Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari FV, David M. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature 2007;449:919-922. Available at: www.nature.com (Reprinted with permission.)
RNA interference through non-coding microRNAs (miRNAs) represents a vital component of the innate antiviral immune response in plants and invertebrate animals; however, a role for cellular miRNAs in the defence against viral infection in mammalian organisms has thus far remained elusive. Here we show that interferon beta (IFNbeta) rapidly modulates the expression of numerous cellular miRNAs, and that eight of these IFNbeta-induced miRNAs have sequence-predicted targets within the hepatitis C virus (HCV) genomic RNA. The introduction of synthetic miRNA-mimics corresponding to these IFNbeta-induced miRNAs reproduces the antiviral effects of IFNbeta on HCV replication and infection, whereas neutralization of these antiviral miRNAs with anti-miRNAs reduces the antiviral effects of IFNbeta against HCV. In addition, we demonstrate that IFNbeta treatment leads to a significant reduction in the expression of the liver-specific miR-122, an miRNA that has been previously shown to be essential for HCV replication. Therefore, our findings strongly support the notion that mammalian organisms too, through the interferon system, use cellular miRNAs to combat viral infections.
Hepatitis C virus (HCV) is a hepatotrophic, positive-stranded RNA virus of the flaviviridae family that causes significant chronic liver disease worldwide. The only available treatment for this chronic infection is pegylated interferon (IFN) in combination with ribavirin. However a sustained virological response is achieved in 40%-50% of patients at best. The factors responsible for success or failure of current treatment regimes are not understood; however, identification of viral and host determinants that dictate treatment response represent a major challenge in HCV biology. Similarly the mechanism(s) responsible for the anti-HCV action of IFN are not well understood. It is most likely a multifactorial process involving modulation of the innate and adaptive immune response and expression of IFN-stimulated genes (ISGs), some whose role is to directly limit HCV replication. The specific ISGs which act as antiviral effectors against HCV remain relatively unknown, although this is an active area of HCV research. MicroRNAs (miRNAs) are a class of small RNA molecules, 21 to 22 nucleotides in length, that function in posttranslational regulation of gene expression through an RNA interference (RNAi) mechanism. Originally identified in plants, it is now clear that in mammals more than 500 miRNAs are expressed. These miRNAs are processed from noncoding regions of the genome by an enzymatic cascade (Fig. 1) that results in the formation of an RNA-induced silencing complex (RISC) (reviewed in Cullen1 and Berkhout and Jeang2). Homology between the miRNA and the target mRNA guides the RISC to the mRNA, thus inhibiting either translation or promoting cleavage of the mRNA; this process represents a powerful specific mechanism for posttranslational regulation of gene expression. Although RNAi mechanisms have been described to control viral infection in plants and invertebrates, it was thought in mammals that the IFN system superseded the need for an RNAi mechanism to control viral infection. This could not be further from the truth, as shown by the recent exciting work from Pederson and colleagues,3 revealing that IFN-β can stimulate expression of miRNAs that specifically target the HCV genome and limit replication.
Using microarray technology, Pederson and colleagues describe induction of approximately 30 miRNAs following stimulation of cells with either IFN-α/β or IFN-γ. Screening these miRNAs for complementarity to viral genomes in the public domain revealed eight IFN-β induced miRNAs (miRNA-1, miRNA-30, miRNA-128, miRNA-196, miRNA-296, miRNA-351, miRNA-431, and miRNA-448) with near perfect complementarity to the HCV genome. These miRNAs were induced by IFN-α/β in a dose-dependent manner with rapid induction (30 minutes) after stimulation. This is certainly a rapid response compared to induction of ISG mRNA expression (∼8 hours), and strongly suggests that this novel miRNA antiviral pathway is indeed the first line of defense against invading pathogens. Furthermore, they also observed that miRNA-122, which has recently been shown to enhance HCV replication, was down-regulated by IFN-β.4 These results are significant in that they provide direct evidence for the connectivity between the immune response and miRNAs with potential control of viral infection.
This work also raises the possibility that other innate sensing pathways within the cell are able to induce miRNA expression. Consistent with this hypothesis, it has recently been discovered that miRNA-146 is up-regulated following stimulation of TLR 2, TLR 4, and TLR 5.5 Clearly the innate response to viral infection is complex and miRNAs may work at multiple levels not only to control viral replication but also to modulate ISG expression through posttranslational regulation. Recent studies have shown that the stability of a number of ISGs is regulated by miRNAs via AU-rich elements present in their 3′-untranslated region.6 Overall, this response may help to balance the host innate immune response and protect infected tissues from the effects of deleterious IFN-induced cytokines.
Direct evidence that these miRNAs could actually regulate HCV replication was demonstrated by transfection of synthetic miRNA-mimics corresponding to the miRNAs in question into Huh-7 cells that harbor HCV replication in the form of a full-length replicon. Of the eight miRNAs identified, only five could inhibit HCV replication (miRNA-196, miRNA-296, miRNA-351, miRNA-431, and miRNA-448) in the order of 50%-75%, whereas a mix of all five miRNAs revealed approximately 80% reduction in HCV replication. However, when all five miRNAs were mixed with an anti-miRNA-122 (miRNA-122 enhances HCV replication), HCV replication could be decreased by almost 85%, which is comparatively close to the 90% reduction in HCV replication shown by IFN-β alone.
The real test of the importance of these miRNAs in the antiviral actions of IFN was demonstrated in experiments assessing the ability of IFN-β to limit HCV replication in the JFH-1 replication model, whereby IFN-β was added in combination with miRNA-122 and an anti-miRNA mix (anti-miRNAmix5) corresponding to the five miRNAs outlined above. They were able to demonstrate that the efficacy of IFN was reduced from >90% to approximately 75% in the presence of miRNA-122, and approximately 80% in the presence of anti-miRNAmix5. However in the presence of both miRNA-122 and anti-miRNAmix5, the authors show a 50% reduction in the ability of IFN to limit HCV replication. Specificity of these miRNAs in control of HCV was demonstrated using a JFH-1 chimeric virus (J6CF) in which single-nucleotide variations exists within the predicted seed sequence for miRNA-196 and miRNA-448 that resulted in abrogation of the miRNAs to limit HCV replication. Clearly, in vitro these miRNAs play an important role in the cellular antiviral state in response to IFN stimulation; however, they are not the complete picture, and other IFN-stimulated factors (the ISGs) also play a role in control of HCV replication. Given the work by Pederson and colleagues, it is conceivable to envisage that IFN-induced miRNAs are the first line of defense in control of viral infection, closely followed by the ISG response to finish things off. However, as we know, in the majority of infected individuals this is not the case with the liver in a state of chronic infection. Although this work suggests that the miRNA system can control HCV replication, further studies are required to determine the relative roles of these miRNAs in the HCV-infected liver from persons that have acute infection to those that develop chronic disease and those that respond to current IFN therapy.
The first miRNA, lin-4, and its target mRNA, lin-14, were described in Caenorhabiditis elegans by Ambros, Ruvkun, and colleagues in 1993.7, 8 However, a role for miRNA in the control of viruses in mammals has not been shown until now. Pedersen and colleagues have certainly uncovered a novel mechanism for the actions of IFN against mammalian viruses, which adds another layer to the already complex host innate defense against viral infection and raises more questions than it answers. In evolutionary terms, it is unlikely that this system is uniquely adapted to control of HCV, and it will be interesting to determine the cellular targets of these miRNAs, and whether they are involved in a feedback mechanism to regulate the cellular IFN response. Furthermore, can the HCV genome evolve adaptations to evade the action of these miRNAs? Pedersen and colleagues identified up to 30 miRNAs that were induced following IFN-α/β or IFN-γ stimulation, although only eight were studied; do the remainder have antiviral activity against other viruses and do the different IFNs induce a mutually exclusive set of miRNAs? They also offer no explanation as to the mechanism of miRNA action, although it is most likely to abrogation of viral protein translation. Future experiments are needed to determine if miRNAs inhibit HCV replication by translational control or via RNA degradation. Finally, with the development of miRNA-based therapeutics and novel delivery options for small RNA molecules, it is possible that in the future these miRNAs may form the basis of novel RNAi therapeutics strategies to combat HCV and other viral infections.9, 10