Interactions of Rice Tungro Bacilliform Pararetrovirus and Its Protein P4 with Plant RNA-Silencing Machinery

Small interfering RNA (siRNA)-directed gene silencing plays a major role in antiviral defense. Virus-derived siRNAs inhibit viral replication in infected cells and potentially move to neighboring cells, immunizing them from incoming virus. Viruses have evolved various ways to evade and suppress siRNA production or action. Here, we show that 21-, 22-, and 24-nucleotide (nt) viral siRNAs together constitute up to 19% of total small RNA population of Oryza sativa plants infected with Rice tungro bacilliform virus (RTBV) and cover both strands of the RTBV DNA genome. However, viral siRNA hotspots are restricted to a short noncoding region between transcription and reverse-transcription start sites. This region generates double-stranded RNA (dsRNA) precursors of siRNAs and, in pregenomic RNA, forms a stable secondary structure likely inaccessible to siRNA-directed cleavage. In transient assays, RTBV protein P4 suppressed cell-to-cell spread of silencing but enhanced cell-autonomous silencing, which correlated with reduced 21-nt siRNA levels and increased 22-nt siRNA levels. Our findings imply that RTBV generates decoy dsRNA that restricts siRNA production to the structured noncoding region and thereby protects other regions of the viral genome from repressive action of siRNAs, while the viral protein P4 interferes with cell-to-cell spread of antiviral silencing.

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Structural organization of upstream exons and distribution of transcription start sites in the chicken c-myb gene

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.837-843.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 837-843

Author(s):

S L Hahn ◽

M Hahn ◽

W S Hayward

Keyword(s):

Binding Sites ◽

Noncoding Region ◽

Open Reading Frames ◽

Transcription Start ◽

S1 Nuclease ◽

Transcription Start Sites ◽

Conserved Sequence ◽

Myb Gene ◽

Putative Transcription Factor ◽

Myb Genes

We mapped and sequenced three upstream exons of the chicken c-myb gene and the regions flanking the first coding exon. We found multiple potential binding sites for transcription factors in the 5'-noncoding region, a T-rich stretch of 78 base pairs (bp) (68% T) in the first intron, and four fairly long open reading frames in the antisense direction of the first coding exon and its flanking regions. Three major transcription start sites, contained within a single 11-bp region, were identified by S1 nuclease analysis and primer extension. A sequence comparison of the avian and murine c-myb genes revealed a highly conserved sequence of 124 bp in the 5'-noncoding region. Its location between the putative transcription factor binding sites and the major transcription start sites suggests that it may play an important regulatory role in c-myb expression.

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

Journal of Virology ◽

10.1128/jvi.00864-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 12914-12923 ◽

Cited By ~ 30

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.

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Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus

Journal of Virology ◽

10.1128/jvi.00009-07 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5449-5459 ◽

Cited By ~ 27

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.

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Identification of Human Herpesvirus 6 Latency-Associated Transcripts

Journal of Virology ◽

10.1128/jvi.76.8.4145-4151.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 4145-4151 ◽

Cited By ~ 37

Author(s):

Kazuhiro Kondo ◽

Kazuya Shimada ◽

Junji Sashihara ◽

Keiko Tanaka-Taya ◽

Koichi Yamanishi

Keyword(s):

Human Herpesvirus ◽

Immediate Early ◽

Human Herpesvirus 6 ◽

Coding Region ◽

Transcription Start ◽

Protein Coding ◽

Transcription Start Sites ◽

Latently Infected ◽

Infected Cells ◽

Herpesvirus 6

ABSTRACT Four kinds of latency-associated transcripts of human herpesvirus 6 were identified which were detected only in latently infected cells. Although they were oriented in the same direction as the immediate-early 1 and 2 (IE1/IE2) genes and shared their protein-coding region with IE1/IE2, their transcription start sites and exon(s) were latency associated.

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Viral Class 1 RNase III Involved in Suppression of RNA Silencing

Journal of Virology ◽

10.1128/jvi.79.11.7227-7238.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 7227-7238 ◽

Cited By ~ 117

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.

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