Figure 1.
HCV RNA replication and virus production in transfected human and mouse cell lines.
(A) Different human and mouse cell lines were transfected with Luc-Jc1 or a replication-deficient construct Luc-Jc1ΔGDD. HCV RNA replication was determined 4, 24, 48, 72 h post transfection using luciferase assays and is expressed as relative light units. (B) In parallel, cell-free culture fluid of transfected cells was harvested 48 h post transfection and infectivity was determined by inoculation of naïve Huh-7.5 cells with cell supernatants and subsequent luciferase assays at 72 h post inoculation. Mean values of quadruplicate measurements of a representative experiment with 3 independent repetitions are shown. The grey horizontal bar represents the background luciferase activity determined in mock infected Huh-7.5 cells.
Figure 2.
Trans-complementation of HCV assembly and release in heterokaryons between human liver cells.
(A) Schematic overview of the experimental procedure of PEG-mediated cell fusion. The JFH1 luciferase reporter replicon Luc-NS3-5B was transfected into naïve Huh-7.5 cells. The next day cells were washed with PBS and co-cultured with naïve Huh-7.5 cells or Huh-7.5 packaging cells constitutively expressing core, E1, E2, p7 and NS2 (Huh-7.5[CE1][E2p7NS2]). After 24 h of incubation, fusion was induced by treating co-cultivated cells with 40% PEG for 5 min at 37°C. Treatment with PBS instead of PEG served as control. After 48 h cell-free supernatant was used to inoculate naïve Huh-7.5 cells following determination of infectivity by luciferase assay. (B) For the detection of cell fusion and heterokaryon formation, cells were immunostained using monoclonal antibodies directed against structural protein E2 and non-structural protein NS5A. Simultaneous expression of both proteins within cells is indicative of heterokaryon formation (compare white arrows). (C) Fusion of Huh-7.5 cells transfected with a subgenomic reporter replicon to either Huh-7.5 naïve or Huh-7.5 packaging cells using PEG or PBS as a negative controls. Production of infectious particles was quantified by inoculation of naïve Huh-7.5 cells followed by luciferase readout 72 h post-infection. Mean values of 4 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.
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.
Figure 4.
Neutralization of virus particles released from heterokaryons by CD81- specific antibodies.
Huh-7.5 cells were inoculated with supernatants derived from heterokaryons between Luc-NS3-5B transfected Huh-7.5 cells and given packaging cell lines in the presence of CD81-specific monoclonal antibodies or CD13 control antibodies. The efficiency of infection was determined 72 h later by luciferase reporter assay and is expressed relative to infection in the presence of CD13-specific antibodies. Mean values of at least two independent experiments and standard deviations of the means are displayed.
Figure 5.
Responsiveness of selected packaging cell lines to viral pathogen associated molecular patterns.
Given cell lines were inoculated with a recombinant La Crosse virus lacking the non-structural protein NSs (rLACVdelNSs;[32]). At 8 or 16 h post inoculation (p.i.) cells were collected and expression levels of IFN-β (left) and ISG56 (right) were determined by quantitative RT-PCR. The relative induction compared to cells that were not inoculated with the virus is given. Mean values of three independent experiments including the standard deviations are given.
Figure 6.
Effect of human and mouse IFN-α treatment on HCV and VSV in human and mouse cells as well as in human-mouse heterokaryons.
(A) Four hours prior to infection with Luc-Jc1, Huh-7.5 cells were treated with the indicated dose of human or mouse IFN-α. HCV infectivity was assessed by measuring luciferase activity 72 h post-infection. (B) Hep56.1D cells were pre-incubated for 4 h with different doses of human or mouse IFN-α following inoculation with Luc-VSV for 4 h. After 72 h infectivity was determined by luciferase assay. (C) Either Huh-7.5 replicon cells or Hep56.1D[CE1][E2p7NS2] packaging cells were incubated with human or mouse IFN-α for 4h before co-culture. Production of trans-complemented particles after PEG-mediated induction of cell fusion was quantified by inoculation of naïve Huh-7.5 cells with cell supernatants of these cultures and determination of luciferase activity 72 h later. Mean values of two independent experiments and standard deviations of the means are displayed. (D) Mouse IFN-α was applied to Hep56.1D packaging cells for 4 h either at indicated time points before co-culture, or to cells co-cultured with Huh-7.5 replicon cells as indicated by cross hatched boxes of the diagram displayed above. Alternatively, mIFN-α was present for 48 h directly after fusion induction and until harvesting of cell culture fluid. In each case infectivity produced was determined by inoculation of naïve Huh-7.5 cells and is expressed relative to the control which was not treated with mouse IFN-α. Mean values of quadruplicate measurements including the standard deviations are shown. The grey horizontal bar indicates the background of the assay based on the luciferase activity determined in Huh-7.5 cells that had not been infected.
Figure 7.
Completion of HCV replication cycle after infection of heterokaryons.
(A) Schematic overview of the experimental procedure. Huh-7 Lunet N cells lacking CD81 (refractory to HCV cell entry; [34]) were co-cultured with the indicated cell lines lacking or expressing human CD81 (see also Table S2). Fusion between these cells was initiated by transfection of a fusogenic viral envelope protein derived from the prototype foamy virus the next day. Thirty hours later, cells were challenged with infectious HCV particles. Cell entry, RNA replication and virus production sustained by these cultures was quantified by determining infectivity titers in the cell-free culture fluid 48 h post inoculation using naïve Huh-7.5 cells as target cells for a limiting dilution assay. (B) For the detection of heterokaryons formed due to expression of the prototype foamy virus envelope protein, cells were stained with CellTracker Green or CellTracker Orange 6 h prior to co-cultivation (and transfection). Thirty hours later, cells were fixed with 3% PFA and stained with DAPI. The white arrow indicates heterokaryons produced upon co-culture of Lunet N cells labeled with CellTracker Orange and Lunet N cells stained with CellTracker Green. (C) Huh-7 Lunet N cells were fused to the different naïve cell lines or cell lines transduced to express human CD81. These cultures were inoculated with cell culture derived HCV particles (MOI of 2.3). Culture fluid was collected 48 h later and de novo particle release from heterokaryons was determined by limiting dilution (TCID50) using naïve Huh-7.5 target cells. Mean values of 3 independent experiments are given. The grey bar represents the detection limit of the limiting dilution assay.