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Highly efficacious antiviral protection of plants by siRNAs identified in vitro

ABSTRACT: Highly efficacious antiviral protection of plants by siRNAs identified in vitro

PROVIDER: PRJNA497995 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8090350.fastq.gz	Fastqsanger.gz
	SRR8090343.fastq.gz	Fastqsanger.gz
	SRR8090344.fastq.gz	Fastqsanger.gz
	SRR8090345.fastq.gz	Fastqsanger.gz
	SRR8090346.fastq.gz	Fastqsanger.gz

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Highly efficacious antiviral protection of plants by siRNAs identified in vitro

Project description:In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, very few of these siRNAs actually interfere with viral replication. A reliable approach to define the characteristics underlying the activity of these immunologically effective siRNAs (esiRNAs) has not been available so far. We developed a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. The approach is essentially based on the use of a cytoplasmic extract from Nicotiana tabacum BY-2 protoplasts (BY-2 lysate, BYL), that shows Dicer-like (DCL) activity and facilitates the assembly of active RNA-induced silencing complexes (RISC) with an in vitro-translated Argonaute (AGO) protein of choice. We exposed double-stranded (ds) RNA of Tomato bushy stunt virus (TBSV) to the BYL to generate viral siRNAs (DCL assay). Total RNA was isolated from the reactions and DCL-generated TBSV siRNAs were identified by NGS. In another approach, AGO1/RISC- or AGO2/RISC-associated siRNAs were isolated using FLAG-AGO immunoprecipitation (AGO-IP) and analyzed by NGS. Subsequently, the antiviral activity of siRNAs with high affinity to AGO proteins was characterized in vitro and in vivo.

2019-10-01 | GSE121644 | GEO

Highly efficacious antiviral protection of plants by small interfering RNAs identified in vitro.

Project description:In response to a viral infection, the plant's RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very few of these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs (esiRNAs) and to define the characteristics underlying their activity has not been available so far. Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens.

| S-EPMC6755098 | biostudies-literature

Virus-derived siRNAs in Arabidopsis thaliana

Project description:We investigated the role of A. thaliana RDRs in the RNAi-mediated viral immunity by using a mutant of cucumber mosaic virus (CMV) that does not express the VSR protein 2b. CMV contains three positive-strand genomic RNAs and the 2b protein encoded by RNA2 is essential for infection by suppressing antiviral silencing initiated by either DCL4 or DCL2. Our results demonstrate an essential role for the amplification of viral siRNAs by either RDR1 or RDR6 in antiviral silencing. Further analyses, including Illumina sequencing of more than 3.5 million viral siRNAs, indicated target specificity of the two antiviral RDRs.

2010-01-20 | GSE19965 | GEO

A new level of RNA-based plant protection - dsRNAs designed from functionally characterized siRNAs highly effective against Cucumber mosaic virus

Project description:dsRNAs composed of multiple functionally characterized, efficient siRNAs provide effective protection against Cucumber mosaic virus

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Antiviral RNA interference in mammalian cells

Project description:Plants and invertebrates protect themselves from viruses through RNA interference (RNAi), yet it remains unknown whether this defense mechanism exists in mammals. Antiviral RNAi involves the processing of viral long double-stranded (ds) RNA molecules into small interfering RNAs (siRNAs) by the ribonuclease (RNAse) III Dicer. These siRNAs are incorporated into effector complex(es) containing members of the Argonaute (Ago) protein family and guide silencing of complementary target viral RNAs. Here, we detect the accumulation of phased Dicer-dependent virus-derived siRNA (viRNAs) and demonstrate their loading into Ago2 after infection of mouse embryonic stem (ES) cells with Encephalomyocarditis virus (EMCV). We further show that the production of these viRNAs is drastically reduced, yet not completely abolished, if ES cells are first induced to differentiate before infection. Finally, we reveal that the mammalian virus Nodamura virus (NoV) encodes for a protein that counteracts such antiviral RNAi in ES cells supporting the existence of an effective RNAi-based immunity in mammals. Infection of wild-type or mutant mouse ES cells and analysis of small RNAs from total extracts or immunoprecipitated components of the RNAi pathway

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Triplet nucleotide repeat-based siRNAs are highly toxic to cancer cells

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Project description:We report the impact of two pairs of GNA nucleotides, the native GNA-C and -G compared with the isomeric GNA-isoC and -isoG, on the on- and off-target activity of chemically modified siRNAs in primary mouse hepatocytes by RNA sequencing. We found that both GNA-C and -isoC mitigate the suppression of off-targets in a similar fashion, however, GNA-isoG was less efficacious in the mitigation of off-targets as the native GNA-G. This study informs the further design of more potent and specific chemically modified siRNAs for potential application in RNAi therapeutics.

2021-09-24 | GSE183164 | GEO

Antiviral RNA interference in mammalian cells

2013-10-10 | GSE43153 | GEO

Pharmacological inhibition of tyrosine kinase 2 preserves interferon-λ-mediated protection (neutrophils)

Project description:Treating inflammatory diseases with Janus kinase 1/2 (JAK1/2) inhibitors bears the risk that patients acquire viral infections due to unwanted immune suppression. Tyrosine kinase 2 (TYK2), a JAK family member, is required for type I interferon (IFN-α/β) signaling, but its role in type III IFN (IFN-λ) signaling is still under debate. We found that the selective TYK2 inhibitor BMS-986165 blocked potentially noxious type I IFN signaling without altering IFN-λ-mediated gene expression. We show that epithelial cells do not require TYK2 for IFN-λ-mediated signaling or antiviral protection. Lack of TYK2 diminished IFN-α-induced protection against lethal influenza virus infection of mice, but did not impair IFN-λ-mediated antiviral protection. Our findings suggest that selective TYK2 inhibitors likely represent a superior treatment option for type I interferonopathies than broadly acting JAK1/2 inhibitors, as selective TYK2 inhibitors may counteract inflammatory responses without abolishing the beneficial antiviral effects of IFN-λ.

2021-04-09 | GSE146103 | GEO

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