Hepatitis C virus RNA functionally sequesters miR-122

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.

Figure 1

Argonaute binding maps on HCV RNA. (A) Mock-subtracted binding map of Ago-CLIP reads across HCV genomic RNA in WT or ΔDrosha Huh-7.5 cells. Data were normalized to total cellular and virus read depth for comparison. Significant peaks per track are named by location and indicated by asterisks. Bottom CIMS track shows location of all deletions (gray) and statistically significant CIMS deletions (red) from the WT track. (B) Ago binding in significant peaks from WT Huh-7.5 cells in (A) shown as normalized read densities calculated per dataset. Data were normalized to background read density of non-peak regions (dashed line). Asterisks, **P<0.01, *P<0.05, Student’s t-test. Error bars, ±SD. (C) Schematic of a miR-122 binding model to S1 and S2 highlighting locations of CIMS deletions. (D) Zoom in view of Ago binding from WT cells in (A) across the viral IRES into the coding sequence. IRES domains (II–IV), associated stemloops (a–d) and the pseudoknot (pk) region are indicated. Upper track displays seeds for the top 50 miRNA seeds, previously proposed miR-122 binding (Pang et al., 2012) highlighted in red. (E–F) Ago binding timecourse of WT (E) and replication deficient (GNN) (F) HCV post-electroporation (n=2). (G) Absolute qPCR measurements of miR-122 and HCV RNA levels at indicated time points post electroporation (n=3). Replication-deficient J6/JFH1-GNN and mock controls are shown. Dashed line indicates lower limit of quantitation. Error bars, ±SD. See also Figure S3.

Figure 2

An HCV mutant resistant to miR-122 antagonism engages Ago and miR-122. (A–B) Timecourse qPCR measurements of WT-Clone2 virus (A) or U3-Clone2 virus (B) in WT cells or in ΔmiR-122 cells with or without 3nM miR-122 supplementation. Error bars, ±SD. (C) Ago binding map (top track) and CIMS locations (bottom track) across the U3 virus 5′UTR corresponding to miR-122 binding at S2. Relevant CIMS deletions are shown in gray (not significant) and red (significant). U3 snoRNA sequence is shown in green. (D) Ago binding map across the U3 virus genome after treatment with increasing doses of LNA122. Significant peaks are named by location and indicated by asterisks. Bottom CIMS track shows location of all deletions (gray) and significant CIMS deletions (red) for the untreated dataset. (E) Ago binding in significant peaks from untreated U3 datasets in (D) shown as normalized read densities calculated per dataset. ****P<0.0001, *P<0.05, one-way ANOVA with bonferroni correction. Error bars, ±SD.

Figure 3

HCV infection de-represses endogenous miR-122 targets. (A) Cumulative density function (CDF) of the log₂ fold change in CLIP binding between infected and uninfected cells for all 3′UTR clusters containing indicated 7–8mer seeds by family, from triplicate experiments. “Top” refers to the top 10 miRNA families, exclusive of miR-122. “All” refers to the top 50 miRNA families, inclusive of miR-122. Two-sided K-S test p-value between miR-122 and all targets shown. (B) The mean log₂ fold change (± ranges) in CLIP binding on miR-122 3′UTR targets versus all targets during HCV infection broken down by seed type. (C) A CDF plot during HCV infection as in (A) but measuring target mRNA expression via RNA-Seq, from duplicate experiments at 72hrs post-infection. Targets with more than one miRNA binding site were collapsed such that no gene is represented more than once per category. (D) The mean log₂ fold change (± ranges) in mRNA expression of CLIP targets during HCV infection broken down by seed type. (E–G) CDF plot as in (A), between treatment over control cells with LNA122 (E) or miravirsen (F) at 30nM or genetic deletion (G) of miR-122 (ΔmiR-122), each from triplicate experiments. (H) Proportional Venn diagram showing the overlap of miR-122 targets with reduced CLIP binding across ΔmiR-122, LNA or miravirsen treatment, and HCV infection conditions. Hypergeometic p-value of overlap shown. Asterisks: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, two-sided Mann-Whitney U-test. See also Figures S2 and S4.

Figure 4

Meta-analysis of published array data suggests HCV induced changes on the miR-122 target network. (A) Miravirsen pre- and post-treatment array data from four HCV infected chimpanzees (Lanford et al., 2010) was binned according to conserved 7–8mer TargetScan (TS) predictions for miR-15 or miR-122, or from miR-122 targets with CLIP support from the current study. Boxplot whiskers denote 1.5 times the inter-quartile distance from the nearest quartile. The mean fold change in expression for miR-122 targets was compared to miR-15 targets or all genes represented on the array, where the number of genes in each bin (n) is indicated. (B) Analysis as in (A) comparing 24 HCV positive to 5 negative liver biopsies (Peng et al., 2009). (C) Analysis as in (A) comparing 41 HCV positive with cirrhosis samples to 19 normal livers (Mas et al., 2009). Asterisks: ****P<0.0001, ***P<0.001, ** P<0.01, *P<0.05, ns P>0.05, two-sided Mann-Whitney U-test.

Figure 5

Validation of HCV induced de-repression of miR-122 targets in bulk and single-cell resolution. (A) Luciferase reporter measurements for synthetic miR-122, miR-17, or cellular 3′UTR target constructs. Data were normalized to “no oligo” p3,4 mutant conditions. Significance testing was performed relative to endogenous “no oligo” repression for each tested construct. Asterisks: *** P<0.001, ** P<0.01, * P<0.05, ANOVA with bonferroni correction. (B) Two-color fluorescent reporter containing a bidirectional Tet promoter that drives expression of blue and red fluorescent proteins (TagBFP and TagRFP). Each fluorescent protein is tagged with a nuclear localization sequence (NLS) to aid in flow cytometric analysis. The 3′UTR of TagRFP is engineered to contain N binding sites for miR-122, or full 3′UTRs of miR-122 targets. (C–F) Log-log transfer functions for N=1 (C), ALDOA 3′UTR (D), N=4 (E) or one perfectly complementary (F) miR-122 site in the presence or absence of 30nM miRNA mimic and/or HCV infection.

Figure 6

Quantitative modeling of miR-122 sequestration by HCV. (A) Illustration of model reactions for miR-122 dynamics, including transcription and translation of a target mRNA, binding to miR-122 and decay of mRNA species. HCV RNA can replicate, be degraded, or bind miR-122, functionally sequestering miR-122 and leading to de-repression of mRNA targets. (B) Increasing amounts of HCV (or a relative increase in binding strength at miR-122 sites) leads to changes in single-cell gene expression as compared to unregulated targets, with stronger effects at the low mRNA expression levels. Parameters used are fitted from data in (C). Each curve, from top to bottom, represents a reduction in the miRNA pool by 20%. Inset displays model on a linear scale. (C) Model fitting of the steady state approximation to experimental data while increasing the number of binding sites corresponding to changes in total binding strength. (D) Model fitting for the N=4 case showing a 50% reduction in the miRNA pool by HCV modeled by a proportional change in the theta parameter. (E) Model fitting for the N=4 construct under 30nM miR-122 mimic addition ± HCV infection. (F) Increasing HCV:miR-122 binding strength or HCV RNA abundance results in functional de-repression of miR-122 targets. The curves (top to bottom) represent 10 percentage-points increases in the available miR-122 pool (10% to 100% availability). (G–H) Experimental HCV induced derepression of synthetic miR-122 binding site constructs (G) or endogenous 3′ UTRs with miR-122 binding sites (H). See also Figure S5 and S6.

Figure 7

Exchanging HCV miRNA tropism redirects functional miRNA sequestration. (A) Base pairing diagram of miR-15a onto the mutated m15 HCV RNA. Base changes from the WT at S1 and S2 are highlighted in blue. (B) Luciferase measurements of supernatants from WT and m15 HCV reporter virus electroporations (±SD). Non-replicating GNN control is shown. (C) Dose response of WT and m15 reporter viruses following pre-treatment with LNA inhibitors of miR-122 or miR-15a/b at indicated concentrations, measured at 96 hours post-infection (±SD). (D) Time course post infection of ΔmiR-122 Huh-7.5 cells of indicated viruses (±SD). (E) Ago binding map of m15 virus infection in WT Huh-7.5 (top panel) or ΔmiR-122 Huh-7.5 cells (bottom panel). Data were normalized to total cellular read depth for cross track comparison. Statistically significant peaks per track are named by location and are indicated by asterisks. (F) Ago binding in significant peaks from (E) shown as normalized read densities calculated per dataset. Two-sided Student’s t-test used. Error bars, ±SD.. (G) CDF plot of the log₂ fold change in mRNA expression between HCV m15 infected and uninfected cells for all 3′UTR clusters containing indicated 7–8mer seeds by family, from duplicate experiments at 96hrs post infection. “Top” refers to the top 10 miRNA families, exclusive of miR-122 and miR-15. “All” refers to the top 50 miRNA families, inclusive of miR-122 and miR-15. Two-sided K-S test P-value comparing miR-15 (blue) or miR-122 (red) clusters to “All” is shown. (H) Infectivity titers of m15 virus in ΔmiR-122 Huh-7.5 complemented with exogenous miR-122 at indicated concentrations. One-way ANOVA with Bonferroni correction, whiskers, ± ranges. Asterisks: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. See also Figure S7.