known to interact with dsRNA (2). As expected, MBP alone did not inhibit dsRNA processing (Figure 6A, lanes 9, 14, 19 and 24). These data indicate that the entomobirnavirus VP3 proteins interfere with siRNA production by Dcr-2. Importantly, inhibition of dsRNA cleavage into siRNAs was also observed in extracts from Drosophila S2 and Culex CT cells infected with CYV. The dsRNA was processed in extracts from mock-infected cells (Figure 6B, lanes 5 and 10), but no dsRNA processing was observed in extracts from CYV-infected cells (Figure 6B, lanes 1 and 6). Titration of CYV-infected cell extracts into mock-infected cell extracts abolished dsRNA processing (Figure 6B, lanes 2–4 and 7–9), which confirms the presence of a Dcr-2 inhibitor in CYV-infected cells.


Mosquito and Drosophila entomobirnaviruses suppress dsRNA- and siRNA-induced RNAi.

van Cleef KW, van Mierlo JT, Miesen P, Overheul GJ, Fros JJ, Schuster S, Marklewitz M, Pijlman GP, Junglen S, van Rij RP - Nucleic Acids Res. (2014)

CYV and DXV VP3 inhibit Dicer, but not Slicer activity. (A) Dicer assays in extracts from Drosophila S2 (first panel), Aedes U4.4 (second panel), Culex Hsu (third panel) and Culex CT (fourth panel) cells. Radioactively-labeled 126-nt dsRNA was incubated in the extracts in the presence of MBP-tagged CYV or DXV VP3. Glutathione S-transferase (GST)-tagged DCV 1A served as positive control and MBP and GST alone as negative controls. Processing of the dsRNA into 21-nt siRNAs was monitored by denaturing polyacrylamide gel electrophoresis followed by autoradiography. Protein concentrations were as follows: 10-fold dilutions starting at 1 μM for CYV VP3 (lanes 6–8, 11–13, 16–18 and 21–23) and DXV VP3 (lanes 3–5); 1 μM for MBP (lanes 9, 14, 19 and 24); 0.5 μM for DCV 1A (lane 1) and GST (lane 2). Reactions with buffer instead of recombinant protein (No protein; lanes 10, 15, 20 and 25) were used as controls for Dcr-2 activity. (B) Dicer assays in extracts from infected S2 (left panel) and CT (right panel) cells. Radioactively-labeled 126-nt dsRNA was incubated in mixtures of mock- and CYV-infected cell extracts. The production of siRNAs was monitored as in (A). Ratios of mock- to CYV-infected cell extracts were as follows: 4:0 (lanes 5 and 10); 3:1 (lanes 4 and 9); 2:2 (lanes 3 and 8); 1:3 (lanes 2 and 7); 0:4 (lanes 1 and 6). (C) Slicer assay in Drosophila embryo lysates. Embryo lysates were first incubated with non-specific control (siCtrl; lane 1) or firefly luciferase (siFLuc; lanes 2–12) siRNAs. MBP-tagged CYV or DXV VP3 was added after 30 min. MBP-tagged Nora virus VP1 (Nora VP1) served as positive control and MBP alone as negative control. After another 30 min, the radioactive 5′ cap-labeled FLuc target RNA was added. Cleavage of the target RNA was monitored by denaturing polyacrylamide gel electrophoresis followed by autoradiography. Protein concentrations were as follows: 2-fold dilutions starting at 1.2 μM for CYV VP3 (lanes 5–7) and DXV VP3 (lanes 8–10); 1.2 μM (lane 3) or 0.3 μM (lanes 4 and 12) for MBP; 0.3 μM for Nora VP1 (lane 11). Reactions with buffer instead of recombinant protein (lanes 1 and 2) were used as controls for Slicer activity.
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Figure 6: CYV and DXV VP3 inhibit Dicer, but not Slicer activity. (A) Dicer assays in extracts from Drosophila S2 (first panel), Aedes U4.4 (second panel), Culex Hsu (third panel) and Culex CT (fourth panel) cells. Radioactively-labeled 126-nt dsRNA was incubated in the extracts in the presence of MBP-tagged CYV or DXV VP3. Glutathione S-transferase (GST)-tagged DCV 1A served as positive control and MBP and GST alone as negative controls. Processing of the dsRNA into 21-nt siRNAs was monitored by denaturing polyacrylamide gel electrophoresis followed by autoradiography. Protein concentrations were as follows: 10-fold dilutions starting at 1 μM for CYV VP3 (lanes 6–8, 11–13, 16–18 and 21–23) and DXV VP3 (lanes 3–5); 1 μM for MBP (lanes 9, 14, 19 and 24); 0.5 μM for DCV 1A (lane 1) and GST (lane 2). Reactions with buffer instead of recombinant protein (No protein; lanes 10, 15, 20 and 25) were used as controls for Dcr-2 activity. (B) Dicer assays in extracts from infected S2 (left panel) and CT (right panel) cells. Radioactively-labeled 126-nt dsRNA was incubated in mixtures of mock- and CYV-infected cell extracts. The production of siRNAs was monitored as in (A). Ratios of mock- to CYV-infected cell extracts were as follows: 4:0 (lanes 5 and 10); 3:1 (lanes 4 and 9); 2:2 (lanes 3 and 8); 1:3 (lanes 2 and 7); 0:4 (lanes 1 and 6). (C) Slicer assay in Drosophila embryo lysates. Embryo lysates were first incubated with non-specific control (siCtrl; lane 1) or firefly luciferase (siFLuc; lanes 2–12) siRNAs. MBP-tagged CYV or DXV VP3 was added after 30 min. MBP-tagged Nora virus VP1 (Nora VP1) served as positive control and MBP alone as negative control. After another 30 min, the radioactive 5′ cap-labeled FLuc target RNA was added. Cleavage of the target RNA was monitored by denaturing polyacrylamide gel electrophoresis followed by autoradiography. Protein concentrations were as follows: 2-fold dilutions starting at 1.2 μM for CYV VP3 (lanes 5–7) and DXV VP3 (lanes 8–10); 1.2 μM (lane 3) or 0.3 μM (lanes 4 and 12) for MBP; 0.3 μM for Nora VP1 (lane 11). Reactions with buffer instead of recombinant protein (lanes 1 and 2) were used as controls for Slicer activity.
Mentions: VSRs of different viruses may target different aspects of the RNAi machinery, such as Dcr-2-mediated cleavage of dsRNA and slicing of target RNAs by AGO2. To characterize the VSR activity of CYV and DXV VP3 in more detail, we performed a series of biochemical assays using maltose-binding protein (MBP)-tagged recombinant proteins purified from Escherichia coli. We first tested whether the recombinant VP3 proteins interfere with dicing of dsRNA, the initiation phase of the RNAi pathway. We incubated radioactively-labeled 126-nt dsRNA in D. melanogaster S2, A. albopictus U4.4, C. quinquefasciatus Hsu and C. tarsalis CT cell extracts and monitored its processing into 21-nt siRNAs on denaturing polyacrylamide gels. The dsRNA was efficiently processed into siRNAs in extracts from all cell types (Figure 6A, lanes 10, 15, 20 and 25). Processing of the dsRNA was, however, inhibited in a dose-dependent manner by the VP3 proteins of both CYV (Figure 6A, lanes 6–8, 11–13, 16–18 and 21–23) and DXV (Figure 6A, lanes 3–5), and in the presence of DCV 1A (Figure 6A, lane 1), a VSR that is known to interact with dsRNA (2). As expected, MBP alone did not inhibit dsRNA processing (Figure 6A, lanes 9, 14, 19 and 24). These data indicate that the entomobirnavirus VP3 proteins interfere with siRNA production by Dcr-2. Importantly, inhibition of dsRNA cleavage into siRNAs was also observed in extracts from Drosophila S2 and Culex CT cells infected with CYV. The dsRNA was processed in extracts from mock-infected cells (Figure 6B, lanes 5 and 10), but no dsRNA processing was observed in extracts from CYV-infected cells (Figure 6B, lanes 1 and 6). Titration of CYV-infected cell extracts into mock-infected cell extracts abolished dsRNA processing (Figure 6B, lanes 2–4 and 7–9), which confirms the presence of a Dcr-2 inhibitor in CYV-infected cells.

Bottom Line: We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs).VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs.Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus