Interferon regulatory factor 3-independent double-stranded RNA-induced inhibition of hepatitis C virus replicons in human embryonic kidney 293 cells

Abstract

The treatment of human embryonic kidney 293 cells harboring a hepatitis C virus (HCV) subgenomic replicon with the double-stranded RNA (dsRNA) mimic poly(I . C) inhibits HCV RNA replication through an undefined mechanism. Interferon regulatory factor 3 (IRF 3) has been widely postulated to mediate various antiviral responses, and its role in mediating the response to dsRNA in 293 cells was examined. Treating the cells with dsRNA did not induce IRF-3 activation, as measured by nuclear localization or the induction of reporter genes. Moreover, the expression of a dominant negative form of IRF-3 did not affect either colony formation upon transfection of subgenomic replicon RNA or the inhibition of the HCV replicon by dsRNA. Our results suggest that the inhibition of HCV RNA replication by poly(I . C) in 293 cells is independent of IRF-3 activation.

FIG. 1.

Induction of IRF-3 nuclear localization by dsRNA. (A) Huh-7 or 293 cells harboring the HCV replicon, i.e., S22.3 and LIP3 cells, respectively, were transfected with a GFP-IRF-3 expression vector and then treated with 20 HAU of SenV per ml or 250 μg of poly(I · C) per ml for 24 h, and the localization of IRF-3 was visualized by fluorescence microscopy. (B) The GFP-IRF-3 construct was transfected into Huh-7 cells. The cells were then treated with 20 HAU of SenV per ml, with the indicated amounts of the dsRNA mimic poly(I · C) or poly(A · U) for 24 h (Dose), or with 250 μg of dsRNA per ml for the indicated durations (Time). (C) 293 cells were treated like the Huh-7 cells described above. DAPI staining is presented to the right sides of selected panels.

FIG. 2.

Induction of IRF-3-responsive reporter genes by dsRNA. Luciferase reporter genes regulated by ISRE (A) or the RANTES promoter (B) were transfected into 293 cells. Cells were treated with 0, 5, 20, and 50 HAU (first to last bars, respectively) of SenV per ml or 0, 50, 250, and 500 μg (first to last bars, respectively) of poly(I · C) and poly(A · U) (A) or poly(I · C) and poly(G · C) per ml (B). The averages of results of three independent experiments are presented.

FIG. 3.

Establishment of dominant negative IRF-3 ΔN 293 cell lines. (A) Stable expression levels of Flag-IRF-3 ΔN (ΔN) were examined using Western blotting of 2IN cells, 293 cells transiently overexpressing IRF-3 (Transient), stable cells lines generated by transfecting an empty vector (2pd), or stable cells lines expressing wild-type (WT) IRF-3 (2IW-5 and 2IW-2). Proteins were detected with an IRF-3-specific polyclonal antibody (upper panel) or a Flag tag-specific monoclonal antibody (α-Flag; lower panel). Molecular size markers (in kilodaltons) are noted at the left. (B) 293, LIP3, or 2IN cells were transfected with the ISRE or RANTES luciferase reporter genes and were treated with SenV as described in the legend for Fig. 2.

FIG. 4.

Dominant negative IRF-3 ΔN has no effect on stable replicon establishment or inhibition by dsRNA. (A) Stable 293 cells expressing dominant negative IRF-3 (2IN) or wild-type IRF-3 (2IW) or stable cells that were generated by introducing an empty vector (2PD) were transfected with either total RNA isolated from replicon-containing 293Rep17 cells, an in vitro-transcribed R3 replicon, or replication-incompetent mutant RNA (GDD). Cells were selected for 3 weeks, and the numbers of colonies generated were quantified (shown in the lower right corners). The panel is representative of two separately performed experiments. (B) Inhibition of HCV RNA replication by dsRNA (3.9 to 1,000 μg/ml) measured by TaqMan real-time reverse transcription-PCR of LIP3 cells or stable 293 cells expressing dominant negative IRF-3 and harboring the replicon (2NR cells). Results are the averages of three independent determinations.