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The 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome forms RNA structures that regulate virus replication and translation. The region contains an internal ribosomal entry site (IRES) and a 5'-terminal region. Binding of the liver-specific microRNA (miRNA) miR-122 to two binding sites in the 5'-terminal region regulates viral replication, translation, and genome stability and is essential for efficient virus replication, but its precise mechanism of action is still unresolved. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5' UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable replication of wild-type HCV genomes in cell culture, several viral variants with 5' UTR mutations exhibit low-level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Further, we provide evidence that translation regulation is the major role for miR-122 and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5' UTR mutations that stimulate translation and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Finally, we show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in promoting HCV. IMPORTANCE The unusual and essential role of miR-122 in promoting HCV propagation is incompletely understood. To better understand its role, we have analyzed HCV mutants capable of replicating independently of miR-122. Our data show that the ability of viruses to replicate independently of miR-122 correlates with enhanced virus translation but that genome stabilization is required to restore efficient HCV replication. This suggests that viruses must gain both abilities to escape the need for miR-122 and impacts the possibility that HCV can evolve to replicate outside the liver., Competing Interests: The authors declare no conflict of interest.

A liver-specific microRNA, miR-122, anneals to the hepatitis C virus (HCV) genomic 5' terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full-length RNAs with specific mutations in the 5' untranslated region (UTR) can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having encephalomyocarditis virus internal ribosomal entry site (EMCV IRES)-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by the amount of viral RNA delivered. HCV RNAs replicating independently of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of an miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication, suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection and also supports new replication complex formation during ongoing infection and after infected cell division. IMPORTANCE The mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as a potential HCV therapy.

Hepatitis C virus (HCV) replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5' untranslated region (UTR). Annealing has been reported to (a) stabilize the genome, (b) stimulate translation and (c) promote the formation of translationally active Internal Ribosome Entry Site (IRES) RNA structure. In this report, we map the RNA element to which small RNA annealing promotes HCV to nucleotides 1-44 and identify the relative impact of small RNA annealing on virus translation promotion and genome stabilization. We mapped the optimal region on the HCV genome to which small RNA annealing promotes virus replication to nucleotides 19-37 and found the efficiency of viral RNA accumulation decreased as annealing moved away from this region. Then, by using a panel of small RNAs that promote replication with varying efficiencies we link the efficiency of lifecycle promotion with translation stimulation. By contrast small RNA annealing stabilized the viral genome even if they did not promote virus replication. Thus, we propose that miR-122 annealing promotes HCV replication by annealing to an RNA element that activates the HCV IRES and stimulates translation, and that miR-122 induced HCV genome stabilization is insufficient alone but enhances virus replication., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hepatitis C virus (HCV) replication requires annealing of a liver specific microRNA, miR-122 to 2 sites on 5' untranslated region (UTR). While, microRNAs downregulate gene expression by binding to the 3' untranslated region of the target mRNA, in this case, the microRNA anneals to the 5'UTR of the viral genomes and upregulates the viral lifecycle. In this review, we explore the current understandings of the mechanisms by which miR-122 promotes the HCV lifecycle, and its contributions to pathogenesis. Annealing of miR-122 has been reported to (a) stimulate virus translation by promoting the formation of translationally active internal ribosome entry site (IRES) RNA structure, (b) stabilize the genome, and (c) induce viral genomic RNA replication. MiR-122 modulates lipid metabolism and suppresses tumor formation, and sequestration by HCV may influence virus pathogenesis. We also discuss the possible use of miR-122 as a biomarker for chronic hepatitis and as a therapeutic target. Finally, we discuss roles for miR-122 and other microRNAs in promoting other viruses.

Annealing of the liver-specific microRNA, miR-122, to the Hepatitis C virus (HCV) 5' UTR is required for efficient virus replication. By using siRNAs to pressure escape mutations, 30 replication-competent HCV genomes having nucleotide changes in the conserved 5' untranslated region (UTR) were identified. In silico analysis predicted that miR-122 annealing induces canonical HCV genomic 5' UTR RNA folding, and mutant 5' UTR sequences that promoted miR-122-independent HCV replication favored the formation of the canonical RNA structure, even in the absence of miR-122. Additionally, some mutant viruses adapted to use the siRNA as a miR-122-mimic. We further demonstrate that small RNAs that anneal with perfect complementarity to the 5' UTR stabilize and promote HCV genome accumulation. Thus, HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5' end annealing or 3' overhanging nucleotides. Replication promotion by perfect-match siRNAs was observed in Ago2 knockout cells revealing that other Ago isoforms can support HCV replication. At last, we present a model for miR-122 promotion of the HCV life cycle in which miRNA annealing to the 5' UTR, in conjunction with any Ago isoform, modifies the 5' UTR structure to stabilize the viral genome and promote HCV RNA accumulation.

Hepatitis C virus (HCV) recruits two molecules of the liver-specific microRNA-122 (miR-122) to the 5' end of its genome. This interaction promotes viral RNA accumulation, but the precise mechanism(s) remain incompletely understood. Previous studies suggest that miR-122 is able to protect the HCV genome from 5' exonucleases (Xrn1/2), but this protection is not sufficient to account for the effect of miR-122 on HCV RNA accumulation. Thus, we investigated whether miR-122 was also able to protect the viral genome from innate sensors of RNA or cellular pyrophosphatases. We found that miR-122 does not play a protective role against recognition by PKR, RIG-I-like receptors, or IFITs 1 and 5. However, we found that knockdown of both the cellular pyrophosphatases, DOM3Z and DUSP11, was able to rescue viral RNA accumulation of subgenomic replicons in the absence of miR-122. Nevertheless, pyrophosphatase knockdown increased but did not restore viral RNA accumulation of full-length HCV RNA in miR-122 knockout cells, suggesting that miR-122 likely plays an additional role(s) in the HCV life cycle, beyond 5' end protection. Overall, our results support a model in which miR-122 stabilizes the HCV genome by shielding its 5' terminus from cellular pyrophosphatase activity and subsequent turnover by exonucleases (Xrn1/2).

Unlabelled: miR-122 is a liver-specific microRNA (miRNA) that binds to two sites (S1 and S2) on the 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome and promotes the viral life cycle. It positively affects viral RNA stability, translation, and replication, but the mechanism is not well understood. To unravel the roles of miR-122 binding at each site alone or in combination, we employed miR-122 binding site mutant viral RNAs, Hep3B cells (which lack detectable miR-122), and complementation with wild-type miR-122, an miR-122 with the matching mutation, or both. We found that miR-122 binding at either site alone increased replication equally, while binding at both sites had a cooperative effect. Xrn1 depletion rescued miR-122-unbound full-length RNA replication to detectable levels but not to miR-122-bound levels, confirming that miR-122 protects HCV RNA from Xrn1, a cytoplasmic 5'-to-3' exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound RNA levels, suggesting that both sites contribute, but their contributions may be unequal when the need for protection from Xrn1 is reduced. miR-122 binding at S1 or S2 also increased translation equally, but the effect was abolished by Xrn1 knockdown, suggesting that the influence of miR-122 on HCV translation reflects protection from Xrn1 degradation. Our results show that occupation of each miR-122 binding site contributes equally and cooperatively to HCV replication but suggest somewhat unequal contributions of each site to Xrn1 protection and additional functions of miR-122., Importance: The functions of miR-122 in the promotion of the HCV life cycle are not fully understood. Here, we show that binding of miR-122 to each of the two binding sites in the HCV 5' UTR contributes equally to HCV replication and that binding to both sites can function cooperatively. This suggests that active Ago2-miR-122 complexes assemble at each site and can cooperatively promote the association and/or function of adjacent complexes, similar to what has been proposed for translation suppression by adjacent miRNA binding sites. We also confirm a role for miR-122 in protection from Xrn1 and provide evidence that miR-122 has additional functions in the HCV life cycle unrelated to Xrn1. Finally, we show that each binding site may contribute unequally to Xrn1 protection and other miR-122 functions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

MicroRNAs (miRNAs) are small non-coding RNAs that function as part of RNA-induced silencing complexes that repress the expression of target genes. Over the past few years, miRNAs have been found to mediate complex regulation of a wide variety of mammalian viral infections, including Hepatitis C virus (HCV) infection. Here, we focus on a highly abundant, liver-specific miRNA, miR-122. In a unique and unusual interaction, miR-122 binds to two sites in the 5' untranslated region (UTR) of the HCV genome and promotes viral RNA accumulation. We will discuss what has been learned about this important interaction to date, provide insights into how miR-122 is able to modulate HCV RNA accumulation, and how miR-122 might be exploited for antiviral intervention., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hepatitis C Virus (HCV) is a serious global health problem, infecting almost 3% of the world's population. The lack of model systems for studying this virus limit research options in vaccine and therapeutic development, as well as for studying the pathogenesis of chronic HCV infection. Herein we make use of the liver-specific microRNA miR-122 to render mouse cell lines permissive to HCV replication in an attempt to develop additional model systems for the identification of new features of the virus and its life cycle. We have determined that some wild-type and knockout mouse cell lines--NCoA6 and PKR knockout embryonic fibroblasts--can be rendered permissive to transient HCV sub-genomic RNA replication upon addition of miR-122, but we did not observe replication of full-length HCV RNA in these cells. However, other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122, and in fact, different NCoA6 and PKR knockout cell line passages and isolates from the same mice demonstrated varying permissiveness phenotypes and eventually complete loss of permissiveness. When we tested knockdown of NCoA6 and PKR in Huh7.5 cells, we saw no substantial impact in sub-genomic HCV replication, which we would expect if these genes were inhibitory to the virus' life cycle. This leads us to conclude that along with the influence of specific gene knockouts there are additional factors within the cell lines that affect their permissiveness for HCV replication; we suggest that these may be epigenetically regulated, or modulated by cell line immortalization and transformation.

The unusual role for miR-122 in promoting the hepatitis C virus (HCV) life cycle was first identified in 2005, but its mechanism of action remains uncharacterized. The virus appears to use the microRNA (miRNA) in a way that is opposed to that of normal miRNAs. Instead of interacting with sequences in the 3'-untranslated region (UTR), miR-122 binds to two sites in the 5'-UTR, and instead of silencing gene expression or promoting the degradation of the viral RNA, it stabilizes the genome and potently augments the efficiency by which HCV RNA accumulates in infected cells. This review discusses the current knowledge and models for the mechanism by which miR-122 promotes the HCV life cycle. Annealing of miR-122 to the HCV genome requires particular base pairing, stimulates translation, and stabilizes the viral genome by blocking degradation by host exonucleases, but these functions are unlikely to be the whole story. We will discuss other possible functions for miR-122, the stages of the HCV life cycle at which miR-122 may influence HCV, and other related viruses that may be similarly regulated by miR-122. Despite our lack of detailed mechanistic information, antagonism of miR-122 is emerging as a powerful method to inhibit HCV infections, and unique to other HCV treatment strategies does not, thus far, appear to induce emergence of escape mutants. Used alone or in combination with other antiviral drugs, miR-122 antagonists could be useful to both inhibit the virus and provide selective pressure to inhibit the development of resistance., (Copyright © 2013 John Wiley & Sons, Ltd.)

Hepatitis C Virus (HCV) infection-induced liver disease is a growing problem worldwide, and is the primary cause of liver failure requiring liver transplantation in North America. Improved therapeutic strategies are required to control and possibly eradicate HCV infections, and to modulate HCV-induced liver disease. Cellular microRNAs anneal to and regulate mRNA translation and stability and form a regulatory network that modulates virtually every cellular process. Thus, miRNAs are promising cellular targets for therapeutic intervention for an array of diseases including cancer, metabolic diseases, and virus infections. In this review we outline the features of miRNA regulation and how miRNAs may be targeted in strategies to modulate HCV replication and pathogenesis. In particular, we highlight miR-122, a miRNA that directly modulates the HCV life cycle using an unusual mechanism. This miRNA is very important since miR-122 antagonists dramatically reduced HCV titres in HCV-infected chimpanzees and humans and currently represents the most likely candidate to be the first miRNA-based therapy licensed for use. However, we also discuss other miRNAs that directly or indirectly alter HCV replication efficiency, liver cirrhosis, fibrosis and the development of hepatocellular carcinoma (HCC). We also discuss a few miRNAs that might be targets to treat HCV in cases of HCV/HIV co-infection. Finally, we review methods to deliver miRNA antagonists and mimics to the liver. In the future, it may be possible to design and deliver specific combinations of miRNA antagonists and mimics to cure HCV infection or to limit liver pathogenesis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hepatitis C virus (HCV) RNA forms an unusual interaction with human microRNA-122 (miR-122) that promotes viral RNA accumulation in cultured human liver cells and in the livers of infected chimpanzees. GB virus B (GBV-B) is a hepatotropic virus and close relative of HCV. Thus, GBV-B has been used as a surrogate system to study HCV amplification in cultured cells and in infected tamarins. It was discovered that the 5'-terminal sequences of GBV-B RNA, like HCV RNA, forms an Argonaute 2-mediated complex with two miR-122 molecules that are essential for accumulation of GBV-B subgenomic replicon RNA. However, sequences in miR-122 that anneal to each viral RNA genome were different, suggesting distinct overall structural features in HCV:miR-122 and GBV-B:miR-122 complexes. Surprisingly, a deletion that removed both miR-122 binding sites from the subgenomic GBV-B RNAs rendered viral RNA amplification independent from miR-122 and Argonaute 2. This finding suggests that structural features at the end of the viral genome dictate whether miR-122 is required to aid in maintaining viral RNA abundance.

DDX6 and other P-body proteins are required for efficient replication of Hepatitis C Virus (HCV) by unknown mechanisms. DDX6 has been implicated in miRNA induced gene silencing, and since efficient HCV replication and translation relies on the cellular microRNA, miR-122, we hypothesized that DDX6 had a role in the mechanism of action of miR-122. However, by using multiple HCV translation and replication assays we have found this is not the case. DDX6 silencing decreased HCV replication and translation, but did not affect the ability of miR-122 to stimulate HCV translation or promote HCV RNA accumulation. In addition, the negative effect of DDX6 silencing on HCV replication and translation was not dependent on miR-122 association with the HCV genome. Thus, DDX6 does not have a role in the activity of miR-122, and it appears that DDX6 and miR-122 modulate HCV through distinct pathways. This effect was seen in both Huh7.5 cells and in Hep3B cells, indicating that the effects are not cell type specific. Since infections by other viruses in the Flaviviridae family, including Dengue and West Nile Virus, also disrupt P-bodies and are regulated by DDX6, we speculate that DDX6 may have a common function that support the replication of several Flaviviruses.

The study of Hepatitis C Virus (HCV) has benefitted from the use of the Huh7 cell culture system, but until recently there were no other widely used alternatives to this cell line. Here we render another human hepatoma cell line, Hep3B, permissive to the complete virus life cycle by supplementation with the liver-specific microRNA miR-122, known to aid HCV RNA accumulation. When supplemented, Hep3B cells produce J6/JFH-1 virus titres indistinguishable from those produced by Huh7.5 cells. Interestingly, we were able to detect and characterize miR-122-independent replication of di-cistronic replicons in Hep3B cells. Further, we show that Argonaute-2 (Ago2) is required for miR-122-dependent replication, but dispensable for miR-122-independent replication, confirming Ago2's role in mediating the activity of miR-122. Thus Hep3B cells are a model system for the study of HCV, and miR-122 independent replication is a model to identify proteins involved in the function of miR-122., (Copyright © 2012 Elsevier Inc. All rights reserved.)

MicroRNA-122 (miR-122) promotes Hepatitis C Virus (HCV) RNA stability, accumulation, and translation through hybridization with the 5' untranslated region (5' UTR) of the HCV genome. Depletion of Dicer and TRBP, proteins involved in miRNA biogenesis, reduced HCV RNA accumulation, mature duplex miR-122 abundance, and miR-122 directed mRNA translation suppression, suggesting roles in miR-122 processing. HCV RNA accumulation independent of endogenous mature duplex miR-122 was not affected by Dicer knockdown, suggesting that Dicer is required solely for miR-122 biogenesis, but TRBP knockdown reduced HCV RNA accumulation in this system, suggesting an additional role in supporting HCV RNA accumulation. Mature duplex miR-122 and pre-miR-122 hairpin, but not single-stranded miR-122 (guide or * strand), augmented HCV RNA accumulation and translation, and Dicer and TRBP were essential for the activity of pre-miR-122 in mouse fibroblasts. Thus, canonical miRNA processing and strand selection is essential for the activity of miR-122 on HCV translation and RNA accumulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

MicroRNA 122 (miR-122) increases the accumulation and translation of hepatitis C virus (HCV) RNA in infected cells through direct interactions with homologous sequences in the 5' untranslated region (UTR) of the HCV genome. Argonaute 2 (Ago2) is a component of the RNA-induced silencing complex (RISC) and mediates small interfering RNA (siRNA)-directed mRNA cleavage and microRNA translational suppression. We investigated the function of Ago2 in HCV replication to determine whether it plays a role in enhancing the synthesis and translation of HCV RNA that is associated with miR-122. siRNA-mediated depletion of Ago2 in human hepatoma cells reduced HCV RNA accumulation in transient HCV replication assays. The treatment did not adversely affect cell viability, as assessed by cell proliferation, capped translation, and interferon assays. These data are consistent with complementary roles for Ago2 and miR-122 in enhancing HCV RNA amplification. By using a transient HCV replication assay that is dependent on an exogenously provided mutant miR-122, we determined that Ago2 depletion still reduced luciferase expression and HCV RNA accumulation, independently of miR-122 biogenesis. miR-122 has previously been found to stimulate HCV translation. Similarly, Ago2 knockdown also reduced HCV translation, and its depletion reduced the ability of miR-122 to stimulate viral translation. These data suggest a direct role for Ago2 in miR-122-mediated translation. Finally, Ago2 was also necessary for efficient miR-122 enhancement of HCV RNA accumulation. These data support a model in which miR-122 functions within an Ago2-containing protein complex to augment both HCV RNA accumulation and translation.

Hepatitis C virus (HCV) infection causes significant morbidity, and efficient mouse models would greatly facilitate virus studies and the development of effective vaccines and new therapeutic agents. Entry factors, innate immunity, and host factors needed for viral replication represent the initial barriers that restrict HCV infection of mouse cells. Experiments in this paper consider early postentry steps of viral infection and investigate the roles of interferon regulatory factors (IRF-3 and IRF-9) and microRNA (miR-122) in promoting HCV replication in mouse embryo fibroblasts (MEFs) that contain viral subgenomic replicons. While wild-type murine fibroblasts are restricted for HCV RNA replication, deletion of IRF-3 alone can facilitate replicon activity in these cells. This effect is thought to be related to the inactivation of the type I interferon synthesis mediated by IRF-3. Additional deletion of IRF-9 to yield IRF-3(-/-) IRF-9(-/-) MEFs, which have blocked type I interferon signaling, did not increase HCV replication. Expression of liver-specific miR-122 in MEFs further stimulated the synthesis of HCV replicons in the rodent fibroblasts. The combined effects of miR-122 expression and deletion of IRF-3 produced a cooperative stimulation of HCV subgenome replication. miR-122 and IRF-3 are independent host factors that are capable of influencing HCV replication, and our findings could help to establish mouse models and other cell systems that support HCV growth and particle formation.