Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity

Agricultural Biotechnology Center, Plant Science Institute, P.O. Box 411, H-2101 Gödöllö, Hungary.
Journal of Virology (Impact Factor: 4.44). 07/2005; 79(11):7217-26. DOI: 10.1128/JVI.79.11.7217-7226.2005
Source: PubMed


RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.

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Available from: Zoltan Kerenyi, Oct 13, 2015
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    • "Both of these proteins are required for genomic RNA replication and subgenomic (sg) mRNA transcription (Reade et al., 2003). The capsid protein (p41) is translated from sg mRNA1 while overlapping silencing suppressor (p27) and movement (p19) proteins are translated from sg mRNA2; both of which are transcribed during infections (Merai et al., 2005; Xu and White, 2008, 2009). Interestingly, although aureusviruses are similar in genomic structure to tombusvirues , no DI RNAs have been found associated with any aureusvirus, even when passaged at high moi (Rubino and Russo, 1997). "
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    ABSTRACT: Defective RNAs (D RNAs) are small RNA replicons derived from viral RNA genomes. No D RNAs have been found associated with members of the plus-strand RNA virus genus Aureusvirus (family Tombusviridae). Accordingly, we sought to construct a D RNA for the aureusvirus Cucumber leaf spot virus (CLSV) using the known structure of tombusvirus defective interfering RNAs as a guide. An efficiently accumulating CLSV D RNA was generated that contained four non-contiguous regions of the viral genome and this replicon was used as a tool to studying viral cis-acting RNA elements. The results of structural and functional analyses indicated that CLSV contains counterparts for several of the major RNA elements found in tombusviruses. However, although similar, the CLSV D RNA and its components are distinct and provide insights into RNA-based specificity and mechanisms of function.
    Virology 03/2014; s 452–453:67–74. DOI:10.1016/j.virol.2013.12.033 · 3.32 Impact Factor
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    • "The NMD test constructs were agroinfiltrated into N. benthamiana leaves only with a P14 RNA silencing suppressor or were co-infiltrated with P14 and U1DN (referred to as control and U1DN co-infiltrated samples, respectively). P14 was co-infiltrated in these experiments for two reasons: (i) to suppress agroinfiltration-induced intense RNA silencing (39) and (ii) to serve as an internal control for RNA gel blot assays. (Although P14 was co-infiltrated in all assays shown in this article, it will not be mentioned later in the main text. "
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    ABSTRACT: Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3'untranslated region (UTR). In yeasts, unusually long 3'UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3'UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3'UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD.
    Nucleic Acids Research 05/2013; 41(13). DOI:10.1093/nar/gkt366 · 9.11 Impact Factor
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    • "During the last few years, the p69 encoded by Turnip yellow mosaic virus has been identified as a silencing suppressor preventing host RDRdependent secondary dsRNA synthesis (Chen et al., 2004). P14 protein encoded by Aureus viruses suppressed both virus and transgene-induced silencing by sequestering both long dsRNA and siRNA without size specificity (Merai et al., 2005). Multiple suppressors have been reported in the Citrus tristeza virus, where p20 and coat protein (CP) play important roles in suppression of the silencing signal, and p23 inhibited intracellular silencing (Lu et al., 2004). "
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    ABSTRACT: Plant diseases pose a huge threat to crop production globally. Variations in their genomes cause selection to favor those who can survive pesticides and Bacillus thuringiensis (Bt) crops. Though plant breeding has been the classical means of manipulating the plant genome to develop resistant cultivar for controlling plant diseases, the advent of genetic engineering provides an entirely new approach being pursued to render plants resistant to fungi, bacteria, viruses and nematodes. RNA interference (RNAi) technology has emerged to be a promising therapeutic weapon to mitigate the inherent risks such as the use of a specific transgene, marker gene, or gene control sequences associated with development of traditional transgenics. Silencing specific genes by RNAi is a desirable natural solution to this problem as disease resistant transgenic plants can be produced within a regulatory framework. Recent studies have been successful in producing potent silencing effects by using target doublestranded RNAs through an effective vector system. Transgenic plants expressing RNAi vectors, as well as, dsRNA containing crop sprays have been successful for efficient control of plant pathogens affecting economically important crop species. The present paper discusses strategies and applications of this novel technology in plant disease management for sustainable agriculture production.
    AFRICAN JOURNAL OF BIOTECHNOLOGY 05/2013; 12((18)):2303-2312. DOI:10.5897/AJB12.2791 · 0.57 Impact Factor
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