The Tat protein of human immunodeficiency virus-1 enhances hepatitis C virus replication through interferon gamma-inducible protein-10

Abstract

Background: Co-infection with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is associated with faster progression of liver disease and an increase in HCV persistence. However, the mechanism by which HIV-1 accelerates the progression of HCV liver disease remains unknown.

Results: HIV-1/HCV co-infection is associated with increased expression of interferon gamma-induced protein-10 (IP-10) mRNA in peripheral blood mononuclear cells (PBMCs). HCV RNA levels were higher in PBMCs of patients with HIV-1/HCV co-infection than in patients with HCV mono-infection. HIV-1 Tat and IP-10 activated HCV replication in a time-dependent manner, and HIV-1 Tat induced IP-10 production. In addition, the effect of HIV-1 Tat on HCV replication was blocked by anti-IP-10 monoclonal antibody, demonstrating that the effect of HIV-1 Tat on HCV replication depends on IP-10. Taken together, these results suggest that HIV-1 Tat protein activates HCV replication by upregulating IP-10 production.

Conclusions: HIV-1/HCV co-infection is associated with increased expression of IP-10 mRNA and replication of HCV RNA. Furthermore, both HIV-1 Tat and IP-10 activate HCV replication. HIV-1 Tat activates HCV replication by upregulating IP-10 production. These results expand our understanding of HIV-1 in HCV replication and the mechanism involved in the regulation of HCV replication mediated by HIV-1 during co-infection.

Figure 1

Analysis of IP-10 mRNA and HCV RNA levels in PBMCs isolated from healthy individuals, HCV mono-infected patients, and HIV-1/HCV co-infected patients. (A) Transcriptional expression of IP-10 in PBMCs isolated from HCV mono-infected patients, HIV-1/HCV co-infected patients, and healthy individuals. Data were normalized to the amount of GAPDH-specific mRNA as a reference transcript. Numbers below each patient group (e.g. n = 24) indicate the number of persons in that group. (B) Replication of HCV RNA in PBMCs isolated from patients with HCV mono-infection and HIV-1/HCV co-infection. Data were normalized to the amount of GAPDH-specific mRNA as a reference transcript. (C) Correlations between the expression of IP-10 mRNA and HCV RNA in PBMCs isolated from patients with HCV mono-infection and HIV-1/HCV co-infection. (D) Correlations between the expression of IP-10 mRNA and HCV RNA in PBMCs isolated from patients with HIV-1/HCV co-infection. Two groups of patients were analyzed for IP-10 mRNA and HCV RNA expression. Data were subjected to linear regression analysis. Correlation coefficients are indicated.

Figure 2

Determination of the roles of IP-10 protein in the activation of HCV replication. (A) Huh7.5.1 cells were infected with FL-J6/JFH-5'C19Rluc2Aubi virus for 24 h, and then incubated with PBS, IP-10 (1 μg/ml), or heat-inactivated IP-10 (HI-IP-10) (1 μg/ml) in 24-well plates for different times, as indicated. Treated cells were harvested and analyzed for luciferase activity. (B) Huh7.5.1 cells were infected with JFH1 virus, and then incubated with PBS, IP-10, or HI-IP-10 for 9 days. Culture supernatants were harvested and HCV copy numbers were determined by real-time PCR (RT-PCR). (C) Huh7.5.1 cells were infected with JFH1 virus, and then incubated with PBS, IP-10, or HI-IP-10 for 9 days. Cells were harvested and analyzed for HCV core protein expression by Western blot. β-Actin expression was measured as a loading control. (D) Huh7.5.1 cells were infected with FL-J6/JFH-5'C19Rluc2Aubi virus and then incubated with PBS, 1 μg/ml MSIgG, or 1 μg/ml anti-IP-10 neutralizing antibody for 144 h. Cells were harvested at different times as indicated and luciferase activity was assayed.

Figure 3

Analysis of the role of HIV-1 Tat protein in the regulation of IP-10 expression. (A) Huh7.5.1 cells were infected with or without JFH1 virus and then incubated with PBS, 1 μg/ml Tat, or 1 μg/ml heat-inactivated Tat (HI-Tat) for different times, as indicated. Cell culture supernatants were collected and IP-10 levels were quantified by ELISA. (B) Huh7.5.1 cells were infected with or without JFH1 virus and then incubated with PBS, 1 μg/ml Tat, or 1 μg/ml heat-inactivated Tat (HI-Tat) for different times, as indicated. Cells were collected and the levels of IP-10 mRNA were quantified by real-time PCR (RT-PCR).

Figure 4

Determination of the role of HIV-1 Tat protein in the activation of HCV replication. (A) Huh7.5.1 cells were infected with FL-J6/JFH-5'C19Rluc2Aubi virus and then incubated with PBS, 1 μg/ml Tat, or 1 μg/ml HI-Tat in 24-well plates for different times, as indicated. Cells were harvested and analyzed for luciferase activity. (B) Huh7.5.1 cells were infected with JFH1 virus and then incubated with PBS, Tat, or HI-Tat for 9 days. Cell culture supernatants were harvested and HCV copy numbers were determined by real-time PCR (RT-PCR). (C) Huh7.5.1 cells were infected with JFH1 virus and then incubated with PBS, Tat, or HI-Tat for 9 days. Cells were harvested and HCV core protein levels were detected by Western blot analyses. β-actin level was determined as a loading control.

Figure 5

Role of endogenous IP-10 in the activation of HCV replication regulated by HIV-1 Tat protein determined by antibody depletion. Huh7.5.1 cells were infected with FL-J6/JFH-5'C19Rluc2Aubi virus and then incubated with PBS, Tat, Tat plus MSIgG, and Tat plus anti-IP-10 neutralizing antibody, respectively, in 24-well plates for 120 h. Cells were then harvested at different times (as indicated) and luciferase activity was assayed.