Abstract
Following acute HCV infection, the virus establishes a chronic disease in the majority of patients whilst few individuals clear the infection spontaneously. The precise mechanisms that determine chronic HCV infection or spontaneous clearance are not completely understood but are proposed to be driven by host and viral genetic factors as well as HCV encoded immunomodulatory proteins. Using the HIV-1 LTR as a tool to measure NF-κB activity, we identified that the HCV E1E2 glycoproteins and more so the E2 protein down-modulates HIV-1 LTR activation in 293T, TZM-bl and the more physiologically relevant Huh7 liver derived cell line. We demonstrate this effect is specifically mediated through inhibiting NF-κB binding to the LTR and show that this effect was conserved for all HCV genotypes tested. Transcriptomic analysis of 293T cells expressing the HCV glycoproteins identified E1E2 mediated stimulation of the endoplasmic reticulum (ER) stress response pathway and upregulation of stress response genes such as ATF3. Through shRNA mediated inhibition of ATF3, one of the components, we observed that E1E2 mediated inhibitory effects on HIV-1 LTR activity was alleviated. Our in vitro studies demonstrate that HCV Env glycoprotein activates host ER Stress Pathways known to inhibit NF-κB activity. This has potential implications for understanding HCV induced immune activation as well as oncogenesis.
Highlights
Hepatitis C Virus (HCV) is a member of the Flaviviridae family which infects approximately 58 million people worldwide and can mediate severe hepatic injury and progressive liver fibrosis through stimulating persistent inflammation and oxidative stress [1–5]
Through generating pseudo-typed viral stocks expressing variant Env proteins [Human Immunodeficiency Virus Type 1 (HIV-1) (R5), HIV-1 (X4), HCV Env and Ebola virus glycoprotein (GP)] a consistent decrease in viral p24 production was observed for the HCV E1E2 and TZM-bl-soluble E2 (sE2) cell lines were generated (E1E2) stocks in comparison to other viruses
We confirmed HCV E1E2 Env expression from analysing stably transfected TZM-bl cells with a plasmid expressing a tagged E1E2 molecule using FACS (Supplementary Figure 1). These findings identify that expression of the HCV E1E2 Env protein down-modulates pseudo-typed viral production in 293T producer cells
Summary
Hepatitis C Virus (HCV) is a member of the Flaviviridae family which infects approximately 58 million people worldwide and can mediate severe hepatic injury and progressive liver fibrosis through stimulating persistent inflammation and oxidative stress [1–5]. Infection with HCV in vitro stimulates endoplasmic reticulum (ER) stress and leads to the induction of the Unfolded Protein Response (UPR) and autophagy. These cryoprotective responses function to resolve ER stress by reducing protein synthesis and increasing the protein processing capacity of the infected cell [34–36]. Several reports have identified that individual proteins of HCV are potent activators of the UPR independent of HCV replication, including the envelope (Env) glycoproteins E1 and E2 [40–42] Both E1 and E2 are highly glycosylated type 1 transmembrane glycoproteins which use membrane anchored C-terminal tails to remain tethered in the endoplasmic reticulum (ER) prior to a series of protein maturation events [43]. There is limited understanding regarding the molecular mechanisms through which HCV Env glycoproteins may modulate cellular immune responses and contribute to HCV mediated immune regulation
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