Limits...
Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila.

Nayak A, Berry B, Tassetto M, Kunitomi M, Acevedo A, Deng C, Krutchinsky A, Gross J, Antoniewski C, Andino R - Nat. Struct. Mol. Biol. (2010)

Bottom Line: In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing.In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A.We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of California, San Francisco, California, USA.

ABSTRACT
Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature, Drosophila melanogaster C virus (DCV) infection causes low mortality and persistent infection, whereas the closely related cricket paralysis virus (CrPV) causes a lethal infection. We show that these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing. We examined the link between viral RNAi suppressors and the outcome of infection using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a marked increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

Show MeSH

Related in: MedlinePlus

CrPV-1A is a potent RNAi suppressor and does not interfere in the microRNA pathway. (a) Transgenic flies expressing CrPV-1A148 or CrPV-1A108, or DCV-1A99 or GFP and an inverted repeat [IR] directed against the white gene were analyzed for their ability to carry out efficient RNAi. Loss of red eye pigments in presence of indicated suppressor proteins is expressed as % silencing compared to GFP control fly. (b) Transgenic flies expressing CrPV-1A and DCV-1A were injected intra-thoracically with 100 TCID50 DCV or Tris buffer control and survival rate was monitored daily. (c) Luciferase reporter plasmid carrying miR2b target sequences in sense and antisense orientation (Supplementary Fig. 1b) in the 3’ untranslated region (UTR) was transfected into S2 cells. Luciferase expression in presence of CrPV-1A, DCV-1A was expressed as ratio of luciferase counts produced by luciferase miR2b antisense over luciferase miR2b sense reporter transfection. (d) S2 cells were transfected with miR-2b ASO followed by trasnsfection of reporter system (miR2b antisense). miR-2b ASO restored luciferease expression up to 50% compared to Ctrl ASO (e) The effect of CrPV-1A on bantam mediated translational repression in the imaginal disc was analyzed in flies expressing GFP mRNA containing three complementary bantam miRNA target sites. (f) Fold changes in retrotransposons RNA levels were calculated relative to 297, mdg1 and 412 RNA levels measured in control flies. Fold changes in homozygous Ago2414 mutants were calculated relative to heterozygous ago2414 flies. Data in (a), (c), (d) and (f) indicate standard deviations from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815677&req=5

Figure 2: CrPV-1A is a potent RNAi suppressor and does not interfere in the microRNA pathway. (a) Transgenic flies expressing CrPV-1A148 or CrPV-1A108, or DCV-1A99 or GFP and an inverted repeat [IR] directed against the white gene were analyzed for their ability to carry out efficient RNAi. Loss of red eye pigments in presence of indicated suppressor proteins is expressed as % silencing compared to GFP control fly. (b) Transgenic flies expressing CrPV-1A and DCV-1A were injected intra-thoracically with 100 TCID50 DCV or Tris buffer control and survival rate was monitored daily. (c) Luciferase reporter plasmid carrying miR2b target sequences in sense and antisense orientation (Supplementary Fig. 1b) in the 3’ untranslated region (UTR) was transfected into S2 cells. Luciferase expression in presence of CrPV-1A, DCV-1A was expressed as ratio of luciferase counts produced by luciferase miR2b antisense over luciferase miR2b sense reporter transfection. (d) S2 cells were transfected with miR-2b ASO followed by trasnsfection of reporter system (miR2b antisense). miR-2b ASO restored luciferease expression up to 50% compared to Ctrl ASO (e) The effect of CrPV-1A on bantam mediated translational repression in the imaginal disc was analyzed in flies expressing GFP mRNA containing three complementary bantam miRNA target sites. (f) Fold changes in retrotransposons RNA levels were calculated relative to 297, mdg1 and 412 RNA levels measured in control flies. Fold changes in homozygous Ago2414 mutants were calculated relative to heterozygous ago2414 flies. Data in (a), (c), (d) and (f) indicate standard deviations from three independent experiments.

Mentions: Next, we evaluated the ability of the CrPV sequence upstream of the DVExNPGP motif to suppress RNAi in S2 cells. Using the dual reporter assay, luciferase (Fluc, Rluc) expression plasmids were co-transfected with dsRNA that specifically targets firefly luciferase and plasmids encoding C-terminal deletions of the putative CrPV RNAi suppressor protein (N-terminal 168, 148, 128, or 108 amino acids). Both CrPV-1A168 and CrPV-1A148 efficiently blocked RNA silencing (Fig.1d). In contrast, CrPV-1A128 and CrPV-1A108 expression were unable to suppress RNAi (Fig.1d). Hence, the N-terminal 148 amino acids of CrPV ORF1 are sufficient to block RNAi in S2 cells. To examine the effects of CrPV-1A suppressor in vivo, we generated transgenic flies expressing the CrPV-1A148 protein, the inactive CrPV-1A108 fragment, or GFP. In order to monitor the effects of the CrPV-1A148 suppressor in vivo, these transgenic flies were crossed with flies expressing a hairpin dsRNA (inverted repeat) targeting the white gene (IR[white]). The expression of two mini white genes would be suppressed by expression of this hairpin dsRNA, thus providing a colorimetric readout for RNA silencing. The white gene was efficiently silenced in flies expressing GFP and the IR[white] hairpin causing an orange eye color due to the loss of red eye pigmentation (Fig. 2a). In contrast, RNA silencing was suppressed in flies expressing CrPV-1A148, resulting in expression of the white gene and, thus, a red eye color. Flies expressing the inactive CrPV-1A108 were able to suppress the white gene similar to the GFP flies (Fig. 2a). Therefore, our observations demonstrate that CrPV-1A148 is a potent RNAi suppressor in Drosophila.


Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila.

Nayak A, Berry B, Tassetto M, Kunitomi M, Acevedo A, Deng C, Krutchinsky A, Gross J, Antoniewski C, Andino R - Nat. Struct. Mol. Biol. (2010)

CrPV-1A is a potent RNAi suppressor and does not interfere in the microRNA pathway. (a) Transgenic flies expressing CrPV-1A148 or CrPV-1A108, or DCV-1A99 or GFP and an inverted repeat [IR] directed against the white gene were analyzed for their ability to carry out efficient RNAi. Loss of red eye pigments in presence of indicated suppressor proteins is expressed as % silencing compared to GFP control fly. (b) Transgenic flies expressing CrPV-1A and DCV-1A were injected intra-thoracically with 100 TCID50 DCV or Tris buffer control and survival rate was monitored daily. (c) Luciferase reporter plasmid carrying miR2b target sequences in sense and antisense orientation (Supplementary Fig. 1b) in the 3’ untranslated region (UTR) was transfected into S2 cells. Luciferase expression in presence of CrPV-1A, DCV-1A was expressed as ratio of luciferase counts produced by luciferase miR2b antisense over luciferase miR2b sense reporter transfection. (d) S2 cells were transfected with miR-2b ASO followed by trasnsfection of reporter system (miR2b antisense). miR-2b ASO restored luciferease expression up to 50% compared to Ctrl ASO (e) The effect of CrPV-1A on bantam mediated translational repression in the imaginal disc was analyzed in flies expressing GFP mRNA containing three complementary bantam miRNA target sites. (f) Fold changes in retrotransposons RNA levels were calculated relative to 297, mdg1 and 412 RNA levels measured in control flies. Fold changes in homozygous Ago2414 mutants were calculated relative to heterozygous ago2414 flies. Data in (a), (c), (d) and (f) indicate standard deviations from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815677&req=5

Figure 2: CrPV-1A is a potent RNAi suppressor and does not interfere in the microRNA pathway. (a) Transgenic flies expressing CrPV-1A148 or CrPV-1A108, or DCV-1A99 or GFP and an inverted repeat [IR] directed against the white gene were analyzed for their ability to carry out efficient RNAi. Loss of red eye pigments in presence of indicated suppressor proteins is expressed as % silencing compared to GFP control fly. (b) Transgenic flies expressing CrPV-1A and DCV-1A were injected intra-thoracically with 100 TCID50 DCV or Tris buffer control and survival rate was monitored daily. (c) Luciferase reporter plasmid carrying miR2b target sequences in sense and antisense orientation (Supplementary Fig. 1b) in the 3’ untranslated region (UTR) was transfected into S2 cells. Luciferase expression in presence of CrPV-1A, DCV-1A was expressed as ratio of luciferase counts produced by luciferase miR2b antisense over luciferase miR2b sense reporter transfection. (d) S2 cells were transfected with miR-2b ASO followed by trasnsfection of reporter system (miR2b antisense). miR-2b ASO restored luciferease expression up to 50% compared to Ctrl ASO (e) The effect of CrPV-1A on bantam mediated translational repression in the imaginal disc was analyzed in flies expressing GFP mRNA containing three complementary bantam miRNA target sites. (f) Fold changes in retrotransposons RNA levels were calculated relative to 297, mdg1 and 412 RNA levels measured in control flies. Fold changes in homozygous Ago2414 mutants were calculated relative to heterozygous ago2414 flies. Data in (a), (c), (d) and (f) indicate standard deviations from three independent experiments.
Mentions: Next, we evaluated the ability of the CrPV sequence upstream of the DVExNPGP motif to suppress RNAi in S2 cells. Using the dual reporter assay, luciferase (Fluc, Rluc) expression plasmids were co-transfected with dsRNA that specifically targets firefly luciferase and plasmids encoding C-terminal deletions of the putative CrPV RNAi suppressor protein (N-terminal 168, 148, 128, or 108 amino acids). Both CrPV-1A168 and CrPV-1A148 efficiently blocked RNA silencing (Fig.1d). In contrast, CrPV-1A128 and CrPV-1A108 expression were unable to suppress RNAi (Fig.1d). Hence, the N-terminal 148 amino acids of CrPV ORF1 are sufficient to block RNAi in S2 cells. To examine the effects of CrPV-1A suppressor in vivo, we generated transgenic flies expressing the CrPV-1A148 protein, the inactive CrPV-1A108 fragment, or GFP. In order to monitor the effects of the CrPV-1A148 suppressor in vivo, these transgenic flies were crossed with flies expressing a hairpin dsRNA (inverted repeat) targeting the white gene (IR[white]). The expression of two mini white genes would be suppressed by expression of this hairpin dsRNA, thus providing a colorimetric readout for RNA silencing. The white gene was efficiently silenced in flies expressing GFP and the IR[white] hairpin causing an orange eye color due to the loss of red eye pigmentation (Fig. 2a). In contrast, RNA silencing was suppressed in flies expressing CrPV-1A148, resulting in expression of the white gene and, thus, a red eye color. Flies expressing the inactive CrPV-1A108 were able to suppress the white gene similar to the GFP flies (Fig. 2a). Therefore, our observations demonstrate that CrPV-1A148 is a potent RNAi suppressor in Drosophila.

Bottom Line: In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing.In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A.We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of California, San Francisco, California, USA.

ABSTRACT
Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature, Drosophila melanogaster C virus (DCV) infection causes low mortality and persistent infection, whereas the closely related cricket paralysis virus (CrPV) causes a lethal infection. We show that these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing. We examined the link between viral RNAi suppressors and the outcome of infection using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a marked increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

Show MeSH
Related in: MedlinePlus