Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; family Closteroviridae) consists of a bipartite single-stranded RNA (ssRNA) genome. Open reading frames in RNA1 encode a Class 1 RNase III-like protein (designated as RNase3) and a 22 kDa protein (designated as p22). RNase3 and p22 suppress RNA silencing, the basal antiviral defense mechanism in plants. RNase3 is sufficient to render sweet potato (Ipomoea batatas) virus-susceptible and leads to the development of a severe disease (sweet potato virus disease - SPVD) following infection with unrelated viruses. This study aimed to identify how the genetic variability of silencing supressor genes in different SPCSV isolates affected the ability to suppress RNAi. No differences in silencing suppression capacities were detected for p22 and RNase3 proteins of tested SPCSV isolates. Furthermore, a new RNA virus (that belongs to the genus Crinivirus) was found to encode an RNase3 homolog functional in the suppression of RNAi. --- The double-stranded RNA (dsRNA)-specific RNase3 eliminates antiviral defense in sweet potato in an endoribonuclease activity dependent manner. RNase3 cleaves long dsRNA molecules, synthetic small-interfering RNAs (siRNAs), and plant- and virus-derived siRNAs. Studies in Nicotiana benthamiana have indicated that RNase3 inhibits only sense-transcript-induced post-transcriptional gene silencing (S-PTGS). In this study, conditions for efficient expression and purification of recombinant RNase3 were determined to establish activity assays for characterization of substrate specificity in vitro. RNA-binding was dependent on the dimerization of the protein confirming the affiliation of RNase3 to functional Class1 of RNase III enzymes. RNA-processing was assessed on a range of small dsRNA molecules consisting of mature double-stranded siRNA (ds-siRNA) and microRNA (miRNA) and revealed that small RNA (sRNA) containing asymmetric bulges (as they often appear in miRNA) and methylation did not support cleavage. ------ Some viruses infecting insects, fish and poikilothermic animals encode Class 1 RNase III-like molecules among which ascovirus RNase III was recently characterized as a silencing suppressor. It is still unknown how the different viral RNase III-type molecules act in the host cells. In vivo experiments carried out in N. benthamiana, Drosophila melanogaster and Caenorhabditis elegans revealed that a putative RNase3 homolog encoded by pike-perch iridovirus (designated as PPIV RNase3; genus Ranavirus; family Iridoviridae) was a Class 1 RNase III protein and suppressed RNAi in C. elegans and plant tissues in an endoribonuclease-dependent manner. However, SPCSV RNase3 only suppressed RNAi (in detail, S-PTGS) in plants. The viral Class 1 RNase III-like enzymes appear unique as homologous proteins produced by RNA and DNA viruses and are able to suppress RNAi in plants and animals. -------- The function of SPCSV RNase3 in RNAi suppression (limited to S-PTGS in plants) was postulated to be facilitated through localization or interaction with plant-host factors rather than substrate specificity. Experiments in N. benthamiana revealed that RNase3 localized in sub-cellular structures (punctate granules), which co-localized with characteristic granules that are implicated in antiviral defense and contain SGS3 and RDR6 proteins. Bimolecular fluorescence complementation studies revealed interaction of RNase3 with SGS3. However, no interference with associated trans-acting siRNA production or transitivity was detected.
Keywords: Ipomoea, RNase III, RNAi, suppression of silencing, plant virus, Ranavirus, siRNA, miRNA, protein-protein interaction, SGS3, C. elegans.available shortl
