scholarly journals Viral Class 1 RNase III Involved in Suppression of RNA Silencing

2005 ◽  
Vol 79 (11) ◽  
pp. 7227-7238 ◽  
Author(s):  
Jan F. Kreuze ◽  
Eugene I. Savenkov ◽  
Wilmer Cuellar ◽  
Xiangdong Li ◽  
Jari P. T. Valkonen
Keyword(s):  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Rna Virus ◽  
Antiviral Defense ◽  
Rnase Iii ◽  
Double Stranded Rna ◽  
Class 1 ◽  
Heterologous Virus ◽  
Silencing Suppression ◽  
Interfering Rna

ABSTRACT Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.

scholarly journals Multiple Functions of Rice Dwarf Phytoreovirus Pns10 in Suppressing Systemic RNA Silencing

2010 ◽  
Vol 84 (24) ◽  
pp. 12914-12923 ◽  
Author(s):  
Bo Ren ◽  
Yuanyuan Guo ◽  
Feng Gao ◽  
Peng Zhou ◽  
Feng Wu ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Rna Stability ◽  
Antiviral Defense ◽  
Double Stranded Rna ◽  
Systemic Spread ◽  
Dsrna Viruses ◽  
Systemic Rna ◽  
Interfering Rna ◽  
Diverse Groups

ABSTRACT RNA silencing is a potent mechanism of antiviral defense response in plants and other organisms. For counterdefense, viruses have evolved a variety of suppressors of RNA silencing (VSRs) that can inhibit distinct steps of a silencing pathway. We previously identified Pns10 encoded by Rice dwarf phytoreovirus (RDV) as a VSR, the first of its kind from double-stranded RNA (dsRNA) viruses. In this study we investigated the mechanisms of Pns10 function in suppressing systemic RNA silencing in the widely used Nicotiana benthamiana model plant. We report that Pns10 suppresses local and systemic RNA silencing triggered by sense mRNA, enhances viral replication and/or viral RNA stability in inoculated leaves, accelerates the systemic spread of viral infection, and enables viral invasion of shoot apices. Mechanistically, Pns10 interferes with the perception of silencing signals in recipient tissues, binds double-stranded small interfering RNA (siRNAs) with two-nucleotide 3′ overhangs, and causes the downregulated expression of RDR6. These results significantly deepen our mechanistic understanding of the VSR functions encoded by a dsRNA virus and contribute additional evidence that binding siRNAs and interfering with RDR6 expression are broad mechanisms of VSR functions encoded by diverse groups of viruses.

scholarly journals The Consensus N-Myristoylation Motif of a Geminivirus AC4 Protein Is Required for Membrane Binding and Pathogenicity

2007 ◽  
Vol 20 (4) ◽  
pp. 380-391 ◽  
Author(s):  
Vincent N. Fondong ◽  
R. V. Chowda Reddy ◽  
Cheng Lu ◽  
Bertrand Hankoua ◽  
Christian Felton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Membrane Binding ◽  
Confocal Imaging ◽  
East African ◽  
Intrinsic Signals ◽  
Pathogenicity Determinant ◽  
Silencing Suppression

Some geminiviruses encode a small protein, AC4, whose role in pathogenesis has only recently attracted attention. A few studies have shown that this protein is involved in pathogenesis and suppresses RNA silencing. Here, using Nicotiana benthamiana, we show that East African cassava mosaic Cameroon virus (EACMCV) AC4 is a pathogenicity determinant and that it suppresses the systemic phase of RNA silencing. Furthermore, confocal imaging analyses show that it binds preferentially to the plasma membrane as well as to cytosolic membranes including the perinucleus but is excluded from the nucleus. A computational examination of the AC4 protein encoded by the EACMCV, a bipartite geminivirus, shows that it encodes a consensus N-myristoylation motif and is likely posttranslationally myristoylated and palmitoylated. Replacement of Gly-2 and Cys-3 (sites of posttranslational attachment of myristic and palmatic acids, respectively) with alanine affected AC4 membrane binding and pathogenesis. Furthermore, replacement of Ile-5, a nonessential myristoylation residue, with alanine did not affect AC4 function. Together, these data indicate that EACMCV AC4 is likely dually acylated at Gly-2 and Cys-3 and that these modifications are intrinsic signals for membrane targeting and pathogenesis. This is the first report of a membrane protein to be involved in pathogenesis and RNA silencing suppression.

scholarly journals Aspergillus Mycoviruses Are Targets and Suppressors of RNA Silencing

2007 ◽  
Vol 7 (2) ◽  
pp. 350-357 ◽  
Author(s):  
T. M. Hammond ◽  
M. D. Andrewski ◽  
M. J. Roossinck ◽  
N. P. Keller
Keyword(s):  
Rna Silencing ◽  
Agaricus Bisporus ◽  
Defense Mechanism ◽  
Rna Virus ◽  
Physiological Changes ◽  
Button Mushroom ◽  
Diverse Range ◽  
The Third ◽  
Sphaeropsis Sapinea ◽  
Interfering Rna

ABSTRACT RNA silencing can function as a virus defense mechanism in a diverse range of eukaryotes, and many viruses are capable of suppressing the silencing machinery targeting them. However, the extent to which this occurs between fungal RNA silencing and mycoviruses is unclear. Here, three Aspergillus dsRNA mycoviruses were partially characterized, and their relationship to RNA silencing was investigated. Aspergillus virus 1816 is related to Agaricus bisporus white button mushroom virus 1 and suppresses RNA silencing through a mechanism that alters the level of small interfering RNA. Aspergillus virus 178 is related to RNA virus L1 of Gremmeniella abietina and does not appear to affect RNA silencing. The third virus investigated, Aspergillus virus 341, is distantly related to Sphaeropsis sapinea RNA virus 2. Detection of mycovirus-derived siRNA from this mycovirus demonstrates that it is targeted for degradation by the Aspergillus RNA silencing machinery. Thus, our results indicate that Aspergillus mycoviruses are both targets and suppressors of RNA silencing. In addition, they suggest that the morphological and physiological changes associated with some mycoviruses could be a result of their antagonistic relationship with RNA silencing.

scholarly journals Antiviral Defense Involves AGO4 in an Arabidopsis–Potexvirus Interaction

2016 ◽  
Vol 29 (11) ◽  
pp. 878-888 ◽  
Author(s):  
Chantal Brosseau ◽  
Mohamed El Oirdi ◽  
Ayooluwa Adurogbangba ◽  
Xiaofang Ma ◽  
Peter Moffett
Keyword(s):  
Gene Expression ◽  
Nuclear Localization ◽  
Rna Silencing ◽  
Defense Mechanism ◽  
Rna Viruses ◽  
Antiviral Defense ◽  
Double Stranded Rna ◽  
Micro Rnas ◽  
Plantago Asiatica ◽  
Antiviral Activities

In plants, RNA silencing regulates gene expression through the action of Dicer-like (DCL) and Argonaute (AGO) proteins via micro RNAs and RNA-dependent DNA methylation (RdDM). In addition, RNA silencing functions as an antiviral defense mechanism by targeting virus-derived double-stranded RNA. Plants encode multiple AGO proteins with specialized functions, including AGO4-like proteins that affect RdDM and AGO2, AGO5, and AGO1, which have antiviral activities. Here, we show that AGO4 is also required for defense against the potexvirus Plantago asiatica mosaic virus (PlAMV), most likely independent of RdDM components such as DCL3, Pol IV, and Pol V. Transient assays showed that AGO4 has direct antiviral activity on PlAMV and, unlike RdDM, this activity does not require nuclear localization of AGO4. Furthermore, although PlAMV infection causes a decrease in AGO4 expression, PlAMV causes a change in AGO4 localization from a largely nuclear to a largely cytoplasmic distribution. These results indicate an important role for AGO4 in targeting plant RNA viruses as well as demonstrating novel mechanisms of regulation of and by AGO4, independent of its canonical role in regulating gene expression by RdDM.

scholarly journals Apple chlorotic leaf spot virus 50 kDa movement protein acts as a suppressor of systemic silencing without interfering with local silencing in Nicotiana benthamiana

2007 ◽  
Vol 88 (1) ◽  
pp. 316-324 ◽  
Author(s):  
Hajime Yaegashi ◽  
Tsubasa Takahashi ◽  
Masamichi Isogai ◽  
Takashi Kobori ◽  
Satoshi Ohki ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Silencing Suppressor ◽  
Spot Virus ◽  
Double Stranded Rna ◽  
Chlorotic Leaf ◽  
Green Fluorescent

Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa protein (P216) involved in replication, a 50 kDa movement protein (P50) and a 21 kDa coat protein (CP). In this study, it was investigated whether these proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single- and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals.

scholarly journals Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus

2007 ◽  
Vol 81 (11) ◽  
pp. 5449-5459 ◽  
Author(s):  
Beau J. Fenner ◽  
Winnie Goh ◽  
Jimmy Kwang
Keyword(s):  
Rna Silencing ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Alanine Scanning Mutagenesis ◽  
Marine Aquaculture ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Fish Cells ◽  
Interfering Rna

ABSTRACT Betanodaviruses are small RNA viruses that infect teleost fish and pose a considerable threat to marine aquaculture production. These viruses possess a small protein, termed B2, which binds to and protects double-stranded RNA. This prevents cleavage of virus-derived double-stranded RNAs (dsRNAs) by Dicer and subsequent production of small interfering RNA (siRNA), which would otherwise induce an RNA-silencing response against the virus. In this work, we have performed charged-to-alanine scanning mutagenesis of the B2 protein in order to identify residues required for dsRNA binding and protection. While the majority of the 19 mutated B2 residues were required for maximal dsRNA binding and protection in vitro, residues R53 and R60 were essential for both activities. Subsequent experiments in fish cells confirmed these findings by showing that mutations in these residues abolished accumulation of both the RNA1 and RNA2 components of the viral genome, in addition to preventing any significant induction of the host interferon gene, Mx. Moreover, an obvious positive correlation was found between dsRNA binding and protection in vitro and RNA1, RNA2, and Mx accumulation in fish cells, further validating the importance of the selected amino acid residues. The same trend was also demonstrated using an RNA silencing system in HeLa cells, with residues R53 and R60 being essential for suppression of RNA silencing. Importantly, we found that siRNA-mediated knockdown of Dicer dramatically enhanced the accumulation of a B2 mutant. In addition, we found that B2 is able to induce apoptosis in fish cells but that this was not the result of dsRNA binding.

scholarly journals Identification of a movement protein of Mirafiori lettuce big-vein ophiovirus

2013 ◽  
Vol 94 (5) ◽  
pp. 1145-1150 ◽  
Author(s):  
Akihiro Hiraguri ◽  
Shoko Ueki ◽  
Hideki Kondo ◽  
Koji Nomiyama ◽  
Takumi Shimizu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Protein Gene ◽  
Movement Protein ◽  
Rna Virus ◽  
Infectious Clone ◽  
Microprojectile Bombardment ◽  
Tomato Mosaic Virus ◽  
Intercellular Movement ◽  
Heterologous Virus

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP.

scholarly journals Argonaute 2 Controls Antiviral Activity against Sweet Potato Mild Mottle Virus in Nicotiana benthamiana

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 867
Author(s):  
Erzsébet Kenesi ◽  
Juan-Jose Lopez-Moya ◽  
László Orosz ◽  
József Burgyán ◽  
Lóránt Lakatos
Keyword(s):  
Sweet Potato ◽  
Nicotiana Benthamiana ◽  
Small Rnas ◽  
Rna Silencing ◽  
Mottle Virus ◽  
Antiviral Defense ◽  
Biological Processes ◽  
Catalytic Subunits ◽  
Argonaute 2

RNA silencing is a sequence specific post-transcriptional mechanism regulating important biological processes including antiviral defense in plants. Argonaute (AGO) proteins, the catalytic subunits of the silencing complexes, are loaded with small RNAs to execute the sequence specific RNA cleavage or translational inhibition. Plants encode several AGO proteins and a few of them, especially AGO1 and AGO2, have been shown to be required for antiviral silencing. Previously, we have shown that the P1 protein of the sweet potato mild mottle virus (SPMMV) suppresses the primary RNA silencing response by inhibiting AGO1. To analyze the role of AGO2 in antiviral defense against the SPMMV, we performed a comparative study using a wild type and ago2−/− mutant Nicotiana benthamiana. Here we show that the AGO2 of N. benthamiana attenuates the symptoms of SPMMV infection. Upon SPMMV infection the levels of AGO2 mRNA and protein are greatly increased. Moreover, we found that AGO2 proteins are loaded with SPMMV derived viral small RNAs as well as with miRNAs. Our results indicate that AGO2 protein takes over the place of AGO1 to confer antiviral silencing. Finally, we provide a plausible explanation for the AGO2 mediated recovery of an SPMMV-infected sweet potato.

scholarly journals Double-Stranded RNA Binding May Be a General Plant RNA Viral Strategy To Suppress RNA Silencing

2006 ◽  
Vol 80 (12) ◽  
pp. 5747-5756 ◽  
Author(s):  
Zsuzsanna Mérai ◽  
Zoltán Kerényi ◽  
Sándor Kertész ◽  
Melinda Magna ◽  
Lóránt Lakatos ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Rna Binding ◽  
Binding Protein ◽  
Turnip Crinkle Virus ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Beet Yellows Virus ◽  
Dsrna Binding Protein ◽  
Silencing Suppression ◽  
Suppression Strategy

ABSTRACT In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3′ overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus γB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.

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