title: Impaired Nrf1-dependent gene expression in cells replicating HCV results in elevated cholesterol levels and impacts the size of lipid droplets
Creators: name: Schorsch, Patrycja
Publisher: name: UNSPECIFIED
Year: 2024

description: Hepatitis C virus (HCV) infection may lead to chronic hepatitis. Currently, there are more than 57,8 million individuals globally who experience persistent infection, enduring the consequences of chronic hepatitis, which can often progress to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). One mechanism to protect against oxidative stress is the Nrf2/Keap1 pathway. Nrf2/ARE signalling is impaired in HCV replicating cells, due to withdrawal of sMaf proteins from nucleus and binding to NS3 on the cytoplasmic site of the ER, as the integral part of replicon complex. The NS3-bound sMaf proteins bind to Nrf2, which prevents Nrf2 from entering the nucleus to trigger the expression of the genes responsible for protection against oxidative stress. Another factor involved in redox homeostasis is the ubiquitously expressed transcription factor Nrf1 of the Cap´N´Collar family. Nrf1 is located in the ER and upon stimulus, the inactive 120 kDa glycoprotein is selectively processed in the ER to gain distinct multiple isoforms (between ~25-kDa and ~140-kDa). Besides, Nrf1 has been recently described as a cholesterol sensor that protects the liver from excess cholesterol.
In this thesis, the impact of HCV infection on Nrf1 expression, localization, antioxidant response and cholesterol sensing ability were studied. Additionally, the relevance of the Nrf1-HCV crosstalk for the viral life cycle and virus-associated pathogenesis was examined. The data indicated that, HCV infection reduced Nrf1 protein levels while increasing Nrf1 transcript expression in both cell culture models and liver sections of patients with chronic HCV infection. However, the HCV-induced decrease in Nrf1 protein levels was not attributed to a shortened half-life. Nrf1 knockdown experiments showed an impact on HCV protein levels and viral titers. Overexpression of Nrf1 fragments, specifically the 25kDa (dominant-negative inhibitor of longer NRF1 isoforms and NRF2) and 85kDa (cleaved and transcriptionally active isoform of Nrf1) isoforms, led to negative effects on the HCV life cycle, including
replication, accumulation and release of viral particles. Additionally, the interplay between Nrf1 fragments and sMaf proteins influenced Nrf1 fragments localization. Overexpressing Nrf1 fragments could not rescue the decreased expression of the cytoprotective gene NQO1, suggesting the complexity of Nrf1's regulation in HCV infection.
HCV infection impacts Nrf1 expression, localization and activity, disrupting host-virus interactions essential for efficient replication. The observed decrease in viral replication and impaired release of viral particles suggest Nrf1's potential regulatory role in modulating HCV infection. Additionally, the crosstalk between HCV and Nrf1 has a direct impact on the activation of Nrf1-ARE dependent gene expression including genes related to controlling of the lipid metabolism. HCV infection affects LXR promoter activity and Nrf1 fragments' overexpression has an impact on cholesterol regulation. The reduced activation of the LXR expression in HCV positive cells due to an impaired Nrf1-dependent
activation of the LXR-promoter may be reflected by an impaired cholesterol export leading to an elevated intracellular cholesterol level. Inhibited activity of Nrf1 in HCV-positive cells influences the lipid content and therefore the number and size of lipid droplets. Targeting Nrf1 or its associated pathways may offer promising therapeutic strategies to disrupt the HCV life cycle and inhibit viral replication. The use of cell culture models is one of the study's limitations, which emphasizes the necessity of validation in in vivo systems or clinical samples. Taken together, these data provide the relevance of Nrf1's role as an antiviral factor in response to HCV infection in addition to a well-known crucial role in oxidative stress and cholesterol removal program.
This study described for the first-time extensive work on the various mechanisms and explores the fragments of Nrf1 specific fragments in affecting HCV replication and host cells. Further studies in addressing the question about the about the Nrf1-HCV crosstalk, could be of interest to gain a better understanding of how Nrf1 modulates the oxidative stress response and cholesterol removal program in the context of viral infections.