Most metazoan miRNAs (microRNAs) bind to sites in the 3′-UTRs (untranslated regions) of mRNA targets and negatively regulate protein synthesis. The liver-specific miR-122, however, exerts a positive effect on HCV (hepatitis C virus) RNA levels by binding directly to a site in the 5′-UTR of the viral RNA. HCV translation and RNA stability are unaffected, and therefore miR-122 is likely to act at the level of viral replication. The miR-122-binding site in HCV RNA was examined to determine whether the nature of the site is responsible for the unusual mode of action for a miRNA. When the site was placed in the 3′-UTR of a reporter mRNA, miR-122 repressed translation, and therefore the location of the miR-122-binding site dictates its effect on gene expression. Additionally, a second binding site for miR-122 was identified in the HCV 5′-UTR, and miR-122 binding to both sites in the same viral RNA was found to be necessary for viral replication. The two sites are adjacent and are separated by a short spacer, which is largely conserved between HCV genotypes. The binding site requirements for miR-122 to positively regulate HCV replication provide an insight into this unusual mode of miRNA action.
- hepatitis C virus (HCV)
- internal ribosome entry site (IRES)
- viral replication
miRNAs (microRNAs) are non-coding RNAs expressed by a wide range of eukaryotic organisms that are important in the control of gene expression . Several hundred miRNAs are expressed in mammals, initially as part of large precursor transcripts, which subsequently undergo nuclear and cytoplasmic processing to yield 21–23 nt single-stranded mature miRNA molecules. miRNAs can mediate cleavage of exactly complementary RNA targets, but in metazoan systems most miRNAs have been shown to function by binding with imperfect complementarity to 3′-UTR (untranslated region) sites as part of a complex of proteins known as the miRNP. This binding resulted in inhibition of gene expression, which occurred at the level of translation initiation or by a post-initiation block in different studies. Localization of mRNA to processing (P) bodies and subsequent degradation has also been observed, and it is likely that all three mechanisms of repression may function in different circumstances .
In higher organisms miRNAs show a high degree of specificity of expression, both according to tissue type and developmental stage. miR-122 was identified as a highly liver-specific miRNA and was found to account for approx. 70% of total liver miRNA content, with approx. 66000 copies per cell .
HCV (hepatitis C virus) is a hepatotropic positive-sense RNA virus that establishes persistent infections in liver tissue . Infection can eventually lead to cirrhosis of the liver and hepatocellular carcinoma. Current antiviral therapies are frequently ineffective, and a greater understanding of the virus is important for the development of new treatments. We examined liver cell lines and found that miR-122 is expressed in Huh7 cells, which are supportive of HCV replication, but not in a different human liver cell line, HepG2 . Huh7 cells stably expressing a dicistronic HCV replicon, in which the EMCV (encephalomyocarditis virus) IRES (internal ribosome entry site) directs translation of the complete HCV polyprotein, whereas HCV 5′- and 3′-UTRs allow replication to occur , were also examined, and miR-122 was expressed in these cells. We were interested to see what impact the presence of high levels of miR-122 had on HCV replication, and used a 2′-O-methylated antisense oligonucleotide to sequester miR-122 in Huh7 cells. Both in Huh7 cells stably containing the dicistronic genotype 1b HCV replicon described above, and in cells transiently expressing a full-length type 1a HCV replicating RNA , sequestration of miR-122 resulted in a dramatic reduction in HCV RNA levels .
Mutagenesis indicated that miR-122 binds to a site in the 5′-UTR of HCV RNA and that its effect on HCV is due to this binding. The miR-122-binding site we identified is in an unstructured region of the 5′-UTR, upstream of the HCV IRES, and is conserved across all six genotypes of the virus (Figure 1A, seed match 1). We observed no effect of miR-122 binding on HCV translation or RNA stability, and concluded that miR-122 positively regulates HCV at the level of viral replication .
The miR-122-binding site from HCV mediates repression of gene expression when placed in a heterologous 3′-UTR
The discovery of this novel mode of action for a miRNA suggested that particular features of miR-122 or its binding site in HCV might cause this unusual mode of regulation. A major determinant of productive miRNA binding has been found to be exact Watson–Crick complementarity to nt 2–7 of the miRNA, known as the ‘seed’. This seed match should ideally be flanked by A residues pairing to nt 1 and 8 of the miRNA . Further pairing to the 3′ region of the miRNA contributes to binding affinity, but is less crucial. The miR-122-binding site in HCV fits this model (Figure 1B, seed match 1), despite the fact that miR-122 binding to this site does not lead to repression of HCV translation .
The region spanning the miR-122-binding site was amplified from the HCV 5′-UTR by PCR, and inserted in the 3′-UTR of the firefly luciferase gene. Transfection of this firefly luciferase plasmid into Huh7 cells and sequestration of miR-122 showed that miR-122 mediates repression of luciferase expression from this mRNA, and quantitative RT (reverse transcriptase)–PCR indicated that luciferase mRNA levels are unaffected, so repression occurs at the level of translation . Mutagenesis of the miR-122-binding site, and introduction of mutant miR-122 that can bind to the mutant site, showed that the miR-122 response is partly mediated by binding to this site. However, some miR-122-dependent down-regulation of luciferase expression persisted when the site was mutated. A second miR-122 seed match exists adjacent to, and downstream of, the first one. This seed match (seed match 2) extends from nt 2 to 7 of miR-122 and lacks the A residues pairing to positions 1 and 8 that are seen in seed match 1 (Figure 1B). However, this second seed match is conserved across HCV genotypes (Figure 1A, seed match 2), suggesting a possible functional role. Mutagenesis of both seed matches in the 3′-UTR of luciferase abolished miR-122-dependent repression of luciferase expression, which was restored by introduction of mutant miR-122 that binds to the mutant site . This indicated that miR-122 mediates repression of gene expression via both these adjacent sites in the context of a 3′-UTR.
miR-122 regulates HCV replication via two adjacent binding sites
These findings raised the question as to whether miR-122 binds to both adjacent seed matches in the context of HCV RNA. Point mutations were introduced into the miR-122 seed match 2 in a plasmid encoding an HCV type 1a (H77) genomic RNA, containing five adaptive mutations that allow efficient replication in Huh7 cells . Following the introduction of the mutant RNA into cells and a five-day culture period to allow replication to occur, no HCV RNA was detectable. Moreover, the introduction of a miR-122 mutant that can bind to this mutant site led to the restoration of HCV RNA levels, indicating that miR-122 binding to this second site is necessary for HCV replication to occur. Levels of HCV RNA containing mutations in both miR-122-binding sites could also be restored by transfection of mutant miR-122 molecules that can bind to the sites. Importantly, when different mutations were introduced into each of the two sites, individual miR-122 mutants that could bind to both sites had to be introduced into cells in combination for HCV replication to occur. This indicates that miR-122 must bind to both sites within the same HCV molecule . However, it is not yet known whether this is due to a requirement for overlapping binding to the two sites (Figure 1B), or for a sequential binding process.
Multiple binding sites for the same or different miRNAs are frequently observed in 3′-UTRs, and some co-operativity occurs when miRNAs bind to adjacent sites, with an optimal distance of 13–35 nt between the start of two seed matches . However, the essential requirement for binding to the two adjacent miR-122 seed matches that we find in HCV RNA has not been observed in other systems. An additive, but inessential, effect of multiple copies of the miR-122-binding site from HCV was observed in the context of the 3′-UTR of luciferase mRNA, indicating that the absolute requirement for the double binding site is specific to HCV replication. The sites are separated by an 8 nt spacer that is largely conserved across HCV genotypes (Figure 1A), and preliminary data indicate that the length and sequence of the spacer are important for HCV replication . It is possible that miR-122 binding to the adjacent sites allows the recruitment of an oligomeric protein complex to the 5′-UTR of HCV and that this is required for viral replication.
Implications for miRNA function
The finding that a miRNA can bind to the same site in different contexts and mediate different events suggests that miRNA activity is more flexible than was previously thought. Most efforts, both experimental and computational, to detect miRNA-binding sites have been directed towards the 3′-UTR. Several recent studies, however, suggest that miRNA activity may extend beyond repression of gene expression via 3′-UTR sites. It was shown that miR-369-3 binds to AU-rich elements in the 3′-UTR of the TNFα (tumour necrosis factor α) mRNA and mediates a switch from repression to activation of mRNA translation upon serum starvation. Similar effects were observed with a let-7 target, and recruitment of the FXR1 (fragile X mental retardation-related protein 1) protein as part of the miRNP was responsible for this change in activity . Further study is needed to indicate how general a mechanism this is, but it provides an indication that the cellular environment and the composition of the miRNP may have a considerable effect on miRNA activity.
Very recently, miR-10a was shown to interact with sites in the 5′-UTR of 5′-TOP (5′-terminal oligopyrimidine tract) mRNAs . These mRNAs encode components of the ribosome and translational machinery. The TOP sequence at the 5′-end of these mRNAs governs a rapid increase in translation in response to growth signals. miR-10a was shown to bind downstream of, and adjacent to, the TOP sequence, and this binding was required for activation of translation. Interestingly, the binding sites for miR-10a in TOP mRNAs did not have extensive seed matches, although the overall thermal stability of the miRNA–mRNA duplex was good. Other target sites that are similar to this, and may not be present in 3′-UTRs, are likely to have been missed by current bioinformatic site identification tools. As with miR-122, miR-10a can mediate different processes by binding to sites in different locations, as 3′-UTR-binding sites for this miRNA were also identified, and miR-10a binding to these sites results in repression of gene expression. The mechanism by which miR-10a positively regulates translation of TOP mRNAs is not yet known.
The miRNA pathway and viruses
The role for miR-122 in HCV replication fits in with increasing evidence of interplay between viral infection and the miRNA pathway. Several DNA viruses express miRNAs, and these can regulate both viral and cellular transcripts and play a role in viral infection or latency. Other studies have suggested that cellular miRNAs directly target and repress PFV-1 (primate foamy virus 1) and VSV (vesicular-stomatitis virus), but the role of these miRNAs in viral infection has not been established, and it is likely that viruses would evolve to escape deleterious host miRNA interactions . As yet, a positive effect on viral replication by direct binding of a cellular miRNA has only been observed in HCV, but it is possible that this mechanism may extend to different viruses.
A recent paper showed that IFN (interferon) β treatment of cells results in up-regulation of a number of miRNAs . Five of these have predicted targets in the HCV RNA genome, and their overexpression exerts a negative effect on HCV. Mutagenesis indicated that direct binding to their sites in HCV RNA mediates this effect for two of these miRNAs. Interestingly, miR-122 expression decreased upon IFN treatment, and overexpression of miR-122 partially alleviated the antiviral effects of IFN .
Further research has indicated that miR-122 is required for HCV replication in the genotype 2a JFH-1 system, which permits production of infectious virus in Huh7 cells. Therefore, it appears that the conservation of the miR-122-binding sites across genotypes of HCV is functionally important and that the miR-122 requirement for HCV replication extends across genotypes. Moreover, RNAi (RNA interference)-mediated knockdown of components of the miRNA and RNAi pathways resulted in inhibition of HCV replication . This indicates that the positive effect of miR-122 on HCV replication overrides the effects of any negatively acting miRNAs or siRNAs.
The finding that a miRNA is required for HCV infection has great potential for the development of novel antiviral therapies. Excitingly, intravenous injection of LNA (locked nucleic acid)-modified oligonucleotides complementary to miR-122 into African green monkeys led to effective, reversible inhibition of miR-122 activity in the liver . The relief of miR-122 inhibition of cellular targets, most of which are involved in cholesterol metabolism, resulted in a decrease in total plasma cholesterol levels. No toxicity was observed over the course of the study, suggesting that targeting miR-122 is a promising avenue to pursue for the treatment of HCV infection. A greater understanding of the role for miR-122 binding to the two adjacent sites we have identified in HCV is important for the future development of novel therapies.
MicroRNAs and the Regulation of Biological Function: A Biochemical Society Focused Meeting held at Imperial College London, U.K., 8 July 2008. Organized and Edited by Tamas Dalmay (University of East Anglia, U.K.), Mark Lindsay (Imperial College London, U.K.) and Sterghios Moschos (Imperial College London, U.K.).
Abbreviations: HCV, hepatitis C virus; IFN, interferon; IRES, internal ribosome entry site; miRNA, microRNA; TOP, terminal oligopyrimidine tract; UTR, untranslated region
- © The Authors Journal compilation © 2008 Biochemical Society