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

Isabel Weinheimer,Rui Lu,Jukka Kallijärvi,Olli Matilainen,Jussi Jäntti,Jari P T Valkonen,Carina I Holmberg,Wilmer J Cuellar,Mart Saarma,Yaming Jiu,Minna-Liisa Rajamäki

doi:10.1371/journal.ppat.1004711

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.

Highlights

Eukaryotic RNA interference (RNAi) pathways are activated by double-stranded RNA and function in gene regulation and antiviral defense [1,2,3,4,5]
RNA interference (RNAi) is a cellular mechanism activated by double-stranded RNA
Our results underscore that the active viral RNaseIII enzymes suppress RNAi

Summary

Introduction

Eukaryotic RNA interference (RNAi) pathways are activated by double-stranded RNA (dsRNA) and function in gene regulation and antiviral defense [1,2,3,4,5]. Genes can be silenced via dsRNA as demonstrated in the nematode Caenorhabditis elegans [6] and the fruit fly Drosophila melanogaster [7], whereas in plants post-transcriptional gene silencing can be induced by homologous antisense or positive-sense single-stranded RNA (ssRNA) [8]. Induction of sense-mediated RNAi typically requires the activity of a cellular RNA-dependent RNA polymerase (RdRp) for synthesis of dsRNA on the sense RNA transcript [9]. Dicers recognize dsRNA and process it into double-stranded small interfering RNAs (ds-siRNAs) that are 21–25 nucleotides (nt) long [1, 12]. RdRp helps to amplify RNAi via production of secondary triggers of RNAi derived from cleaved RNA in plants and nematodes (C. elegans) [12, 15] and contributes to the generation of secondary siRNAs acting as mobile signals for systemic RNAi in plants, nematodes, and possibly insects (D. melanogaster) [5, 16]

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Discussion

Conclusion