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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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Suppression of RNAi in C. elegans by PPR3 and PPR3-Ala.The gfp-transgenic strain RT476 was transformed to express PPR3 or PPR3-Ala under a heat shock—inducible promoter. Sense-mediated silencing of gfp expression was induced in the nematodes by feeding them E. coli expressing gfp mRNA (RNAi). In Control, gfp-transgenic nematodes were fed bacteria harboring an insert-less plasmid. After 24 h, expression of PPR3 and PPR3-Ala was induced by heat shock in the gfp-silenced nematodes, which recovered GFP fluorescence by 72 h post-induction. (A) gfp-transgenic C. elegans (strain RT476) in bright field (BF) to observe morphology or under UV illumination to observe GFP fluorescence. The anterior intestine and posterior intestine are oriented to the left and right, respectively (see also Fig. 3B). (B) Normalized average GFP fluorescence intensity (FI) shown using arbitrary units (A.U.) in four independent experiments. Two transgenic lines expressing each protein were included in the experiments. Error bars indicate S.E. (n = 38–50). GFP fluorescence intensity is significantly higher in the nematodes induced to express PPR3 than PPR3-Ala, but in both it is higher than in uninduced controls (Student’s t-test, p < 0.001). Dashed line indicates the level of GFP fluorescence in control.
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ppat.1004711.g004: Suppression of RNAi in C. elegans by PPR3 and PPR3-Ala.The gfp-transgenic strain RT476 was transformed to express PPR3 or PPR3-Ala under a heat shock—inducible promoter. Sense-mediated silencing of gfp expression was induced in the nematodes by feeding them E. coli expressing gfp mRNA (RNAi). In Control, gfp-transgenic nematodes were fed bacteria harboring an insert-less plasmid. After 24 h, expression of PPR3 and PPR3-Ala was induced by heat shock in the gfp-silenced nematodes, which recovered GFP fluorescence by 72 h post-induction. (A) gfp-transgenic C. elegans (strain RT476) in bright field (BF) to observe morphology or under UV illumination to observe GFP fluorescence. The anterior intestine and posterior intestine are oriented to the left and right, respectively (see also Fig. 3B). (B) Normalized average GFP fluorescence intensity (FI) shown using arbitrary units (A.U.) in four independent experiments. Two transgenic lines expressing each protein were included in the experiments. Error bars indicate S.E. (n = 38–50). GFP fluorescence intensity is significantly higher in the nematodes induced to express PPR3 than PPR3-Ala, but in both it is higher than in uninduced controls (Student’s t-test, p < 0.001). Dashed line indicates the level of GFP fluorescence in control.

Mentions: Strain RT476 was stably transformed with a gene encoding PPR3, PPR3-Ala, CSR3, or CSR3-Ala placed under the heat shock—inducible promoter mtl-2 [28]. Two independent transgenic lines of the same strain expressing each protein were used for the experiments. Silencing of gfp was induced by feeding the nematodes with bacteria expressing gfp mRNA, and 72 h later expression of the viral protein was induced by heat shock. GFP fluorescence was observed 24 h after inducing viral protein expression. In four independent experiments, PPR3 restored GFP expression, as indicated by GFP fluorescence intensity that was similar to the gfp-transgenic strain RT476 fed bacteria containing a plasmid lacking an insert (control; Fig. 4). PPR3-Ala partially restored GFP expression, with GFP fluorescence intensity attaining ~75% of the level measured in the control. In contrast, no enhancement of GFP fluorescence was observed in the gfp-silenced nematodes that were transformed with PPR3 or PPR3-Ala but not induced to express these proteins (Fig. 4).


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)

Suppression of RNAi in C. elegans by PPR3 and PPR3-Ala.The gfp-transgenic strain RT476 was transformed to express PPR3 or PPR3-Ala under a heat shock—inducible promoter. Sense-mediated silencing of gfp expression was induced in the nematodes by feeding them E. coli expressing gfp mRNA (RNAi). In Control, gfp-transgenic nematodes were fed bacteria harboring an insert-less plasmid. After 24 h, expression of PPR3 and PPR3-Ala was induced by heat shock in the gfp-silenced nematodes, which recovered GFP fluorescence by 72 h post-induction. (A) gfp-transgenic C. elegans (strain RT476) in bright field (BF) to observe morphology or under UV illumination to observe GFP fluorescence. The anterior intestine and posterior intestine are oriented to the left and right, respectively (see also Fig. 3B). (B) Normalized average GFP fluorescence intensity (FI) shown using arbitrary units (A.U.) in four independent experiments. Two transgenic lines expressing each protein were included in the experiments. Error bars indicate S.E. (n = 38–50). GFP fluorescence intensity is significantly higher in the nematodes induced to express PPR3 than PPR3-Ala, but in both it is higher than in uninduced controls (Student’s t-test, p < 0.001). Dashed line indicates the level of GFP fluorescence in control.
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Related In: Results  -  Collection

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ppat.1004711.g004: Suppression of RNAi in C. elegans by PPR3 and PPR3-Ala.The gfp-transgenic strain RT476 was transformed to express PPR3 or PPR3-Ala under a heat shock—inducible promoter. Sense-mediated silencing of gfp expression was induced in the nematodes by feeding them E. coli expressing gfp mRNA (RNAi). In Control, gfp-transgenic nematodes were fed bacteria harboring an insert-less plasmid. After 24 h, expression of PPR3 and PPR3-Ala was induced by heat shock in the gfp-silenced nematodes, which recovered GFP fluorescence by 72 h post-induction. (A) gfp-transgenic C. elegans (strain RT476) in bright field (BF) to observe morphology or under UV illumination to observe GFP fluorescence. The anterior intestine and posterior intestine are oriented to the left and right, respectively (see also Fig. 3B). (B) Normalized average GFP fluorescence intensity (FI) shown using arbitrary units (A.U.) in four independent experiments. Two transgenic lines expressing each protein were included in the experiments. Error bars indicate S.E. (n = 38–50). GFP fluorescence intensity is significantly higher in the nematodes induced to express PPR3 than PPR3-Ala, but in both it is higher than in uninduced controls (Student’s t-test, p < 0.001). Dashed line indicates the level of GFP fluorescence in control.
Mentions: Strain RT476 was stably transformed with a gene encoding PPR3, PPR3-Ala, CSR3, or CSR3-Ala placed under the heat shock—inducible promoter mtl-2 [28]. Two independent transgenic lines of the same strain expressing each protein were used for the experiments. Silencing of gfp was induced by feeding the nematodes with bacteria expressing gfp mRNA, and 72 h later expression of the viral protein was induced by heat shock. GFP fluorescence was observed 24 h after inducing viral protein expression. In four independent experiments, PPR3 restored GFP expression, as indicated by GFP fluorescence intensity that was similar to the gfp-transgenic strain RT476 fed bacteria containing a plasmid lacking an insert (control; Fig. 4). PPR3-Ala partially restored GFP expression, with GFP fluorescence intensity attaining ~75% of the level measured in the control. In contrast, no enhancement of GFP fluorescence was observed in the gfp-silenced nematodes that were transformed with PPR3 or PPR3-Ala but not induced to express these proteins (Fig. 4).

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