Fig. 5

Production of two-layers enveloped VLPs by HCV4a-HEK293T stable cells. a A schematic diagram showing the principal of proteinase K protection assay. The structure of HCV-VLPs contains a central capsid of core protein surrounded by an outer lipid-bilayer originated from ER-derived membranes named the envelope and contains the HCV E1 and E2 glycoproteins. Because the VLPs contain both inner protein and outer lipid layers, selective solubilization of the outer envelope layer by Triton-X100 and the western blotting analysis for the presence or absence of the HCV core protein after Proteinase K digestion can be used to study the VLPs assembly process. This diagram was created with BioRender.com. b The assembly and envelopment of HCV4a-VLPs in the developed HCV4a-HEK293T stable cells. Enough number of HCV4a-HEK293T stable cells were cultured and harvested. The cell lysate was prepared and subjected to the treatment with either proteinase K alone or in combination with the detergent, Triton X100. The cell lysate was divided into three equal parts in separate tubes for the following different treatments; (1) Tube 1 for untreated cell lysate, (2) Tube 2 for the treatment with Triton-X100 followed by Proteinase K treatment of the cell lysate to cleave the core protein after dissolving the envelope with Triton X100, (3) Tube 3 for the treatment of cell lysate with only Proteinase K where the core is protected within the outer lipid layer of the envelope of the HCV4a-VLPs. After the treatments, all lysates were mixed with SDS-loading dye and subjected to the SDS-PAGE and WB analysis for detection of core protein using the HCV core antigen mouse monoclonal antibodies (C7-50) and goat anti-mouse IgG (H+L) secondary antibodies, HRP