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Abstract

Hepatotropic viruses constitute a global health concern, as the WHO estimates over 300 million people worldwide suffering from chronic viral hepatitis. One of the causative agents is the Hepatitis C virus (HCV). In many infected, HCV is able to evade clearance by the immune system and establish a persistent infection. Here, the characteristics of a constantly activated immune response turn from antiviral to pro-inflammatory, thereby establishing a chronic inflammation of the liver, which promotes increasingly severe levels of liver damage, liver failure, and the development of liver cancer. In this study, we investigated the consequences of prolonged activation of antiviral signaling pathways in a virus-free cell culture system, using the lung adenocarcinoma cell line A549 and the immortalized hepatocyte cell line PH5CH. Ectopic expression of NS5B, the RNA-dependent RNA polymerase of HCV, produced double-stranded RNA (dsRNA) that was sensed by the pattern recognition receptor retinoic acid-inducible gene I (RIG-I). This induced the expression and secretion of interferons (IFNs) beta and lambda 1, pro-inflammatory cytokines tumor necrosis factor and interleukin 6, and chemokines CCL3, -4, -17, and CXCL10. Cells expressed interferon-stimulated genes (ISGs) and exhibited reduced proliferation, which we could attribute to a concerted effect of IFN-beta and IFN-lambda 1. We showed that IFN-lambda 1 expression correlated with NS5B expression on a single cell level, while ISG induction and impairment of proliferation was exerted through paracrine IFN signaling. NS5B expression was sufficiently high to trigger the RIG-I pathway for over 14 days, before unspecific downregulation of the transgene, likely in combination with counterselection of NS5B high-expressing cells, led to ceasing production of IFNs. We compared the supernatant from NS5B-expressing cells to a defined mix of IFN-beta and IFN-lambda 1 in elicited changes in global gene transcription of exposed cells. In A549 and in PH5CH, both treatments triggered a very similar response in the upregulation of, for the most part, well-defined ISGs. Long-term exposure of naïve A549 cells to NS5B-derived supernatant for one month showed only subtle differences in ISG gene expression, suggesting that cells do not become refractory to IFN-beta or IFN-lambda 1. Of three genes that were significantly upregulated only after treatment for one month, the tetraspanin TM4SF4 warrants further investigation due to its implication in hepatocellular carcinoma and a possible link to SARS-CoV2 infection. In summary, this study describes a cell culture system that enables to study the effects of continuous antiviral signaling for over two weeks, thereby mimicking a persistent viral infection while excluding virus-mediated interference. Furthermore, the system allows to produce a mix of secreted factors that resembles the epithelial-derived secretome present in an infected organ. Further elucidating the composition of this mix as well as long-term co-culture experiments of NS5B-expressing cells and non-parenchymal liver-resident or -infiltrating cells may help elucidate the complex intercellular dynamics that form the basis of HCV-associated chronic infection, inflammation, and liver deterioration.