Hepatitis C virus addiction to liver miR‐122 takes its Toll on the host
Authors: Amir Shlomai
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. Hepatitis C virus (HCV) is a positive‐strand RNA virus that recruits the liver‐specific microRNA‐122 (miR‐122) to its genome to support its replication. In a recent article Luna et al.1 employed high‐throughput sequencing and crosslinking immunoprecipitation (HITS‐CLIP) of the microRNA (miRNA) regulator Argonaute (Ago) in HCV‐infected cells. They show that the robust recruitment of miR‐122 by HCV results in reduced availability of miR‐122 to bind and repress its cellular messenger RNA (mRNA) targets. Several of the derepressed host genes are implicated in liver fibrosis and carcinogenesis, suggesting that by recruitment of a large fraction of cellular miR‐122, HCV alters the host's gene expression in a way that might promote liver pathologies. As an obligatory parasite, HCV utilizes the host's resources for its needs. A prominent example is the dependence of HCV on the liver‐specific miR‐122, which is recruited to two distinct regions at the 5′‐untranslated region (5′UTR) of the viral genomic RNA to support its replication.2 In contrast to the positive role of miR‐122 in the HCV life cycle, miRNAs, including miR‐122, usually attenuate cellular gene expression. This is done through binding to complementary regions at the 3′UTR of their target mRNAs, resulting in mRNA degradation and/or translational silencing. Thus, one can envision a self‐perpetuating process in which miR‐122 promotes HCV replication and increases the viral RNA pool that in turn binds additional miR‐122 molecules. Theoretically, miR‐122 recruitment by HCV may limit miR‐122 availability to bind and suppress its "natural" cellular target genes, as was recently suggested by a study utilizing a reporter for miR‐122 activity in HCV‐infected hepatoma cells.3 However, because of the minimal requirement of only a six‐ to eight‐nucleotide base pairing match between an miRNA and its target mRNA, a detailed transcriptome analysis of miR‐122 targets in the context of HCV infection, based solely on bioinformatics prediction, is extremely challenging. To address this, Luna and colleagues exploited the powerful HITS‐CLIP technology to biochemically map miR‐122 recruitment to HCV as well as to its cellular targets. The Ago HITS‐CLIP takes advantage of the close proximity between an miRNA, its target mRNA, and the Ago protein, an essential component of the RNA‐induced silencing complex that facilitates mRNA silencing. Following ultraviolet cross‐linking and coprecipitation with Ago‐specific antibodies, the Ago‐bound RNA fragments are isolated and a complementary cDNA library is produced. A high‐throughput sequencing generates a genome‐wide data set of miRNAs and their mRNA target sites that can be further paired using bioinformatics analyses.4 By first mapping Ago binding to HCV RNA in infected hepatoma cells, the authors show that Ago is robustly recruited mostly, but not exclusively, to the two predicted miR‐122 binding sites at the 5′UTR of the plus strand HCV RNA. Furthermore, comparison of Ago binding to HCV RNA in wild‐type versus Drosha knockout cells, depleted from most cellular miRNAs but enriched with exogenous miR‐122, shows that miR‐122 is by far the dominant cellular miRNA recruited by HCV. On the host side, HCV replication resulted in a significant reduction in Ago binding to the 3′UTR of mRNAs targeted by miR‐122 but not by other miRNAs. Functionally, an RNA‐seq analysis showed increased levels of transcripts targeted by miR‐122 relative to their levels in HCV‐null cells. Thus, the authors concluded that HCV RNA massively recruits miR‐122, thereby reducing its availability to bind and suppress the expression of its target genes. A wide variety of elegant controls strongly support the paper's main conclusions. For one, Ago HITS‐CLIP reads of miR‐122 target genes obtained in HCV‐infected cells mostly overlapped those obtained in HCV‐null cells treated with the miR‐122 antagonist miravirsen5 or in cells knocked out for miR‐122 by CRISPR/Cas9. In addition, to directly link viral recruitment of miRNA to derepression of its cellular target genes, the authors created the "perfect control" by swapping the miR‐122 with an miR‐15 binding site on the HCV RNA. The resulting mutant virus (m15) can replicate and bind miR15, but not miR‐122, to its 5′UTR. Importantly, m15 viral replication leads to specific derepression of miR‐15 but not miR‐122 target genes, thereby reversing the miR‐122 derepression observed with wild‐type virus (Fig. 1).Figure 1: The HCV "sponge effect" on miR‐122 is redirected to miR15 by the m15 mutant virus. In HCV null liver cells, both miR15 and miR‐122 bind to the 3′UTR of their target mRNAs to suppress their expression (left). In HCV‐infected cells, miR‐122 is robustly recruited mainly to the 5′UTR of the viral RNA. This results in detachment of a large fraction of miR‐122 from its target mRNAs, resulting in their derepression, thereby possibly promoting pathologies such as malignant transformation (middle). In cells infected with a recombinant mutant virus that contains a miR‐15 binding site at its 5′UTR (m15 HCV mutant), miR15, rather than miR‐122, is sponged by the virus, resulting in derepression of miR‐15 target mRNAs (right).The idea of using the Ago HITS‐CLIP to elucidate the interplay between a virus and cellular miRNAs is not new. An Ago HITS‐CLIP analysis previously performed in cells infected with Kaposi's sarcoma–associated herpesvirus revealed that the majority of Ago‐bound miRNAs are virus‐derived and therefore may "hijack" the RNA‐induced silencing complex to reduce its accessibility for cellular miRNAs. This results in a global derepression of host mRNAs that might undermine normal cellular physiology.6 Albeit mediated through a different mechanism, the outcomes of miR‐122 sequestration by HCV could be far‐reaching as well. Indeed, miR‐122 has a major role in maintaining normal liver homeostasis as evident by miR‐122 knockout mice that develop liver steatosis, inflammation and cancer,7 pathologies that are also associated with HCV infection. Furthermore, several miR‐122 targets found in this study to be derepressed upon HCV infection, such as P4HA1, PKM2, and MASP1, are up‐regulated in liver fibrosis and carcinogenesis. Therefore, it is tempting to speculate that miR‐122 sequestration by HCV and the resulting deregulation of its cellular target genes might link HCV infection to liver cirrhosis and cancer. Still, most experiments performed in this study were done in hepatoma cells; and therefore, the clinical relevance of this study's intriguing findings is not straightforward. First, based on the "sponge effect," one could predict that the risk of developing liver pathologies in HCV patients will correlate to some extent with viral replication. However, in contrast to the case of hepatitis B virus, where the risk for liver cancer correlates well with viral load,8 this is not the case in HCV patients. Second, primary human liver cells contain much higher levels of miR‐122 and support lower rates of HCV replication compared to hepatoma cells; and therefore, the "sponge effect" observed in hepatoma cells might not necessarily hold true in vivo. To partially address this caveat, the authors performed single‐cell analyses using a reporter for miR‐122 activity, showing that upon supplementation of exogenous miR‐122 to reach physiological, in vivo‐like levels, HCV's ability to rescue miR‐122 repression is not abrogated. Yet, the relative low percentage of HCV‐infected hepatocytes in vivo compared to the situation in Huh‐7.5 hepatoma cells calls into question whether the global derepression of miR‐122 targets seen in vitro is also valid in vivo. The authors rely on microarrays performed in liver samples taken from HCV‐infected and uninfected donors, suggesting specific de‐depression of miR‐122 target genes in the presence of HCV. However, a direct comparison of Ago HITS‐CLIP analyses performed on liver biopsies from HCV‐infected or uninfected patients and correlation with viral replication could be much more convincing. Nevertheless, given the large heterogeneity in HCV replication in vivo, it is still possible that the so‐called sponge effect and the resulting derepression of miR‐122 targets are mainly maintained at the single‐cell level but might be largely masked upon bulk cell analysis. In summary, this study not only shows how a virus might alter its host's gene expression by massive recruitment of its chief miRNA but also suggests a novel mechanism by which HCV might promote pathological changes at the cellular level. Future studies should better define the miR‐122 target genes derepressed by HCV, which could be imperative for promoting intracellular pathologies, especially malignant transformation. That way, the addiction of HCV for cellular miR‐122 could be a platform not only for understanding viral biology but also for elucidating the pathophysiology of its host. Author names in bold denote shared co‐first authorship.