Establishment of a subgenomic replicon for bovine viral diarrhea virus in Huh-7 cells and modulation of interferon-regulated factor 3-mediated antiviral response

Abstract

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.

FIG. 1.

Schematic representation of the BVDV genome and subgenomic replicons. (A) Genome organization of BVDV. (B) Structural genes C, E^rns, E1, E2, P7, and NS2 were replaced with a luciferase-ubiquitin-neomycin phosphotransferase cassette (Luc-Ubi-Neo) to create pLN-BR. (C) Further deletion of the luciferase and ubiquitin genes produced pN-BD.

FIG. 2.

Establishment of the BVDV replicon in Huh-7 cells. (A) Colony-forming abilities of RNA transcribed from, left to right, pLN-BR, pN-BR, and HCV replicon I₃₈₉luc-ubi-neo/NS3-3′/wt/5.1. (B) TaqMan quantitative PCR analysis of RNA samples isolated from the indicated cell clones. A specific probe for the neomycin phosphotransferase sequence (neo) was used to quantitate replicon RNA, and a GAPDH probe was used to quantitate the host gene. Higher Ct values indicate lower RNA abundance. Respective replicon RNA was detected in BVDV and HCV cell lines but not in parental Huh-7 cells. Similar levels of GAPDH mRNA were present in all samples. (C) Northern blot analysis of total RNA isolated from the replicon cells. A radiolabeled probe corresponding to a portion of the neomycin phosphotransferase gene was used to detect the replicon RNA, and a GAPDH probe was used as RNA control. One cell clone from pLN-BR (BVDV Luc-Ubi-Neo) (lane 1), two from pN-BR (BVDV Neo1 and Neo2) (lanes 2 and 3), and two from HCV NK5.1 (HCV Neo1 and Neo2) (lanes 4 and 5) were analyzed. 5-15 is a previously established HCV replicon cell line.

FIG. 3.

Effects of interferons on BVDV and HCV replicon RNA replication in Huh-7 cells. Luciferase-expressing BVDV/Huh7 or HCV/Huh7 cells in 96-well plates were treated with the indicated amounts of IFN-α (A), IFN-β (B), or IFN-γ (C) for 48 h. Luciferase signal detected in the absence of interferons was defined as 100% for each data set, and the remaining data points were expressed as a percentage of the response in untreated cells.

FIG. 4.

Demonstration of viral specificity with known inhibitor compounds. BVDV/Huh7 or HCV/Huh7 cells expressing luciferase were plated in white 96-well plates and allowed to attach overnight (∼18 h) before treatment with known small-molecule inhibitors for a further 48 h. (A and B) 2′-β-C-methyl adenosine (A) and 2′-β-C-methyl cytosine (B) showed comparable EC₅₀ values in the parallel replicon assays. (C) VP32947, a triazinoindole derivative, showed potent activity against BVDV/Huh-7, with an EC₅₀ of <0.14 μM, but was not active against HCV/Huh-7. (D) GSK-4, a benzothiadiazine derivative, showed specific activity against HCV/Huh7 but much lower activity against BVDV/Huh7.

FIG. 5.

BVDV replicon blocks IRF-3-dependent transcription after SV infection. Parental Huh-7 cells and several BVDV/Huh7 or HCV/Huh7 cell lines (40,000 cells/well in a 24-well plate) were transfected with a firefly luciferase reporter plasmid under the control of an IRF-3-responsive promoter. Cells were infected with SV 24 h after transfection. After an 18-h incubation postinfection, the cells were lysed and the production of luciferase was quantified. Luciferase signals were normalized for transfection efficiency against a constitutively active Renilla luciferase reporter cotransfected into the cells, and the data are expressed as the fold increase before and after SV infection in firefly luciferase signal relative to that in uninfected Huh-7 cells. Both BVDV/Huh-7 and HCV/Huh-7 blocked SV-induced transcription from ISRE (A), ISG56 (B), RANTES (C), and IFN-β (D) promoters. Three independent HCV replicon clones (HCV Neo1, Neo2, and Neo6) and four independent BVDV replicon clones (BVDV Neo1, Neo2, Neo3, and Neo5) were tested in addition to parental Huh-7 and 5-15 cells. Error bars represent the standard deviations.

FIG. 6.

Suppression of IRF-3-dependent transcription after SV infection requires the presence of replicon RNA. Control Huh-7 cells and replicon-containing cells (BVD Neo and HCV Neo) were treated with either VP32947 (0.25 μM) or GSK-4 (4 μM) or mock treated for 2 weeks in the absence of G418 selection. Medium was replaced every 3 to 4 days with freshly added inhibitor compound, and cells were split before reaching confluence. At the end of treatment, cells were assayed for SV-induced IRF-3 activation by using an ISRE reporter plasmid (see the legend to Fig. 5). Equal amounts of total RNA isolated from the untreated and treated cells were analyzed for levels of replicon RNA by using sequence-specific (neo) real-time quantitative RT-PCR analysis (TaqMan) as described in Materials and Methods. (A) Fold induction of ISRE-mediated luciferase signal after SV infection. The cell origin and inhibitor used are indicated at the bottom of the graph. Luciferase signals were normalized for transfection efficiency against a constitutively active Renilla luciferase reporter cotransfected into the cells, and the data are expressed as the fold increase before and after SV infection in firefly luciferase signal relative to that in the uninfected Huh-7 cells. (B) Relative levels of replicon RNA after inhibitor treatments, expressed as Ct values. Higher Ct values indicate lower RNA abundance. Cell origin and the inhibitor used are indicated below.

FIG. 7.

BVDV N^pro autoprotease partially blocks SV-induced IRF-3 activation. (A) Effect of HCV NS3/4A, BVDV NS3/4A, or BVDV N^pro on IRF-3-dependent transcription in Huh-7 cells infected with SV. Expression of HCV NS3/4A serine protease, but not BVDV NS3/4A, blocked the up-regulation of ISRE-mediated transcription in a dose-dependent manner. Expression of N^pro autoprotease also showed a dose-dependent suppression of ISRE-directed transcription. (B) N^pro-mediated inhibition of SV-induced IRF-3 activation in Huh-7 cells was confirmed by mutating two of its catalytic residues (Glu₂₂ and His₄₉) and creating an inactive form of N^pro (mtN^pro). Expression of the inactive form of N^pro did not suppress the ISRE-mediated transcription induced by SV. Data were analyzed with GraphPad Prism software (GraphPad Software) using a paired t test to determine the significance of observed differences between control groups and the indicated treatment groups. **, P < 0.005; ***, P < 0.0001. Error bars represent the standard deviations.