Analysis of the Domains of Hepatitis C Virus Core and NS5A Proteins that Activate the Nrf2/ARE Cascade

Abstract

The hepatitis C virus (HCV) triggers a chronic disease that is often accompanied by a spectrum of liver pathologies and metabolic alterations. The oxidative stress that occurs in the infected cells is considered as one of the mechanisms of HCV pathogenesis. It is induced by the viral core and NS5A proteins. It is already known that both of these proteins activate the antioxidant defense system controlled by the Nrf2 transcription factor. Here, we show that this activation is mediated by domain 1 of the NS5A protein and two fragments of the core protein. In both cases, this activation is achieved through two mechanisms. One of them is mediated by reactive oxygen species (ROS) and protein kinase C, whereas the other is triggered through ROS-independent activation of casein kinase 2 and phosphoinositide 3-kinase. In the case of the HCV core, the ROS-dependent mechanism was assigned to the 37-191 a.a. fragment, while the ROS-independent mechanism was assigned to the 1-36 a.a. fragment. Such assignment of the mechanisms to different domains is the first evidence of their independence. In addition, our data revealed that intracellular localization of HCV proteins has no impact on the regulation of the antioxidant defense system.

Keywords: Hepatitis C virus; Nrf2; oxidative stress; regulation; transcription factor.

Fig. 1

Activation of the Nrf2/ARE cascade by domain 1 of the NS5A protein results in the translocation of the Nrf2 transcription factor from the cytoplasm into the nucleus (A) and induction of human NAD(P)H:quinone oxidoreductase 1 (Nqo1) and heme oxygenase 1 (HO-1) (B, C). Intracellular localization of factor Nrf2 was determined by separation of cytoplasmic (Cyt) and nuclear (Nu) protein fractions, with subsequent detection of Nrf2 by immunoblotting. Quantification of the Nqo1 and HO-1 expression levels was performed by reverse transcription and real-time PCR (B) and immunoblotting (C). * p < 0.01 and **p < 0.05 compared to pVax1.

Fig. 2

Fragments 1–36 and 37–191 a.a. of the HCV core protein activate the Nrf2/ARE cascade by triggering the translocation of transcription factor Nrf2 from the cytoplasm into the nucleus (A) with subsequent induction of human NAD(P)H:quinone oxidoreductase 1 (Nqo1) and heme oxygenase 1 (HO-1) (B, C). Intracellular localization of factor Nrf2 was determined by separation of cytoplasmic (Cyt) and nuclear (Nu) protein fractions, with subsequent detection of Nrf2 by immunoblotting. Quantification of the Nqo1 and HO-1 expression levels was performed by reverse transcription and real-time PCR (B) and immunoblotting (C). *p < 0.01 and **p < 0.05 compared to pVax1.

Fig. 3

Fragment 1–36 a.a. of the HCV core protein activates the Nrf2/ARE cascade by the ROS-independent mechanism involving casein kinase 2 and phosphoinositide 3-kinase; the 37–191 a.a. fragment – by the ROS-dependent mechanism involving protein kinase C. The role of protein kinases and reactive oxygen species was estimated by measuring luciferase expression in Huh7 cells co-transfected with the reporter pP-ARE plasmid with constructs encoding the full-length core protein (A) or its fragments 1–36 a.a. (B) and 37–191 a.a. (C) or by an analysis of the intracellular localization of Nrf2 (D). The latter was studied by separation of cytoplasmic (Cyt) and nuclear (Nu) protein fractions, with subsequent detection of Nrf2 by immunoblotting. Inhibitors of protein kinases Ro 31-8220 (Ro, protein kinase C inhibitor), DRB (casein kinase 2 inhibitor), or wortmannin (Wo, phosphoinositide 3-kinase inhibitor) in the absence or presence of an antioxidant pyrrolidine dithiocarbamate (PDTC) were added into the culture medium 18 h post-transfection. *p < 0.01.