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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 interacts with Argonaute 2 in S2 cells. (a) S2 cells were transfected with plasmids transiently expressing CrPV-1A-3FH (lane 2, 4) or empty vector control (lane 1, 3). Flag-His tagged CrPV-1A was immunopurified with anti-Flag antibody and western blotted with Drosophila Argonaute 2 (α-Ago2, right panel) or Argonaute 1 (α-Ago1, left panel) polyclonal antibodies. Two isoforms of Ago2 (Ago2a and Ago2b) were detected with Ago2 specific antibodies. Asterisks (*) indicate cross-reacting host-protein band. Lane 6 and 8 show the positive detection of E.coli expressed Drosophila Ago1 and endogenous Drosophila Ago2 with Ago1 and Ago2 antibody respectively (Ab control). (b) Flag-His tagged CrPV-1A148, CrPV-1A108 were transfected in to S2 cells, immunopurified using anti-Flag antibody and probed with Ago2 antibody (α-Ago2). To detect the expression of 1A protein, the blot was stripped and further probed with anti-Flag antibody (α-Flag) (lane 3 and lane 4, bottom panel). Asterisks (*) indicates a cross-reacting host-protein band (c) Stable S2 cell lines either expressing CrPV-1A-3FH (induced with CuSO4) or uninduced control was lysed, immunopurified with anti-Flag antibody followed by Talon magnetic bead purification. The silver stained gel shows the co-purification of the CrPV-1A and Argonaute 2 (Ago2b isoform) from S2 cells (right lane). Left lane represents the molecular weight marker. Asterisk (*) indicates non-specific contaminant band (see Supplementary figure 3).
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Figure 4: CrPV-1A interacts with Argonaute 2 in S2 cells. (a) S2 cells were transfected with plasmids transiently expressing CrPV-1A-3FH (lane 2, 4) or empty vector control (lane 1, 3). Flag-His tagged CrPV-1A was immunopurified with anti-Flag antibody and western blotted with Drosophila Argonaute 2 (α-Ago2, right panel) or Argonaute 1 (α-Ago1, left panel) polyclonal antibodies. Two isoforms of Ago2 (Ago2a and Ago2b) were detected with Ago2 specific antibodies. Asterisks (*) indicate cross-reacting host-protein band. Lane 6 and 8 show the positive detection of E.coli expressed Drosophila Ago1 and endogenous Drosophila Ago2 with Ago1 and Ago2 antibody respectively (Ab control). (b) Flag-His tagged CrPV-1A148, CrPV-1A108 were transfected in to S2 cells, immunopurified using anti-Flag antibody and probed with Ago2 antibody (α-Ago2). To detect the expression of 1A protein, the blot was stripped and further probed with anti-Flag antibody (α-Flag) (lane 3 and lane 4, bottom panel). Asterisks (*) indicates a cross-reacting host-protein band (c) Stable S2 cell lines either expressing CrPV-1A-3FH (induced with CuSO4) or uninduced control was lysed, immunopurified with anti-Flag antibody followed by Talon magnetic bead purification. The silver stained gel shows the co-purification of the CrPV-1A and Argonaute 2 (Ago2b isoform) from S2 cells (right lane). Left lane represents the molecular weight marker. Asterisk (*) indicates non-specific contaminant band (see Supplementary figure 3).

Mentions: We hypothesized that CrPV-1A inhibits RISC activity by associating with one or more components of holo-RISC. To examine this possibility, CrPV-1A carrying a C-terminal tandem affinity (Flag and His) purification tag (CrPV-1A-3FH) was immunoisolated from S2 cells. Western blot analysis using Ago2 specific antibody revealed that CrPV-1A co-immunoprecipitated with Ago2 (Fig. 4a, lane 4, right panel). In contrast, an antibody directed against Ago1 did not detect any Ago1 association with CrPV1A (Fig. 4a. lane 2, left panel), consistent with our results showing that CrPV-1A does not affect the Ago1-dependent miRNA pathway. The Ago2 antibody used detected two Ago2 forms of 130 kDa and 95 kDa (Obbard et al., 2006). It is possible that the smaller form is the product of partial proteolysis during affinity tag purification, as we often detect 95 kDa isoform (Fig 4b and Fig 4c). To further analyze the interaction between CrPV-1A and Ago2, we transiently expressed CrPV-1A148-3FH and CrPV-1A108-3FH in S2 cells. While full length CrPV-1A148-3HF co-precipitated with Ago2, the truncated CrPV-1A108 was unable to interact with Ago2 (Fig 4b, lane 2). The interaction of CrPV-1A with Ago2 was further analyzed by two-step affinity purification from S2 extracts stably expressing CrPV-1A148-3FH. Commassie staining of the purified materials revealed the presence of only two distinct bands not present in the control sample (Supplementary Fig. 3). Mass-spectroscopy analysis identified the two bands as CrPV-1A and Ago2. Furthermore, silver staining of CrPV-1A associated proteins uncovered no additional proteins (Fig. 4c, lane 2). These experiments suggested that CrPV-1A blocks catalytic activity of the RISC by direct interaction with endonuclease Ago2.


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 interacts with Argonaute 2 in S2 cells. (a) S2 cells were transfected with plasmids transiently expressing CrPV-1A-3FH (lane 2, 4) or empty vector control (lane 1, 3). Flag-His tagged CrPV-1A was immunopurified with anti-Flag antibody and western blotted with Drosophila Argonaute 2 (α-Ago2, right panel) or Argonaute 1 (α-Ago1, left panel) polyclonal antibodies. Two isoforms of Ago2 (Ago2a and Ago2b) were detected with Ago2 specific antibodies. Asterisks (*) indicate cross-reacting host-protein band. Lane 6 and 8 show the positive detection of E.coli expressed Drosophila Ago1 and endogenous Drosophila Ago2 with Ago1 and Ago2 antibody respectively (Ab control). (b) Flag-His tagged CrPV-1A148, CrPV-1A108 were transfected in to S2 cells, immunopurified using anti-Flag antibody and probed with Ago2 antibody (α-Ago2). To detect the expression of 1A protein, the blot was stripped and further probed with anti-Flag antibody (α-Flag) (lane 3 and lane 4, bottom panel). Asterisks (*) indicates a cross-reacting host-protein band (c) Stable S2 cell lines either expressing CrPV-1A-3FH (induced with CuSO4) or uninduced control was lysed, immunopurified with anti-Flag antibody followed by Talon magnetic bead purification. The silver stained gel shows the co-purification of the CrPV-1A and Argonaute 2 (Ago2b isoform) from S2 cells (right lane). Left lane represents the molecular weight marker. Asterisk (*) indicates non-specific contaminant band (see Supplementary figure 3).
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Figure 4: CrPV-1A interacts with Argonaute 2 in S2 cells. (a) S2 cells were transfected with plasmids transiently expressing CrPV-1A-3FH (lane 2, 4) or empty vector control (lane 1, 3). Flag-His tagged CrPV-1A was immunopurified with anti-Flag antibody and western blotted with Drosophila Argonaute 2 (α-Ago2, right panel) or Argonaute 1 (α-Ago1, left panel) polyclonal antibodies. Two isoforms of Ago2 (Ago2a and Ago2b) were detected with Ago2 specific antibodies. Asterisks (*) indicate cross-reacting host-protein band. Lane 6 and 8 show the positive detection of E.coli expressed Drosophila Ago1 and endogenous Drosophila Ago2 with Ago1 and Ago2 antibody respectively (Ab control). (b) Flag-His tagged CrPV-1A148, CrPV-1A108 were transfected in to S2 cells, immunopurified using anti-Flag antibody and probed with Ago2 antibody (α-Ago2). To detect the expression of 1A protein, the blot was stripped and further probed with anti-Flag antibody (α-Flag) (lane 3 and lane 4, bottom panel). Asterisks (*) indicates a cross-reacting host-protein band (c) Stable S2 cell lines either expressing CrPV-1A-3FH (induced with CuSO4) or uninduced control was lysed, immunopurified with anti-Flag antibody followed by Talon magnetic bead purification. The silver stained gel shows the co-purification of the CrPV-1A and Argonaute 2 (Ago2b isoform) from S2 cells (right lane). Left lane represents the molecular weight marker. Asterisk (*) indicates non-specific contaminant band (see Supplementary figure 3).
Mentions: We hypothesized that CrPV-1A inhibits RISC activity by associating with one or more components of holo-RISC. To examine this possibility, CrPV-1A carrying a C-terminal tandem affinity (Flag and His) purification tag (CrPV-1A-3FH) was immunoisolated from S2 cells. Western blot analysis using Ago2 specific antibody revealed that CrPV-1A co-immunoprecipitated with Ago2 (Fig. 4a, lane 4, right panel). In contrast, an antibody directed against Ago1 did not detect any Ago1 association with CrPV1A (Fig. 4a. lane 2, left panel), consistent with our results showing that CrPV-1A does not affect the Ago1-dependent miRNA pathway. The Ago2 antibody used detected two Ago2 forms of 130 kDa and 95 kDa (Obbard et al., 2006). It is possible that the smaller form is the product of partial proteolysis during affinity tag purification, as we often detect 95 kDa isoform (Fig 4b and Fig 4c). To further analyze the interaction between CrPV-1A and Ago2, we transiently expressed CrPV-1A148-3FH and CrPV-1A108-3FH in S2 cells. While full length CrPV-1A148-3HF co-precipitated with Ago2, the truncated CrPV-1A108 was unable to interact with Ago2 (Fig 4b, lane 2). The interaction of CrPV-1A with Ago2 was further analyzed by two-step affinity purification from S2 extracts stably expressing CrPV-1A148-3FH. Commassie staining of the purified materials revealed the presence of only two distinct bands not present in the control sample (Supplementary Fig. 3). Mass-spectroscopy analysis identified the two bands as CrPV-1A and Ago2. Furthermore, silver staining of CrPV-1A associated proteins uncovered no additional proteins (Fig. 4c, lane 2). These experiments suggested that CrPV-1A blocks catalytic activity of the RISC by direct interaction with endonuclease Ago2.

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