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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 rescues VSR-deficient FHV replication. (A) Schematic representation of the WT and ΔB2 FHV replicons. FHV RNA1 is self-replicating and encodes the RNA-dependent RNA polymerase (RdRP). B2, the viral suppressor of RNAi, is expressed from a subgenomic RNA (upper panel). The FHV ΔB2 replicon lacks B2 expression due to point mutations (triangles) that disrupt the start codon (M1S) and introduce a premature stop codon (S58*) (bottom panel) (49). pMT, metallothionein promoter; HdV, Hepatitis delta virus. (B) FHV RNA levels in S2 cells co-transfected with the FHV ΔB2 replicon and expression plasmids encoding the indicated viral proteins. FHV RNA levels were analyzed by qRT-PCR, normalized to Rp49 and presented as fold change relative to the empty vector control. Bars represent the means and standard deviations of three independent samples. Viral RNA levels were compared to the control (empty plasmid) with one-way ANOVA followed by a post hoc Dunnett's test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.
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Figure 6: 340R rescues VSR-deficient FHV replication. (A) Schematic representation of the WT and ΔB2 FHV replicons. FHV RNA1 is self-replicating and encodes the RNA-dependent RNA polymerase (RdRP). B2, the viral suppressor of RNAi, is expressed from a subgenomic RNA (upper panel). The FHV ΔB2 replicon lacks B2 expression due to point mutations (triangles) that disrupt the start codon (M1S) and introduce a premature stop codon (S58*) (bottom panel) (49). pMT, metallothionein promoter; HdV, Hepatitis delta virus. (B) FHV RNA levels in S2 cells co-transfected with the FHV ΔB2 replicon and expression plasmids encoding the indicated viral proteins. FHV RNA levels were analyzed by qRT-PCR, normalized to Rp49 and presented as fold change relative to the empty vector control. Bars represent the means and standard deviations of three independent samples. Viral RNA levels were compared to the control (empty plasmid) with one-way ANOVA followed by a post hoc Dunnett's test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.

Mentions: To analyze whether the 340R-mediated VSR activity is sufficient to suppress an antiviral RNAi response, we investigated whether 340R can rescue replication of a VSR-defective FHV replicon (12,34). The WT replicon consists of RNA1 of FHV, which encodes the viral RNA-dependent RNA polymerase (RdRP) and expresses the B2 suppressor protein that antagonizes RNAi by binding dsRNA and siRNA duplexes (Figure 6A) (12,14,15). In the VSR-defective FHV replicon (FHV ΔB2), two-point mutations were introduced that abolish B2 expression, resulting in an RNAi-dependent replication defect (Figure 6A) (12,43,49).


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 rescues VSR-deficient FHV replication. (A) Schematic representation of the WT and ΔB2 FHV replicons. FHV RNA1 is self-replicating and encodes the RNA-dependent RNA polymerase (RdRP). B2, the viral suppressor of RNAi, is expressed from a subgenomic RNA (upper panel). The FHV ΔB2 replicon lacks B2 expression due to point mutations (triangles) that disrupt the start codon (M1S) and introduce a premature stop codon (S58*) (bottom panel) (49). pMT, metallothionein promoter; HdV, Hepatitis delta virus. (B) FHV RNA levels in S2 cells co-transfected with the FHV ΔB2 replicon and expression plasmids encoding the indicated viral proteins. FHV RNA levels were analyzed by qRT-PCR, normalized to Rp49 and presented as fold change relative to the empty vector control. Bars represent the means and standard deviations of three independent samples. Viral RNA levels were compared to the control (empty plasmid) with one-way ANOVA followed by a post hoc Dunnett's test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.
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Figure 6: 340R rescues VSR-deficient FHV replication. (A) Schematic representation of the WT and ΔB2 FHV replicons. FHV RNA1 is self-replicating and encodes the RNA-dependent RNA polymerase (RdRP). B2, the viral suppressor of RNAi, is expressed from a subgenomic RNA (upper panel). The FHV ΔB2 replicon lacks B2 expression due to point mutations (triangles) that disrupt the start codon (M1S) and introduce a premature stop codon (S58*) (bottom panel) (49). pMT, metallothionein promoter; HdV, Hepatitis delta virus. (B) FHV RNA levels in S2 cells co-transfected with the FHV ΔB2 replicon and expression plasmids encoding the indicated viral proteins. FHV RNA levels were analyzed by qRT-PCR, normalized to Rp49 and presented as fold change relative to the empty vector control. Bars represent the means and standard deviations of three independent samples. Viral RNA levels were compared to the control (empty plasmid) with one-way ANOVA followed by a post hoc Dunnett's test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, not significant.
Mentions: To analyze whether the 340R-mediated VSR activity is sufficient to suppress an antiviral RNAi response, we investigated whether 340R can rescue replication of a VSR-defective FHV replicon (12,34). The WT replicon consists of RNA1 of FHV, which encodes the viral RNA-dependent RNA polymerase (RdRP) and expresses the B2 suppressor protein that antagonizes RNAi by binding dsRNA and siRNA duplexes (Figure 6A) (12,14,15). In the VSR-defective FHV replicon (FHV ΔB2), two-point mutations were introduced that abolish B2 expression, resulting in an RNAi-dependent replication defect (Figure 6A) (12,43,49).

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