Fig. 8

A schematic representation summarizes the establishment of HCV4a-HEK293T stable cells for persistent production of HCV4a-VLPs. a A workflow showing the in vitro generation of HCV4a-HEK293T stable cells that was carried out on two stages. The first stage (Top Panel) was performed in the packaging cells, HEK293T, to produce lentivectors carrying HCV4a structural genes (HCV4a-Lentiviruses). The second stage (Bottom Panel) was conducted in the target cells, HEK293T, by the transduction of target cells with the produced HCV4a-Lentiviruses to insert HCV4a four genes, Core, E1, E2 and P7 into the genome of the target cell for stable expression of VLPs. b A schematic diagram showing the in vivo lentiviral-based strategy for stable cells development. In the packaging cells, Left Side (1) Cells were co-transfected with three lentiviral plasmids, transfer (pLV-HCV4a C–P7), packaging (psPAX2) and envelope (pMD2.G) plasmids by the calcium phosphate precipitation method. (2, 3) Transfected plasmids were translocated to the nucleus and transcribed into mRNAs that were transported to the cytoplasm for the translation. (4, 5) The mRNAs were translated into lentiviruses components that can build the complete functional lentiviruses with a genome encoding HCV structural genes. (6, 7) The assembled HCV4a-lentiviruses exit the cells and released to the medium. The lentiviruses were purified, concentrated and used to infect new normal HEK293T cells as target cells. In the target cells, Right Side, the stable cell line was generated for persistent expression of HCV4a structural proteins, Core, E1, E2 and P7 and production of intact enveloped VLPs for HCV genotype 4a. (1, 2) The target cells were infected with the harvested HCV4a-Lentiviruses by binding to cell surface receptors and the fusion with the cell membrane for internalization. (3, 4, 5) The lentiviruses RNA genome was released into the cytoplasm and reverse transcribed into viral DNA which was translocated to the nucleus [87]. In the nucleus, the viral DNA was inserted and integrated into the genome of the target cells. (7, 8) The integrated HCV genes were transcribed into mRNAs of the HCV4a structural genes which were translocated to the cytoplasm for the translation on the surface of the ER. (9, 10, 11) The translated HCV proteins oligomerized and self-assembled into HCV4a-VLPs mimicking the HCV native virus. This diagram was created with BioRender.com