Fig. 3

Production of functional lentivectors encoding HCV4a structural genes. a Production of functional EGFP-lentivectors, EGFP-LVs. HEK293T cells were co-transfected with the three lentiviral plasmids, pWPXL, psPAX2 and pMD2G. The produced EGFP-LVs were collected 48, 72 and 96 h post-transfection, centrifuged, filtered and used to infect cells. The expression of EGFP in the infected cells was investigated 48 h post-infection using the Zoe Fluorescent Cell Imager (BioRad, USA). Non-infected cells were used as a negative control for the EGFP expression. b Production of functional HCV4a-lentivectors (HCV4a-Lentiviruses). HEK293T cells were co-transfected with pLV-HCV4a C–P7, psPAX2 and pMD2G. HCV4a-Lentiviruses -containing medium was collected, centrifuged, filtered and used for transduction of fresh HEK293T cells. 48 h post-transduction, the expression of HCV core gene in the infected cells was examined by the immunostaining using the HCV core antigen mouse monoclonal antibodies (C7-50) and the goat anti-mouse Alexa Fluor 555 secondary antibodies. The nuclei of cells were counter-stained with the Hoechst 33342 blue stain. Non-transduced cells were used as a negative control. c The titration of EGFP-LVs was conducted by the flow cytometric analysis of EGFP-expressing cells that were infected with the harvested EGFP-LVs-containing medium. On the top side, the flow cytometric analysis of non-infected control cells (negative control), while on the bottom side, HEK293T cells were infected with EGFP-LVs and the number of EGFP-expressing cells was counted by the flow cytometry using Alexa Fluor 488 FITC-A channel for detecting the fluorescence of EGFP protein. P2 represents the percentage of EGFP-expressing cells compared to the total number of cells. d The integration of HCV genes into the genome of infected cells. The genes integration was investigated by isolation of total genomic DNA from normal, HCV4a-LVs, and EGFP-LVs infected cells. The isolated DNA was subjected to PCR analysis to examine the presence of HCV or EGFP genes in the DNA samples using specific primers, GT4-E1–F and GT4-E2–R (Table 1) for detection of 1665 bp DNA fragment of HCV4a E1E2. While the integration of EGFP ORF was investigated using vector-specific primers, pWPXL-Seq-For and pWPXL-Seq-Rev to amplify a DNA fragment of about 1011 bp comprising the EGFP ORF. The gel is showing the presence of both HCV E1E2 and EGFP bands in the total genome of cells suggesting their integration in the infected cells