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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.

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CrPV-1A interferes with the function of pre-programmed holo-RISC in vitro and in vivo. (a) S2 extracts were programmed with siRNA for 45 min followed by addition of 1A [(0.35 µM CrPV-1A, lane 3), (0.35 µM DCV-1A, lane 4) or (BSA, lane 5)] for 10 min. RISC assay was initiated by incubating 32P-mRNA substrate for additional 3hrs and 5′ cleaved product was analyzed using denaturing polyacrylamide gel electrophorsis (PAGE). (b) Silencing of firefly luciferase in stable S2 cells was programmed by co-transfecting firefly and renila reporter plasmid with specific FLuc dsRNA (dsLuc) or control eGFP dsRNA (dsGFP). 16 hrs post-transfection, luciferase expression was monitored either in presence (dsLuc+CrPV-1A, dsGFP+ CrPV-1A) or absence (dsLuc) of CrPV-1A for a period of 24 hrs. (c) Stable S2 cells were transfected with FLuc dsRNA for 48 hrs followed by induction of suppressor for additional 20 hrs. The post-nuclear cytoplasmic extract was centrifuged at 200,000×g to separate the ribosomal pellet (P200) from the supernatant (S200). (d) P200 and S200 fractions were western blotted using Drosophila Argonaute 2 (α-Ago2) polyclonal and anti-Flag (α-Flag) monoclonal antibodies. RNA extracted from P200 fraction was analyzed for Luc siRNA by northern blot. P200 ethidium bromide (Et. Br) represents RNA isolated from soluble high salt P200 extraction. (e) DCV-1A and FHV B2 either inhibits Dicer 2 processing of long dsRNA or siRNA incorporation in to RISC by virtue of their dsRNA or siRNA binding activity. Conversely, CrPV-1A acts at the level of holo-RISC by protein-protein interaction with Drosophila Argonaute 2.
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Figure 5: CrPV-1A interferes with the function of pre-programmed holo-RISC in vitro and in vivo. (a) S2 extracts were programmed with siRNA for 45 min followed by addition of 1A [(0.35 µM CrPV-1A, lane 3), (0.35 µM DCV-1A, lane 4) or (BSA, lane 5)] for 10 min. RISC assay was initiated by incubating 32P-mRNA substrate for additional 3hrs and 5′ cleaved product was analyzed using denaturing polyacrylamide gel electrophorsis (PAGE). (b) Silencing of firefly luciferase in stable S2 cells was programmed by co-transfecting firefly and renila reporter plasmid with specific FLuc dsRNA (dsLuc) or control eGFP dsRNA (dsGFP). 16 hrs post-transfection, luciferase expression was monitored either in presence (dsLuc+CrPV-1A, dsGFP+ CrPV-1A) or absence (dsLuc) of CrPV-1A for a period of 24 hrs. (c) Stable S2 cells were transfected with FLuc dsRNA for 48 hrs followed by induction of suppressor for additional 20 hrs. The post-nuclear cytoplasmic extract was centrifuged at 200,000×g to separate the ribosomal pellet (P200) from the supernatant (S200). (d) P200 and S200 fractions were western blotted using Drosophila Argonaute 2 (α-Ago2) polyclonal and anti-Flag (α-Flag) monoclonal antibodies. RNA extracted from P200 fraction was analyzed for Luc siRNA by northern blot. P200 ethidium bromide (Et. Br) represents RNA isolated from soluble high salt P200 extraction. (e) DCV-1A and FHV B2 either inhibits Dicer 2 processing of long dsRNA or siRNA incorporation in to RISC by virtue of their dsRNA or siRNA binding activity. Conversely, CrPV-1A acts at the level of holo-RISC by protein-protein interaction with Drosophila Argonaute 2.

Mentions: The observation that CrPV-1A does not affect siRNA-RISC assembly in vitro (Fig. 3b), but nonetheless inhibits RISC cleavage activity (Fig. 3c), suggests that CrPV-1A interferes with the function of assembled holo-RISC. To directly test this possibility luciferase mRNA target cleavage assay was performed. S2 cell extracts were pre-incubated with duplex siRNA to allow holo-RISC assembly31–33 followed by recombinant CrPV-1A addition. We observed that CrPV-1A efficiently inhibited holo-RISC enzymatic activity even after assembly of holo-RISC (Fig. 5a, lane 3). As expected, DCV-1A and BSA control had no effect on pre-assembled RISC cleavage (Fig. 5a, lanes 4 and 5).


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 interferes with the function of pre-programmed holo-RISC in vitro and in vivo. (a) S2 extracts were programmed with siRNA for 45 min followed by addition of 1A [(0.35 µM CrPV-1A, lane 3), (0.35 µM DCV-1A, lane 4) or (BSA, lane 5)] for 10 min. RISC assay was initiated by incubating 32P-mRNA substrate for additional 3hrs and 5′ cleaved product was analyzed using denaturing polyacrylamide gel electrophorsis (PAGE). (b) Silencing of firefly luciferase in stable S2 cells was programmed by co-transfecting firefly and renila reporter plasmid with specific FLuc dsRNA (dsLuc) or control eGFP dsRNA (dsGFP). 16 hrs post-transfection, luciferase expression was monitored either in presence (dsLuc+CrPV-1A, dsGFP+ CrPV-1A) or absence (dsLuc) of CrPV-1A for a period of 24 hrs. (c) Stable S2 cells were transfected with FLuc dsRNA for 48 hrs followed by induction of suppressor for additional 20 hrs. The post-nuclear cytoplasmic extract was centrifuged at 200,000×g to separate the ribosomal pellet (P200) from the supernatant (S200). (d) P200 and S200 fractions were western blotted using Drosophila Argonaute 2 (α-Ago2) polyclonal and anti-Flag (α-Flag) monoclonal antibodies. RNA extracted from P200 fraction was analyzed for Luc siRNA by northern blot. P200 ethidium bromide (Et. Br) represents RNA isolated from soluble high salt P200 extraction. (e) DCV-1A and FHV B2 either inhibits Dicer 2 processing of long dsRNA or siRNA incorporation in to RISC by virtue of their dsRNA or siRNA binding activity. Conversely, CrPV-1A acts at the level of holo-RISC by protein-protein interaction with Drosophila Argonaute 2.
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Related In: Results  -  Collection

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Figure 5: CrPV-1A interferes with the function of pre-programmed holo-RISC in vitro and in vivo. (a) S2 extracts were programmed with siRNA for 45 min followed by addition of 1A [(0.35 µM CrPV-1A, lane 3), (0.35 µM DCV-1A, lane 4) or (BSA, lane 5)] for 10 min. RISC assay was initiated by incubating 32P-mRNA substrate for additional 3hrs and 5′ cleaved product was analyzed using denaturing polyacrylamide gel electrophorsis (PAGE). (b) Silencing of firefly luciferase in stable S2 cells was programmed by co-transfecting firefly and renila reporter plasmid with specific FLuc dsRNA (dsLuc) or control eGFP dsRNA (dsGFP). 16 hrs post-transfection, luciferase expression was monitored either in presence (dsLuc+CrPV-1A, dsGFP+ CrPV-1A) or absence (dsLuc) of CrPV-1A for a period of 24 hrs. (c) Stable S2 cells were transfected with FLuc dsRNA for 48 hrs followed by induction of suppressor for additional 20 hrs. The post-nuclear cytoplasmic extract was centrifuged at 200,000×g to separate the ribosomal pellet (P200) from the supernatant (S200). (d) P200 and S200 fractions were western blotted using Drosophila Argonaute 2 (α-Ago2) polyclonal and anti-Flag (α-Flag) monoclonal antibodies. RNA extracted from P200 fraction was analyzed for Luc siRNA by northern blot. P200 ethidium bromide (Et. Br) represents RNA isolated from soluble high salt P200 extraction. (e) DCV-1A and FHV B2 either inhibits Dicer 2 processing of long dsRNA or siRNA incorporation in to RISC by virtue of their dsRNA or siRNA binding activity. Conversely, CrPV-1A acts at the level of holo-RISC by protein-protein interaction with Drosophila Argonaute 2.
Mentions: The observation that CrPV-1A does not affect siRNA-RISC assembly in vitro (Fig. 3b), but nonetheless inhibits RISC cleavage activity (Fig. 3c), suggests that CrPV-1A interferes with the function of assembled holo-RISC. To directly test this possibility luciferase mRNA target cleavage assay was performed. S2 cell extracts were pre-incubated with duplex siRNA to allow holo-RISC assembly31–33 followed by recombinant CrPV-1A addition. We observed that CrPV-1A efficiently inhibited holo-RISC enzymatic activity even after assembly of holo-RISC (Fig. 5a, lane 3). As expected, DCV-1A and BSA control had no effect on pre-assembled RISC cleavage (Fig. 5a, lanes 4 and 5).

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