Omarov Rustem T, Ciomperlik Jessica J, Scholthof Herman B
Department of Plant Pathology and Microbiology, Texas A&M University, 2132 TAMU, College Station, TX 77843, USA.
Proc Natl Acad Sci U S A. 2007 Jan 30;104(5):1714-9. doi: 10.1073/pnas.0608117104. Epub 2007 Jan 23.
Tomato bushy stunt virus (TBSV) and other tombusviruses encode a p19 protein (P19), which is a suppressor of RNAi. Wild-type TBSV or p19-defective mutants initially show a similar infection course in Nicotiana benthamiana, but the absence of an active P19 results in viral RNA degradation followed by recovery from infection. P19 homodimers sequester 21-nt virus-derived duplex siRNAs, and it is thought that this prevents the programming of an antiviral RNA-induced silencing complex to avoid viral RNA degradation. Here we report on chromatographic fractionation (gel filtration, ion exchange, and hydroxyapatite) of extracts from healthy or infected Nicotiana benthamiana plants in combination with in vitro assays for ribonuclease activity and detection of TBSV-derived siRNAs. Only extracts of plants infected with p19 mutants provided a source of sequence-nonspecific but ssRNA-targeted in vitro ribonuclease activity that coeluted with components of a wide molecular weight range. In addition, we isolated a discrete approximately 500-kDa protein complex that contained approximately 21-nt TBSV-derived siRNAs and that exhibited ribonuclease activity that was TBSV sequence-preferential, ssRNA-specific, divalent cation-dependent, and insensitive to a ribonuclease inhibitor. We believe that this study provides biochemical evidence for a virus-host system that infection in the absence of a fully active RNAi suppressor induces ssRNA-specific ribonuclease activity, including that conferred by a RNA-induced silencing complex, which is likely the cause for the recovery of plants from infection.
番茄丛矮病毒（TBSV）和其他番茄病毒编码一种p19蛋白（P19），它是RNA干扰的抑制因子。野生型TBSV或p19缺陷型突变体最初在本氏烟草中表现出相似的感染过程，但缺乏活性P19会导致病毒RNA降解，随后从感染中恢复。P19同源二聚体隔离21核苷酸的病毒衍生双链小干扰RNA（siRNA），据认为这可防止抗病毒RNA诱导沉默复合体的编程，从而避免病毒RNA降解。在此，我们报道了对健康或感染的本氏烟草植物提取物进行色谱分离（凝胶过滤、离子交换和羟基磷灰石），并结合体外核糖核酸酶活性测定和TBSV衍生siRNA的检测。只有感染p19突变体的植物提取物提供了一种序列非特异性但靶向单链RNA（ssRNA）的体外核糖核酸酶活性来源，该活性与广泛分子量范围的组分共洗脱。此外，我们分离出一种离散的约500 kDa蛋白质复合体，它包含约21核苷酸的TBSV衍生siRNA，并表现出对TBSV序列有偏好、对ssRNA特异、依赖二价阳离子且对核糖核酸酶抑制剂不敏感的核糖核酸酶活性。我们认为，这项研究为病毒 - 宿主系统提供了生化证据，即在缺乏完全活性的RNA干扰抑制因子的情况下，感染会诱导ssRNA特异性核糖核酸酶活性，包括由RNA诱导沉默复合体赋予的活性，这可能是植物从感染中恢复的原因。
