Abstract
After binding of its ligand transferrin, the transferrin receptor (TfR) is internalized via early endosomes. Ligand and receptor can be recycled. α-Taxilin was identified as an essential factor for TfR recycling. Apart from its role for iron uptake, TfR is a coreceptor for hepatitis C virus (HCV) infection. In HCV-replicating cells, the amount of a-taxilin is decreased. This study aims to investigate the effect of decreased α-taxilin levels in HCV-replicating cells on recycling of TfR, its amount on the cell surface, on iron uptake, and the impact of a disturbed TfR recycling on HCV superinfection exclusion. TfR amount and localization were determined by CLSM and surface biotinylation. α-taxilin expression was modulated by CRISPR-Cas9 knockout, siRNA, and stable or transient overexpression. For analysis of HCV superinfection fluorophor-tagged reporter viruses were used. The amount of α-taxilin is decreased in HCV-infected cells. In accordance to this, the protein amount of TfR is significant lower in HCV-positve cells as compared to the control, while TfR expression is not affected. Due to the impaired recycling, internalized TfR is degraded by the endosomal/lysosomal system. The significant lower number of TfR molecules on the cell surface is reflected by reduced transferrin binding/internalization and strong reduction of intracellular iron level. Overexpression of α-taxilin in HCV-replicating cells rescues TfR recycling, augments TfR on the cell surface, and restores transferrin binding. The block of superinfection in HCV-replicating cells could be overcome by overexpression of α-taxilin. Taken together, the diminished level of α-taxilin in HCV-replicating cells prevents recycling of TfR leading to decreased transferrin binding and iron uptake. Disappearance of TfR from the cell surface could be a factor contributing to the exclusion of superinfection by HCV.
Highlights
The hepatitis C virus (HCV) causes acute and chronic liver diseases
In case of the α-taxilin overexpressing Huh7.5 cells, there is no significant difference. These data further corroborate the observation that the decreased amount of α-taxilin in HCV-replicating cells leads to an impaired recycling of the transferrin receptor (TfR) and increased lysosomal degradation, as reflected by the intracellular accumulation in case of BFLA treated cells
Rescue of the taxilin amount in HCV-replicating cells restores the level of TfR on the surface and overcomes the block of superinfection
Summary
The hepatitis C virus (HCV) causes acute and chronic liver diseases. Currently, about 71 million people worldwide suffer from chronic infection with HCV and approximately 399,000 people die each year on HCV-associated liver cirrhosis and hepatocellular carcinoma (WHO, 2019). The tetraspanin CD81, claudin-1 (CLDN1) and occludin (OCLN), low density lipoprotein receptor (LDLR), as well as the cholesterol transporter Niemann-Pick C1-like protein (NPC1L1) are required for the HCV entry (Pileri et al, 1998; Evans et al, 2007; Ploss et al, 2009; Albecka et al, 2012; Sainz et al, 2012) Another coreceptor mediating the virus entry is the transferrin receptor (TfR) with a monomer size of 90–95 kDa (Daniels et al, 2006; Martin and Uprichard, 2013). We investigate the effect of the decreased level of α-taxilin in HCV-replicating cells on the recycling of TfR, its amount on the cell surface, the capacity to bind transferrin and on the iron uptake, and the impact on HCV superinfection
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