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Suppression of RNAi by dsRNA-degrading RNaseIII enzymes of viruses in animals and plants.

Weinheimer I, Jiu Y, Rajamäki ML, Matilainen O, Kallijärvi J, Cuellar WJ, Lu R, Saarma M, Holmberg CI, Jäntti J, Valkonen JP - PLoS Pathog. (2015)

Bottom Line: Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues.However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans.These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland.

ABSTRACT
Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.

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Ability of CSR3 and CSR3-Ala to interfere with gfp silencing in gfp-transgenic C. elegans (strain RT476) expressing GFP under an intestine-specific promoter.(A, B) Upper panel: representative images of C. elegans intestine using bright field (BF) illumination or UV light to observe fluorescence of GFP or the red fluorescent protein dTomato. Lower panel: normalized average intensity of GFP fluorescence in four independent experiments. Error bars indicated S.E. (n = 35–50). In (A), the gfp-transgenic strain RT476 was transformed to express CSR3 or CSR3-Ala under a heat shock—inducible promoter, and two transgenic lines expressing each protein were included in experiments. Sense-mediated silencing of gfp expression was induced in the nematodes by feeding them E. coli expressing gfp mRNA (RNAi), which reduced GFP fluorescence significantly (gfp-transgenic nematodes fed bacteria harboring an insert-less plasmid were used as a control) (Student’s t-test, p < 0.001). At 72 h, expression of CSR3 and CSR3-Ala was induced in the gfp-silenced nematodes by heat shock. At 24 h post-induction, however, no significant difference (Student’s t-test) in GFP fluorescence was detected in nematodes expressing CSR3 or CSR3-Ala as compared with the gfp-silenced strain (RNAi). In (B), similar experiments as above were carried out with the gfp-transgenic strain RT476 transformed to express CSR3 or CSR3-Ala fused with dTomato. No recovery of GFP fluorescence was observed. (C) Immunoblot analysis using a rabbit polyclonal antibody specific to CSR3 detected the dTomato-CSR3 fusion protein in nematodes (B) in which dTomato-CSR3 expression was induced. (D) Simultaneous induction of CSR3 expression and GFP-silencing. CSR3 was not able to delay silencing.
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ppat.1004711.g005: Ability of CSR3 and CSR3-Ala to interfere with gfp silencing in gfp-transgenic C. elegans (strain RT476) expressing GFP under an intestine-specific promoter.(A, B) Upper panel: representative images of C. elegans intestine using bright field (BF) illumination or UV light to observe fluorescence of GFP or the red fluorescent protein dTomato. Lower panel: normalized average intensity of GFP fluorescence in four independent experiments. Error bars indicated S.E. (n = 35–50). In (A), the gfp-transgenic strain RT476 was transformed to express CSR3 or CSR3-Ala under a heat shock—inducible promoter, and two transgenic lines expressing each protein were included in experiments. Sense-mediated silencing of gfp expression was induced in the nematodes by feeding them E. coli expressing gfp mRNA (RNAi), which reduced GFP fluorescence significantly (gfp-transgenic nematodes fed bacteria harboring an insert-less plasmid were used as a control) (Student’s t-test, p < 0.001). At 72 h, expression of CSR3 and CSR3-Ala was induced in the gfp-silenced nematodes by heat shock. At 24 h post-induction, however, no significant difference (Student’s t-test) in GFP fluorescence was detected in nematodes expressing CSR3 or CSR3-Ala as compared with the gfp-silenced strain (RNAi). In (B), similar experiments as above were carried out with the gfp-transgenic strain RT476 transformed to express CSR3 or CSR3-Ala fused with dTomato. No recovery of GFP fluorescence was observed. (C) Immunoblot analysis using a rabbit polyclonal antibody specific to CSR3 detected the dTomato-CSR3 fusion protein in nematodes (B) in which dTomato-CSR3 expression was induced. (D) Simultaneous induction of CSR3 expression and GFP-silencing. CSR3 was not able to delay silencing.

Mentions: Expression of CSR3 or CSR3-Ala did not result in recovery of GFP fluorescence in gfp-silenced C. elegans, as observed 24 h post-induction of the viral protein expression (Fig. 5A). When CSR3 and CSR3-Ala were expressed as a fusion product with the red fluorescent protein dTomato [29] in C. elegans strain RT476, the readily detectable red fluorescence occurring 24 h after induction indicated high levels of protein expression. As observed before, GFP fluorescence did not recover in the four independent experiments (Fig. 5B). Immunoblotting revealed high amounts of dTomato-CSR3 in the nematodes by 3 h post-induction followed by a gradual decline (Fig. 5C), which is consistent with previous studies carried out using the same promoter [30, 31]. Taken together, these results suggested that CSR3 was unable to reverse sense-mediated silencing in C. elegans.


Suppression of RNAi by dsRNA-degrading RNaseIII enzymes of viruses in animals and plants.

Weinheimer I, Jiu Y, Rajamäki ML, Matilainen O, Kallijärvi J, Cuellar WJ, Lu R, Saarma M, Holmberg CI, Jäntti J, Valkonen JP - PLoS Pathog. (2015)

Ability of CSR3 and CSR3-Ala to interfere with gfp silencing in gfp-transgenic C. elegans (strain RT476) expressing GFP under an intestine-specific promoter.(A, B) Upper panel: representative images of C. elegans intestine using bright field (BF) illumination or UV light to observe fluorescence of GFP or the red fluorescent protein dTomato. Lower panel: normalized average intensity of GFP fluorescence in four independent experiments. Error bars indicated S.E. (n = 35–50). In (A), the gfp-transgenic strain RT476 was transformed to express CSR3 or CSR3-Ala under a heat shock—inducible promoter, and two transgenic lines expressing each protein were included in experiments. Sense-mediated silencing of gfp expression was induced in the nematodes by feeding them E. coli expressing gfp mRNA (RNAi), which reduced GFP fluorescence significantly (gfp-transgenic nematodes fed bacteria harboring an insert-less plasmid were used as a control) (Student’s t-test, p < 0.001). At 72 h, expression of CSR3 and CSR3-Ala was induced in the gfp-silenced nematodes by heat shock. At 24 h post-induction, however, no significant difference (Student’s t-test) in GFP fluorescence was detected in nematodes expressing CSR3 or CSR3-Ala as compared with the gfp-silenced strain (RNAi). In (B), similar experiments as above were carried out with the gfp-transgenic strain RT476 transformed to express CSR3 or CSR3-Ala fused with dTomato. No recovery of GFP fluorescence was observed. (C) Immunoblot analysis using a rabbit polyclonal antibody specific to CSR3 detected the dTomato-CSR3 fusion protein in nematodes (B) in which dTomato-CSR3 expression was induced. (D) Simultaneous induction of CSR3 expression and GFP-silencing. CSR3 was not able to delay silencing.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352025&req=5

ppat.1004711.g005: Ability of CSR3 and CSR3-Ala to interfere with gfp silencing in gfp-transgenic C. elegans (strain RT476) expressing GFP under an intestine-specific promoter.(A, B) Upper panel: representative images of C. elegans intestine using bright field (BF) illumination or UV light to observe fluorescence of GFP or the red fluorescent protein dTomato. Lower panel: normalized average intensity of GFP fluorescence in four independent experiments. Error bars indicated S.E. (n = 35–50). In (A), the gfp-transgenic strain RT476 was transformed to express CSR3 or CSR3-Ala under a heat shock—inducible promoter, and two transgenic lines expressing each protein were included in experiments. Sense-mediated silencing of gfp expression was induced in the nematodes by feeding them E. coli expressing gfp mRNA (RNAi), which reduced GFP fluorescence significantly (gfp-transgenic nematodes fed bacteria harboring an insert-less plasmid were used as a control) (Student’s t-test, p < 0.001). At 72 h, expression of CSR3 and CSR3-Ala was induced in the gfp-silenced nematodes by heat shock. At 24 h post-induction, however, no significant difference (Student’s t-test) in GFP fluorescence was detected in nematodes expressing CSR3 or CSR3-Ala as compared with the gfp-silenced strain (RNAi). In (B), similar experiments as above were carried out with the gfp-transgenic strain RT476 transformed to express CSR3 or CSR3-Ala fused with dTomato. No recovery of GFP fluorescence was observed. (C) Immunoblot analysis using a rabbit polyclonal antibody specific to CSR3 detected the dTomato-CSR3 fusion protein in nematodes (B) in which dTomato-CSR3 expression was induced. (D) Simultaneous induction of CSR3 expression and GFP-silencing. CSR3 was not able to delay silencing.
Mentions: Expression of CSR3 or CSR3-Ala did not result in recovery of GFP fluorescence in gfp-silenced C. elegans, as observed 24 h post-induction of the viral protein expression (Fig. 5A). When CSR3 and CSR3-Ala were expressed as a fusion product with the red fluorescent protein dTomato [29] in C. elegans strain RT476, the readily detectable red fluorescence occurring 24 h after induction indicated high levels of protein expression. As observed before, GFP fluorescence did not recover in the four independent experiments (Fig. 5B). Immunoblotting revealed high amounts of dTomato-CSR3 in the nematodes by 3 h post-induction followed by a gradual decline (Fig. 5C), which is consistent with previous studies carried out using the same promoter [30, 31]. Taken together, these results suggested that CSR3 was unable to reverse sense-mediated silencing in C. elegans.

Bottom Line: Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues.However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans.These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland.

ABSTRACT
Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.

Show MeSH
Related in: MedlinePlus