Asunaprevir Evokes Hepatocytes Innate Immunity to Restrict the Replication of Hepatitis C and Dengue Virus

Abstract

Type I Interferon-mediated innate immunity against Flaviviridae, such as Hepatitis C virus (HCV) and Dengue virus (DENV), involves TLR3, RIG-I-like receptor (RLR) and JAK-STAT signal pathways. Asunaprevir is a newly developed HCV protease inhibitor for HCV treatment. Whether, asunaprevir activates innate immunity to restrict viral infection is unclear. Thus, this study investigates the effect of asunaprevir on innate immunity and its influence on HCV and DENV infection. Huh 7.5.1, Hep-G2 cells, JFH-1 infection model, and DENV-2 infection were used for the analysis. The activity of asunaprevir-regulated innate immunity signal pathway was assessed with IFN-β promoter or IFN-stimulated responsive element (ISRE) reporter assays and immunoblotting of key signal proteins. siRNA-mediated MAVS and TRIF knockdown of cells was performed to assess the effect of asunaprevir-regulated innate immunity against HCV and DENV. Asunaprevir treatment activated ISRE and IFN-β promoter-luciferase activities and signaling proteins in the JAK-STAT, MAVS, and TRIF pathways in Huh 7.5.1 cells. Asunaprevir-mediated signaling activation was decreased in MAVS-knockdown cells. Importantly, both RNA and protein levels of DENV-2 NS3 were decreased in asunaprevir-treated Huh 7.5.1 and HepG2 cells. In MAVS-knockdown cells, the restrictive effect of asunaprevir on HCV and DENV was attenuated. Our findings reveal an unexpected activity of asunaprevir, the activation of MAVS dependent innate immunity to restrict HCV and DENV infection.

Keywords: Dengue virus; asunaprevir; hepatitis C; innate immunity; interferon type I.

Figure 1

Asunaprevir activates ISRE activity and type I IFN and TLR3/RIG-I antiviral signaling pathway. (A) ISRE luciferase reporter assay, plasmids of pISRE-luc expressing firefly luciferase and pRL-TK expressing Renilla luciferase as an internal control were co-transfected to Huh 7.5.1 cells and then treated with 1 or 10 nM of asunaprevir for 3, 6, 24, and 48 h. The 0 nM indicated DMSO vehicle control. The firefly and Renilla luciferase activities were then measured by dual-luciferase assay. Relative firefly luciferase activity was normalized to Renilla luciferase activity. (B) Huh 7.5.1 were treated with different doses of asunaprevir for 48 h and the cell lysates were analyzed by immunoblotting with the indicated antibodies involved in the interferon signaling pathway (upper panels). The protein levels of STAT-1, phosphorylated STAT-1, STAT-2, phosphorylated STAT-2, MxA, and ISG-15 relative to the β-actin were determined by densitometry with ImageJ software (lower, panels). (C) IFN-β luciferase reporter assay, plasmids pIFN-β/Fluc expressing firefly luciferase and pRL-TK expressing Renilla luciferase as an internal control were co-transfected to Huh 7.5.1 cells and then treated with 1, 10, or 100 nM of asunaprevir for 24 h. Firefly and Renilla luciferase activities were then measured. Relative firefly luciferase activity was normalized to Renilla luciferase activity. (D) Huh 7.5.1 cells were treated with different doses of asunaprevir for 48 h and the cell lysates were analyzed by immunoblotting with the indicated antibodies involved in the TLR3/RIG-I signaling pathway (upper panels). The protein levels of MAVS, TRIF, IRF-3, and phosphorylated IRF-3 relative to the β-actin were shown at the bottom of each sample. Immunoblots shown in each figure are representative of three independent experiments. Densitometry was performed with ImageJ software (lower, panels). Values represent the average of three assays ± S.D. Statistical significance was tested by Student's t-test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 2

Effects of Asunaprevir on TLR3/RIG-I signaling pathway in Huh. 7.5.1 cells with MAVS and TRIF knockdown. Huh 7.5.1 cells were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir for 24 h. The key signaling proteins such as MAVS, TRIF, IRF3, and phosphorylated IRF-3 were determined by immunoblotting analysis (left panel). Immunoblots shown in the figure are representative of three independent experiments. The protein levels phosphorylated IRF-3 over total IRF3 was analyzed with ImageJ software (right panel). Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t-test, ^*P < 0.05.

Figure 3

Effects of Asunaprevir on replication of DENV and HCV. (A) Huh 7.5.1 cells were treated with different doses of asunaprevir for 24 h and immunoblotting analysis for NS3 protein of DENV, and real-time PCR for RNA level of DENV were performed (B). (C) HepG2 cells were treated with different doses of asunaprevir for 24 h and immunoblotting analysis for NS3 protein of DENV, and real-time PCR for RNA level of DENV were performed (D). (E) JFH-1 infected Huh 7.5.1 cells were treated with asunaprevir for 24 h, followed by real-time PCR for RNA level of HCV. Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t-test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 4

Asunaprevir inhibits DENV-2 and HCV virus production. (A) The schematic of asunaprevir treatment in DENV and HCV infection in Huh 7.5.1 cells. The asunaprevir-treated Huh 7.5.1 cells were infected by DENV and JFH-1 for 48 h. The supernatant was harvested and transferred to another Huh 7.5.1 cells. After 48 h incubation, the DENV and HCV-infected cells were determined by immunofluorescence assay. (B,C) Immunofluorescence assay, the green fluorescence showed the signals of DENV-2 NS3 protein and HCV core protein, DAPI indicated cell nuclei (left panels). The infectivity of mean ± SD was calculated from 3 observation fields (right panels). About 300 cells in each field were inspected. Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t-test, ^*P < 0.05, ^***P < 0.001.

Figure 5

Effects of asunaprevir on replication of HCV in JFH-1- infected. Huh 7.5.1 cells after knockdown of MAVS and TRIF by siRNA. JFH-1-infected Huh 7.5.1 cells were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir for 24 h. HCV core protein, MAVS and TRIF were determined by immunoblotting analysis. The HCV core protein levels relative to the β-actin were shown at the bottom of each sample. Immunoblots shown in the figure are representative of three independent experiments. Densitometry was performed with ImageJ software. Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t-test, ^*P < 0.05, ^***P < 0.001.

Figure 6

Effects of asunaprevir on replication of DENV in Huh 7.5.1 cells after knockdown of MAVS and TRIF by siRNA. (A) Huh 7.5.1 cells infected with DENV were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir. Immunoblotting analysis for NS3 protein of DENV, MAVS, and TRIF were determined by immunoblotting Analysis (upper panels). The DENV NS3 protein levels relative to the β-actin were shown at the bottom of each sample. Densitometry was performed with ImageJ software (lower panel). Data are mean ± SD from 3 independent tests. Student's t-test was used as statistical test, ^***P < 0.001. (B) Huh 7.5.1 cells were transfected with pcDNA3.1 Flag-DV2 NS2B3 or pcDNA3.1 Flag-DV2 NS2B3 protease dead mutant (2 μg) for 24 h and then treated by asunaprevir for 24 h. Immunoblotting was accessed with anti-NS3 and anti β-actin. Immunoblots shown in each figure are representative of three independent experiments.