Stabilization of hepatitis C virus RNA by an Ago2-miR-122 complex

Lineberger Comprehensive Cancer Center and Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2012; 109(3):941-6. DOI: 10.1073/pnas.1112263109
Source: PubMed


MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3' UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5' UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5' cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5' exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression.

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Available from: Rohit Jangra
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    • "Unique to this virus is a dependence on the liver-specific microRNA-122 (miR-122) (Jopling et al., 2005). Whereas miRNAs typically interact with the 3 0 UTRs of mRNAs to promote mRNA destabilization and/or translational repression (Bartel, 2009), the binding of miR-122 to two binding sites (seed site S1 and S2) in the 5 0 UTR of HCV genomic RNA is critical for viral replication (Jopling et al., 2008; Machlin et al., 2011) by moderately stimulating viral protein translation (Henke et al., 2008) and, in concert with Argonaute (AGO), by stabilizing and protecting the uncapped HCV RNA genome from degradation (Li et al., 2013b; Sedano and Sarnow, 2014; Shimakami et al., 2012). As the predominant miRNA in the liver, miR-122 has multiple roles to regulate lipid metabolism (Esau et al., 2006), iron homeostasis (Castoldi et al., 2011), and circadian rhythms (Gatfield et al., 2009). "
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    ABSTRACT: Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection showed robust AGO binding on the HCV 5'UTR at known and predicted miR-122 sites. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 "sponge" effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV-induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Mar 2015 · Cell
    • "Collectively, these data provide strong justification for our use of Xrn1-depleted cells in the experiments described here. While supplementing infected cells with exogenous miR-122 enhances the expression of HCV proteins (Jangra et al., 2010), the extent to which this results from a direct effect of miR-122 on the viral IRES or is secondary to stabilization of the genome or increased genome synthesis has been unresolved (Conrad et al., 2013; Henke et al., 2008; Roberts et al., 2011; Shimakami et al., 2012a). By developing methods allowing a direct comparison of the influence of miR-122 on viral RNA synthesis versus the synthesis of an essential viral nonstructural protein, NS5A, we have been able to show that transfection of duplex miR- 122 induces significant increases in viral RNA synthesis in Xrn1-depleted cells prior to any increase in viral protein synthesis Figure 5. miR-122 Supplementation and Nascent HCV RNA following Short-Term Shutdown of Cellular Protein Synthesis (A) Experimental design; HJ3–5/NS5A YFP virus-infected cells were transfected with siRNAs, and then treated 2 days later with 50 mg/ml puromycin or cycloheximide for 2 hr prior to transfection of duplex miR-124 or miR-122 (0 hr). "
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    ABSTRACT: The liver-specific microRNA, miR-122, stabilizes hepatitis C virus (HCV) RNA genomes by recruiting host argonaute 2 (AGO2) to the 5' end and preventing decay mediated by exonuclease Xrn1. However, HCV replication requires miR-122 in Xrn1-depleted cells, indicating additional functions. We show that miR-122 enhances HCV RNA levels by altering the fraction of HCV genomes available for RNA synthesis. Exogenous miR-122 increases viral RNA and protein levels in Xrn1-depleted cells, with enhanced RNA synthesis occurring before heightened protein synthesis. Inhibiting protein translation with puromycin blocks miR-122-mediated increases in RNA synthesis, but independently enhances RNA synthesis by releasing ribosomes from viral genomes. Additionally, miR-122 reduces the fraction of viral genomes engaged in protein translation. Depleting AGO2 or PCBP2, which binds HCV RNA in competition with miR-122 and promotes translation, eliminates miR-122 stimulation of RNA synthesis. Thus, by displacing PCBP2, miR-122 reduces HCV genomes engaged in translation while increasing the fraction available for RNA synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Feb 2015 · Cell Host & Microbe
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    • "Nascent RNA genomes are translated to produce new viral proteins, serve as new/additional RNA templates for further RNA replication and are progressively assembled to form infectious virions. HCV replication is dependent on microRNA 122 (miR-122) [97], a liver-specific microRNA that recruits Argonaute 2 to the 5 0 end of the viral genome [98], stabilizing it and slowing its degradation by the 5 0 exonuclease Xrn1 [99]. "
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    ABSTRACT: Hepatitis C virus (HCV) is an important human pathogen that causes hepatitis, liver cirrhosis and hepatocellular carcinoma. It imposes a serious problem to public health in the world as the population of chronically infected HCV patients who are at risk of progressive liver disease is projected to increase significantly in the next decades. However, the arrival of new antiviral molecules is progressively changing the landscape of hepatitis C treatment. The search for new anti-HCV therapies has also been a driving force to better understand how HCV interacts with its host, and major progresses have been made on the various steps of the HCV life cycle. Here, we review the most recent advances in the fast growing knowledge on HCV life cycle and interaction with host factors and pathways.
    Preview · Article · Nov 2014 · Journal of Hepatology
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