Netsu Osamu, Hiraguri Akihiro, Uehara-Ichiki Tamaki, Komatsu Ken, Sasaya Takahide
National Agricultural Research Center, Kannondai, Tsukuba, Ibaraki 305-8666, Japan; Laboratory of Plant Pathology, Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
National Agricultural Research Center, Kannondai, Tsukuba, Ibaraki 305-8666, Japan; Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Virus Res. 2015 May 4;203:10-9. doi: 10.1016/j.virusres.2015.03.010. Epub 2015 Mar 30.
Rice grassy stunt virus (RGSV) is a member of the genus Tenuivirus, which includes rice stripe virus (RSV), as the type species. A viral suppressor of RNA silencing (VSR) of RGSV has not been identified, whereas the p3 protein of RSV (RSVp3) encoded by the viral-sense (v) strand of RNA3 has been reported to act as a VSR. In this study, we examined the VSR function of the p5 protein of RGSV (RGSVp5), encoded by vRNA5. Expecting it to correspond to the vRNA3 of RSV, we compared the VSR function of RGSVp5 with that of RSVp3. In an Agrobacterium-mediated transient expression assay using a transgenic line of Nicotiana benthamiana that expressed green fluorescent protein and the wild type, RGSVp5 suppressed sense transgene-mediated post-transcriptional gene silencing (S-PTGS), inverted-repeat (IR) transgene-induced PTGS (IR-PTGS), and the systemic spread of GFP silencing, as in the case with RSVp3. By contrast, a gel mobility shift assay revealed that RGSVp5 did not have any distinct binding activity with 21-, 22-, or 24-nucleotide small interfering RNA (siRNA) duplexes, whereas RSVp3 binds to all three sizes of siRNA duplexes. Furthermore, the transiently expressed p5 protein fused with GFP was dispersed mainly in the cytoplasm, whereas the GFP-fused p3 protein of RSV was localized both in the nucleus and in the cytoplasm. Our results suggest that RGSVp5 functions as a VSR but that the suppression mechanism of RNA silencing and the subcellular localization of RGSVp5 differ from those of the analogous VSR, RSVp3, even in the same genus.
水稻草状矮化病毒（RGSV）是纤细病毒属的成员，该属包括作为模式种的水稻条纹病毒（RSV）。尚未鉴定出RGSV的RNA沉默病毒抑制子（VSR），而据报道，由RNA3的病毒正义（v）链编码的RSV的p3蛋白（RSVp3）可作为VSR发挥作用。在本研究中，我们检测了由vRNA5编码的RGSV的p5蛋白（RGSVp5）的VSR功能。由于预期它与RSV的vRNA3相对应，我们比较了RGSVp5和RSVp3的VSR功能。在使用表达绿色荧光蛋白的本氏烟草转基因系和野生型进行的农杆菌介导的瞬时表达试验中，RGSVp5抑制了正义转基因介导的转录后基因沉默（S-PTGS）、反向重复（IR）转基因诱导的PTGS（IR-PTGS）以及GFP沉默的系统传播，这与RSVp3的情况相同。相比之下，凝胶迁移率变动分析表明，RGSVp5与21、22或24个核苷酸的小干扰RNA（siRNA）双链体没有任何明显的结合活性，而RSVp3能与所有三种大小的siRNA双链体结合。此外，与绿色荧光蛋白（GFP）融合的瞬时表达的p5蛋白主要分散在细胞质中，而RSV的GFP融合p3蛋白则定位于细胞核和细胞质中。我们的结果表明，RGSVp5作为VSR发挥作用，但即使在同一属中，RGSVp5的RNA沉默抑制机制和亚细胞定位也与类似的VSR即RSVp3不同。
