Argonaute-2 Enhances Suppression of Human Cytomegalovirus Replication by Polycistronic Short Hairpin Rnas Targeting Ul46, Ul70 and Ul122

Shao, Pei-Lan; Lu, M-Y; Liau, Yi-Jen; Chao, Miau-Fen; Chang, Luan‐Yin; Lu, Chun‐Yi; Kao, Chuan-Liang; Chang, Shinn‐Jen; Chi, Ya‐Hui; Huang, Li‐Min

doi:10.3851/imp1808

Cited by 4 publications

(2 citation statements)

References 47 publications

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“…Unlike in the situation where AGO proteins are overexpressed, in the situation where AGO proteins are limiting and the RISCs are already occupied with cellular miRNAs, de novo-synthesized mivaRNAs are likely to become less readily incorporated into RISCs. In analogy, overexpression of AGO2 has been demonstrated to abrogate saturation of RISCs, consequently facilitating RISC incorporation of exogenous siRNAs or shRNAs by abolishing the competition with cellular miRNAs (41)(42)(43)(44). Thus, our data indicate that RISC occupation by mivaRNAs appears to have been overestimated in the past, because under physiologically more relevant conditions, cellular miRNAs are still dominating over mivaRNAs in endogenous RISCs.…”

Section: Discussionmentioning

confidence: 60%

Identification of RISC-Associated Adenoviral MicroRNAs, a Subset of Their Direct Targets, and Global Changes in the Targetome upon Lytic Adenovirus 5 Infection

Bellutti

Kauer

Kneidinger

et al. 2015

J Virol

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Adenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells. IMPORTANCE Viral and cellular miRNAs have been recognized as important players in virus-host interactions.This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

show abstract

Section: Discussionmentioning

confidence: 60%

Identification of RISC-Associated Adenoviral MicroRNAs, a Subset of Their Direct Targets, and Global Changes in the Targetome upon Lytic Adenovirus 5 Infection

Bellutti

Kauer

Kneidinger

et al. 2015

J Virol

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“…As demonstrated, these three methodological avenues can enhance the potency of all conventional RNAi triggers—transfected si or shRNAs or transduced shRNAs—in mammalian cells ex or in vivo . While previous reports had already indicated that ectopic Ago2 expression can enhance si or shRNA activity ( 16 , 17 , 60 , 61 ), the present work represents an important advance in our understanding of Ago2/RNAi biology and its implications for RNAi applications, for three reasons: First, the comprehensive and systematic evaluation of the benefits of Ago2 overexpression in cell lines, primary cells and animals underscores the enormous potential to improve a wide range of typical small- and large-scale RNAi experiments, including high-content screens. Second, the alleviation of shRNA-induced in vivo hepatotoxicities with Ago2-co-encoding vectors reaffirms prior models that a major hurdle for therapeutic RNAi is saturation of the cellular pathway with ecoptic triggers, and concurrently provides a new solution.…”

Section: Discussionmentioning

confidence: 96%

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

Börner

Niopek

Cotugno

et al. 2013

Nucleic Acids Research

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As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.

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Codon usage bias in human cytomegalovirus and its biological implication

Chen

et al. 2014

Gene

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Argonaute-2 Enhances Suppression of Human Cytomegalovirus Replication by Polycistronic Short Hairpin Rnas Targeting Ul46, Ul70 and Ul122

Abstract: Coexpression of Ago2 with shRNA-miRs enhanced the production of mature siRNAs and increased the efficiency of RNA silencing in the suppression of HCMV replication. This strategy may be universally applied to RNA interference-based therapies.

Cited by 4 publications

References 47 publications

Identification of RISC-Associated Adenoviral MicroRNAs, a Subset of Their Direct Targets, and Global Changes in the Targetome upon Lytic Adenovirus 5 Infection

Identification of RISC-Associated Adenoviral MicroRNAs, a Subset of Their Direct Targets, and Global Changes in the Targetome upon Lytic Adenovirus 5 Infection

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

Codon usage bias in human cytomegalovirus and its biological implication

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