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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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RNAi is suppressed during CYV infection. (A) dsRNA-induced RNAi reporter assays in infected Drosophila S2 (left panel) and Culex CT (right panel) cells. Firefly (FLuc) and Renilla (RLuc) luciferase expression plasmids were transfected together with non-specific control (dsCtrl) or FLuc (dsFLuc) dsRNA into mock- and CYV-infected cells. Luciferase activities were measured and FLuc counts were normalized to RLuc counts. The data are presented as fold silencing relative to dsCtrl. (B) siRNA-induced RNAi reporter assay in infected S2 cells. The experiment was performed as in (A), except that RNAi was induced by co-transfection of non-specific control (siCtrl) or FLuc (siFLuc) siRNAs along with the luciferase expression plasmids. The data are presented as fold silencing relative to siCtrl. Bars and error bars in all panels represent the mean and standard deviation of three independent samples.
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Figure 2: RNAi is suppressed during CYV infection. (A) dsRNA-induced RNAi reporter assays in infected Drosophila S2 (left panel) and Culex CT (right panel) cells. Firefly (FLuc) and Renilla (RLuc) luciferase expression plasmids were transfected together with non-specific control (dsCtrl) or FLuc (dsFLuc) dsRNA into mock- and CYV-infected cells. Luciferase activities were measured and FLuc counts were normalized to RLuc counts. The data are presented as fold silencing relative to dsCtrl. (B) siRNA-induced RNAi reporter assay in infected S2 cells. The experiment was performed as in (A), except that RNAi was induced by co-transfection of non-specific control (siCtrl) or FLuc (siFLuc) siRNAs along with the luciferase expression plasmids. The data are presented as fold silencing relative to siCtrl. Bars and error bars in all panels represent the mean and standard deviation of three independent samples.

Mentions: Since CYV is a target of the antiviral RNAi machinery in Culex, we deemed it likely that the virus would encode a VSR. We therefore used well-established reporter assays to determine whether CYV counteracts RNAi (41). In these assays, the effect of virus infection or expression of individual viral proteins on RNAi-mediated silencing of a firefly luciferase (FLuc) reporter is monitored. We first determined whether RNAi is suppressed in cells that are infected with CYV. To this end, we measured luciferase activities in mock- and CYV-infected cells that were co-transfected with the FLuc reporter plasmid and 113-nt in vitro transcribed FLuc dsRNA. A Renilla luciferase (RLuc) reporter plasmid was included as a normalization control. As expected, in mock-infected Drosophila S2 cells, the FLuc reporter was efficiently silenced (∼600-fold) by dsRNA treatment (Figure 2A, left panel). However, dsRNA-mediated silencing of the FLuc reporter was strongly suppressed (to ∼6-fold; P = 0.002) in CYV-infected S2 cells (Figure 2A, left panel). A comparable reduction of FLuc silencing (from ∼300-fold to ∼20-fold; P < 0.001) was observed in CYV-infected Culex CT cells (Figure 2A, right panel). CYV infection also suppressed silencing of the FLuc reporter (from ∼15-fold to ∼3-fold; P = 0.025) when we induced RNAi with 21-nt synthetic siRNA duplexes (Figure 2B). These data show that CYV infection inhibits RNAi induced by dsRNA as well as siRNAs.


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)

RNAi is suppressed during CYV infection. (A) dsRNA-induced RNAi reporter assays in infected Drosophila S2 (left panel) and Culex CT (right panel) cells. Firefly (FLuc) and Renilla (RLuc) luciferase expression plasmids were transfected together with non-specific control (dsCtrl) or FLuc (dsFLuc) dsRNA into mock- and CYV-infected cells. Luciferase activities were measured and FLuc counts were normalized to RLuc counts. The data are presented as fold silencing relative to dsCtrl. (B) siRNA-induced RNAi reporter assay in infected S2 cells. The experiment was performed as in (A), except that RNAi was induced by co-transfection of non-specific control (siCtrl) or FLuc (siFLuc) siRNAs along with the luciferase expression plasmids. The data are presented as fold silencing relative to siCtrl. Bars and error bars in all panels represent the mean and standard deviation of three independent samples.
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Figure 2: RNAi is suppressed during CYV infection. (A) dsRNA-induced RNAi reporter assays in infected Drosophila S2 (left panel) and Culex CT (right panel) cells. Firefly (FLuc) and Renilla (RLuc) luciferase expression plasmids were transfected together with non-specific control (dsCtrl) or FLuc (dsFLuc) dsRNA into mock- and CYV-infected cells. Luciferase activities were measured and FLuc counts were normalized to RLuc counts. The data are presented as fold silencing relative to dsCtrl. (B) siRNA-induced RNAi reporter assay in infected S2 cells. The experiment was performed as in (A), except that RNAi was induced by co-transfection of non-specific control (siCtrl) or FLuc (siFLuc) siRNAs along with the luciferase expression plasmids. The data are presented as fold silencing relative to siCtrl. Bars and error bars in all panels represent the mean and standard deviation of three independent samples.
Mentions: Since CYV is a target of the antiviral RNAi machinery in Culex, we deemed it likely that the virus would encode a VSR. We therefore used well-established reporter assays to determine whether CYV counteracts RNAi (41). In these assays, the effect of virus infection or expression of individual viral proteins on RNAi-mediated silencing of a firefly luciferase (FLuc) reporter is monitored. We first determined whether RNAi is suppressed in cells that are infected with CYV. To this end, we measured luciferase activities in mock- and CYV-infected cells that were co-transfected with the FLuc reporter plasmid and 113-nt in vitro transcribed FLuc dsRNA. A Renilla luciferase (RLuc) reporter plasmid was included as a normalization control. As expected, in mock-infected Drosophila S2 cells, the FLuc reporter was efficiently silenced (∼600-fold) by dsRNA treatment (Figure 2A, left panel). However, dsRNA-mediated silencing of the FLuc reporter was strongly suppressed (to ∼6-fold; P = 0.002) in CYV-infected S2 cells (Figure 2A, left panel). A comparable reduction of FLuc silencing (from ∼300-fold to ∼20-fold; P < 0.001) was observed in CYV-infected Culex CT cells (Figure 2A, right panel). CYV infection also suppressed silencing of the FLuc reporter (from ∼15-fold to ∼3-fold; P = 0.025) when we induced RNAi with 21-nt synthetic siRNA duplexes (Figure 2B). These data show that CYV infection inhibits RNAi induced by dsRNA as well as siRNAs.

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