Regulation of hepatitis C virus translation and infectious virus production by the microRNA miR-122 - PubMed (original) (raw)
Regulation of hepatitis C virus translation and infectious virus production by the microRNA miR-122
Rohit K Jangra et al. J Virol. 2010 Jul.
Abstract
miR-122 is a liver-specific microRNA that positively regulates hepatitis C virus (HCV) RNA abundance and is essential for production of infectious HCV. Using a genetic approach, we show that its ability to enhance yields of infectious virus is dependent upon two miR-122-binding sites near the 5' end of the HCV genome, S1 and S2. Viral RNA with base substitutions in both S1 and S2 failed to produce infectious virus in transfected cells, while virus production was rescued to near-wild-type levels in cells supplemented with a complementary miR-122 mutant. A comparison of mutants with substitutions in only one site revealed S1 to be dominant, as an S2 but not S1 mutant produced high virus yields in cells supplemented with wild-type miR-122. Translation of HCV RNA was reduced over 50% by mutations in either S1 or S2 and was partially rescued by transfection of the complementary miR-122 mutant. Unlike the case for virus replication, however, both sites function equally in regulating translation. We conclude that miR-122 promotes replication by binding directly to both sites in the genomic RNA and, at least in part, by stimulating internal ribosome entry site (IRES)-mediated translation. However, a comparison of the replication capacities of the double-binding-site mutant and an IRES mutant with a quantitatively equivalent defect in translation suggests that the decrement in translation associated with loss of miR-122 binding is insufficient to explain the profound defect in virus production by the double mutant. miR-122 is thus likely to act at an additional step in the virus life cycle.
Figures
FIG. 1.
The replication of infectious HCV is dependent on direct interaction of miR-122 with the RNA genome. (A) Schematic representation of the two miR-122-binding sites, S1 and S2, located between stem-loop (SL) I and II in the 5′UTR of the HJ3-5 genome. At the top is shown the sequence of miR-122. The miR-122 seed sequence and miR-122 complementary sequences in HCV RNA (boxed) are highlighted in bold. The sequences of the S1-p34m and S1-p6m mutants are shown in italic, with base substitutions underlined. (B) Huh-7.5 cells were transfected with the indicated RNA oligonucleotides at 50 nM and 24 h later were infected with HJ3-5 virus, an intergenotypic chimeric HCV, at an MOI of 1. At 72 h postinfection, cell lysates were prepared and subjected to immunoblotting using β-actin, HCV core and NS5B-specific antibodies. (C) Huh-7.5 cells in eight-well chamber slides were transfected with the indicated RNA oligonucleotides at 50 nM and 24 h later were infected with 100 to 120 FFU of chimeric HJ3-5 virus. Seventy-two hours later, cells were labeled for HCV core antigen and individual foci of infected cells enumerated by immunofluorescence microscopy. The results shown represent the means from three independent experiments, each performed in duplicate, ± standard deviations (SD). (D) miR-122 regulates infectious virus production by HCV RNA-transfected cells. FT3-7 cells were transfected with wild-type (WT), p34 mutant, or p6 mutant form of miR-122 or were mock treated. Twenty-four hours later, cells were transfected with wild-type or mutant HJ3-5 HCV RNAs, followed by another transfection of miR-122 and related mutants at 48 h. Supernatant fluids collected 2 and 3 days after HCV RNA transfection were assayed for infectious virus by FFU assay on naïve Huh-7.5 cells (average ± SD; n = 2). (E) Immunoblots (top panel) and Northern blots (bottom panel) of extracts from the cells in panel D prepared 3 days after HCV RNA transfection.
FIG. 2.
Relative importance of the two 5′UTR miR-122-binding sites for production of infectious virus. (A) Schematic representation of the S1 and S2 miR-122-binding site mutant sequences. (B) FT3-7 cells were transfected with the indicated miRNA, retransfected with HJ3-5 RNAs 24 h later, and transfected once more with miRNAs 24 h after that. Samples prepared 3 days after HCV RNA transfection were subjected to immunoblot analysis of HCV core protein, with calnexin run in parallel as a loading control (top panel), and Northern blotting for HCV RNA using β-actin mRNA as a loading control (bottom panel). (C) Infectious virus yields 3 days after HCV RNA transfection of the cells in panel B, as quantified by a fluorescent infectious focus assay using naïve or miR-122p6-supplemented Huh-7.5 cells transfected with miR-122p6 at 50 nM 24 h prior to sample inoculation. Data shown are from a representative experiment.
FIG. 3.
Binding of miR-122 to the S1 site promotes HCV translation. (A) Schematic representation of HJ3-5/RLuc2A-GND (referred to as GND in the text), which contains an in-frame insertion of the RLuc luciferase sequence fused to FMDV 2A (RLuc2A) between p7- and NS2-coding regions of HJ3-5 and a replication-lethal Asn substitution within the active site of the NS5B RNA-dependent RNA polymerase. (B) Duplicate cultures of Huh-7.5 cells in a six-well plate were transfected with miR-122 to supplement endogenous miR-122 levels or with a 2′-O-methyl antisense RNA, anti-miR-122, to functionally sequester endogenous miR-122. Twenty hours later, GND (left panel) or GND-S1-p6m (right panel) HCV RNA (1.25 μg/well) was transfected together with a capped and polyadenylated FLuc mRNA (0.25 μg/well). Dual luciferase assays were carried out on lysates prepared at the intervals noted following HCV RNA transfection, with results expressed as the mean RLuc/FLuc ratio (± SD) at each time point. Solid and dashed lines are best-fit third-order polynomial plots (_r_2 of between 0.78 and 0.99). The data shown are representative of multiple experiments.
FIG. 4.
Contribution of S1 versus S2 binding sites to miR-122 promotion of viral translation versus RNA replication. (A) Huh-7.5 cells were transfected with miR-122, miR-122p6, or miR-124 and cotransfected 20 h later with the indicated GND (see Fig. 3A) and capped control FLuc RNAs. The results shown represent dual luciferase reporter assays of cell lysates prepared 8 h after HCV RNA transfection, presented as the ratio of RLuc to FLuc activity (mean ± SD). Similar results were obtained with 6-h lysates. The data shown are representative of multiple experiments. (B) FT3-7 cells were transfected with miR-122 or miR-122p6 or mock treated and then were retransfected 24 h later with HJ3-5/RLuc2A or related -S1-p6m, -S2-p6m, or -S1-S2-p6m mutant RNAs together with the capped control FLuc RNA. The cells were retransfected with the miRNAs 24 h later. Dual luciferase assays were carried out on lysates prepared 8, 34, 52, and 76 h after transfection of the HCV RNA. RLuc results were normalized to the 8-h FLuc value and are shown as fold increase over RLuc activity at 8 h (mean ± SD; n = 3).
FIG. 5.
Comparison of infectious virus yields from the double binding site mutant and IRES mutants with quantitatively similar and different defects in translation. (A) Schematic representation of IRES mutations constructed in stem-loop (SL) IIId of the HJ3-5 5′UTR. The sequence of stem-loop IIId is depicted, with nucleotide positions numbered according to the prototype genotype 1a H77 HCV sequence (GenBank accession no. NC_004102). (B) Translational activities of IRES mutants constructed in HJ3-5/RLuc2A-GND. Cells were cotransfected with GND or the indicated mutant GND RNAs with the capped, polyadenylated FLuc mRNA as an internal control for transfection and cellular translation efficiency. Dual luciferase reporter assays were carried out at 6 or 8 h following transfection, with results expressed as the RLuc/FLuc ratio. The data shown represent the means ± SD from five independent experiments. (C) Immunoblot assays for core protein expressed in FT3-7 cells 3 days after transfection with wild-type (WT) HJ3-5 RNA or related S1-S2-p6m (double miR-122-binding site) and IRES mutants in the HJ3-5 background. (D) Infectious virus yield in supernatant fluids from the HJ3-5 RNA-transfected cell cultures described for panel C. Infectivity titrations were done on fluids collected at 3 days posttransfection and are shown as mean ± range (n = 2).
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