Competing and noncompeting activities of miR-122 and the 5' exonuclease Xrn1 in regulation of hepatitis C virus replication

Abstract

Hepatitis C virus (HCV) replication is dependent on microRNA 122 (miR-122), a liver-specific microRNA that recruits Argonaute 2 to the 5' end of the viral genome, stabilizing it and slowing its decay both in cell-free reactions and in infected cells. Here we describe the RNA degradation pathways against which miR-122 provides protection. Transfected HCV RNA is degraded by both the 5' exonuclease Xrn1 and 3' exonuclease exosome complex, whereas replicating RNA within infected cells is degraded primarily by Xrn1 with no contribution from the exosome. Consistent with this, sequencing of the 5' and 3' ends of RNA degradation intermediates in infected cells confirmed that 5' decay is the primary pathway for HCV RNA degradation. Xrn1 knockdown enhances HCV replication, indicating that Xrn1 decay and the viral replicase compete to set RNA abundance within infected cells. Xrn1 knockdown and miR-122 supplementation have equal, redundant, and nonadditive effects on the rate of viral RNA decay, indicating that miR-122 protects HCV RNA from 5' decay. Nevertheless, Xrn1 knockdown does not rescue replication of a viral mutant defective in miR-122 binding, indicating that miR-122 has additional yet uncharacterized function(s) in the viral life cycle.

Fig. 1.

Decay of transfected HCV RNA in HeLa cells. (A) Immunoblots of (Left) Xrn1 and Upf1 in HeLa cells following siRNA transfection and (Right) Rrp41 and PmScl100 in HeLa cells stably expressing the indicated shRNA. β-actin was a loading control. (B) HeLa cells were transfected with the indicated siRNAs for 48 h and electroporated with replication-deficient genotype 1a H77S-AAG RNA with or without miR-122 (1 μM). The percentage of HCV RNA remaining at each time point following electroporation without (Left) and with (Right) miR-122 supplementation was determined by qRT-PCR, relative to the abundance of β-actin mRNA. Results shown represent the means of three replicate experiments ± SEM. (C) HeLa cells expressing the indicated shRNAs were electroporated as in B. Percent HCV RNA remaining following electroporation without (Left) and with (Right) miR-122 supplementation was quantified by qRT-PCR relative to the abundance of β-actin mRNA. Results shown represent the means of three replicate experiments ± SEM.

Fig. 2.

Decay of HCV RNA in HeLa S10 lysate. (A) (Upper) miR-122 and mutant miR-122p6 guide strand sequence; (Lower) 5′ terminal sequence of HCV RNA (H77S.3/AAG) with S1 and S2 miR-122 seed sequence-binding sites underlined. Point mutations (*) in the related S1-S2p6m mutant are shown above. (B) H77S.3 RNA was incubated with HeLa S10 lysate containing the indicated duplex miRNA (1 μM). RNAs were extracted at indicated time intervals, stained with SYTO 62, and resolved in 1% agarose. Percent HCV RNA remaining was quantified by the Odyssey Infrared Imaging System relative to the 28S rRNA. Results are the means of three experiments ± SEM. (C) Decay assays were carried out as in B with the H77S.3/S1-S2p6m mutant RNA. (D and E) Decay assays were carried out as in B with lysates from (D) HeLa cells transfected with control or Xrn1 siRNA (mean ± SEM from two replicate experiments) or (E) lysates from HeLa cells stably expressing the indicated shRNA (mean ± SEM from three replicate experiments).

Fig. 3.

Decay of replicating HCV RNA in Xrn1- and PM/Scl-100–depleted cells following PSI-6130 arrest of viral RNA synthesis. (A) Huh-7.5 cells were transfected with replication-competent genotype 1a H77S.3 RNA, and then retransfected 48 h later with siRNAs specific for Xrn1 or PM/Scl-100 or scrambled siCtrl. After an additional 48-h incubation (time = 0), the cells were treated with 10 μM PSI-6130, with (Right) or without (Left) simultaneous miR-122 supplementation. Data shown represent percent HCV RNA remaining following addition of PSI-6130. RNA was quantified by qRT-PCR relative to the abundance of actin mRNA. Results are the means of three experiments ± SEM. (B) Estimated half-life (t1/2) of HCV RNA ± 95% CI under the conditions shown in A. The data were fit to a one-phase decay model (R² = 0.946–0.983). While the decay constant, k, differed significantly between siXrn1-, siPmScl-100–, and siCtrl-transfected cells in the absence of miR-122 supplementation (P < 0.0001 by the extra sum-of-squares F test), there was no significant difference in cells supplemented with miR-122 (P = 0.19). (C) GLuc expression from cells transfected with H77S.3/GLuc2A RNA, followed by transfection of siXrn1 or siCtrl and PSI-6130 arrest of new viral RNA synthesis as in A. Cells were supplemented with miR-122 or miR-124 at the time of addition of PSI-6130, and media were replaced at 4-h intervals thereafter. Data shown are the mean GLuc activity ± SD from four replicate cultures and are representative of multiple experiments. (D) Confocal microscopy demonstrating the absence of colocalization of HCV RNA with Xrn1. Huh-7.5 cells were transfected with H77S.3 RNA for 4 d and then subjected to FISH for detection of HCV RNA (red). Xrn1 was visualized by subsequent immunostaining (green) and is concentrated in P bodies (arrow in center panel). Nuclei (N) are marked by the absence of HCV RNA and Xrn1. Inset represents an enlarged view of a portion of the merged image. An uninfected cell in the lower right quadrant provides an internal control for FISH.

Fig. 4.

Xrn1 knockdown enhances HCV replication in Huh-7.5 cells. Huh-7.5 cells were transfected with siRNAs specific for Xrn1 or PM/Scl-100 or scrambled siCtrl and then 24 h later retransfected with H77S.3/GLuc2A RNA. (A) Immunoblots of Xrn1, PM/Scl-100, and HCV core protein 72 h after HCV RNA transfection, with β-actin as a loading control. (B) GLuc activity in supernatant fluids from Huh-7.5 cells transfected with HCV RNA and the indicated siRNAs. (C) HCV RNA was quantified by qRT-PCR 72 h after HCV RNA transfection relative to β-actin mRNA. (D) Infectious virus titer of supernatant fluids from Huh-7.5 cells transfected with HCV RNA for 48 or 72 h, determined by a fluorescent focus formation assay. (E) GLuc assays were carried out as in B with or without cotransfection of miR-122 (50 nM) and the indicated siRNAs. (F) Cells in E at 72 h posttransfection were harvested, and HCV RNA was quantified by qRT-PCR relative to β-actin mRNA. (G) Graded knockdown of Xrn1 (Upper) by transfection of increasing concentrations of siXrn1 results in proportionate increases in GLuc expression (Lower) that plateaus at the highest siXrn1 concentrations significantly below the level of GLuc expression resulting from miR-122 supplementation. Cells shown as transfected with 0 nM siXrn1 received 80 nM siCtrl. (H) Huh-7.5 cells were transfected with HCV RNA containing p6m mutations in both miR-122 binding sites (HJ3-5/GLuc2A-S1-S2p6m) with cotransfection of the indicated miRNAs. GLuc activity in supernatant fluids was analyzed at indicated time intervals. (I) Graded knockdown of Xrn1 (see G) results in increased transient expression of GLuc from HJ3-5/GLuc2A-S1-S2p6m (due to stabilization of the transfected RNA) but fails to rescue replication of the miR-122 binding mutant. Results shown in G and I represent the mean ± range from replicate cultures, whereas all other results represent the means of three replicate experiments ± SEM.