Completion of Hepatitis C Virus Replication Cycle in Heterokaryons Excludes Dominant Restrictions in Human Non-liver and Mouse Liver Cell Lines
Figure 3
Production of infectious HCV from heterokaryons between human liver cells and human non-hepatic or mouse liver cells.
(A) HCV packaging cell lines constitutively expressing core, E1, E2, p7 and NS2 were created using two lentiviral vectors expressing core, E1 and E2, p7, NS2, respectively (Materials and Methods, and see also Table S1). Core protein expression in the individual packaging cell lines as determined by a commercial core-specific ELISA. (B) Lysates of given packaging cell lines were normalized for equal total protein content, serially diluted and incubated with Galanthus nivalis lectin coated culture plates to capture glycosylated proteins. Bound viral E2 protein was detected using an E2-specific monoclonal antibody (AP33). In each case, lysates of the parental cell line served as negative control. The OD value was plotted against the reciprocal dilution of the cell lysate. Relative expression of E2 protein between different cell lysates was determined by linear regression analysis as described in Material and Methods. The reciprocal dilution of the cell lysate required to reach an OD value of 0.5 for each lysate is given. Raw data of the ELISA are depicted in Figure S1. (C) Huh-7.5 cells transfected with the JFH1 replicon Luc-NS3-5B RNA were fused to the indicated naïve or packaging cell lines using PEG. Forty eight hours later cell-free media of these cultures were collected and used to inoculate naïve Huh-7.5 cells. Viral infectivity was quantified using luciferase assays. Mean values of 3 independent experiments and the standard deviations of the means are given. The grey horizontal bar represents the background luciferase activity determined in mock infected Huh-7.5 cells. Note that in case of heterokaryons involving AML12 and Hepa1-6 packaging cells, culture fluids were concentrated 20-fold to enhance sensitivity of the assay.