Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Cell Fusion by PEG

1. Cell Culture

1. Culture Huh-7.5, HeLa and Hep56.1D naïve or packaging cell lines ¹² on 15 cm cell culture dishes in DMEM complete (DMEM cplt) medium, DMEM supplemented with 2 mM L-glutamine, 1x non-essential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal calf serum. Apply appropriate selection to stable cell lines as indicated in Table 1.

2. Transfection of HCV RNA

1. Wash Huh-7.5 cells (one confluent 15 cm dish) twice with Phosphate Buffered Saline (PBS), trypsinize, and resuspend at a concentration of 1.5×10⁷ cells/mL in Cytomix (120 mM KCl, 0.15 mM CaCl₂, 10 mM K₂HPO₄ [pH 7.6], 25 mM HEPES, 2 mM EGTA, 5 mM MgCl₂, adjusted to pH 7.6 with KOH and sterilized by filtration) containing 2 mM ATP and 5 mM glutathione.
2. Transfect via electroporation 400 μL of the cell suspension (6×10⁶ cells) with 10 μg HCV RNA (pFKi389Luc-EI/NS3-3’_JFH1_dg ¹³) using cuvettes with a gap width of 0.4 cm and a Gene pulser Xcell system (Biorad) set to 975 μF and 270 V as described previously ¹⁴.
3. Immediately transfer transfected Huh-7.5 cells to 10 mL of DMEM cplt medium and seed the entire suspension into a single culture dish of 10 cm diameter. Culture cells for 24 h at 37 °C and 5% CO₂.

3. Induction of Fusion by PEG

1. Harvest cells as described in 2.1, resuspend in DMEM cplt medium and dilute to 5×10⁵ cells/mL.
2. Generate single-cell suspensions of Huh-7.5, HeLa, and Hep56.1D packaging cells, and dilute to 5×10⁵ cells/mL, 2×10⁵ cells/mL, and 2×10⁵ cells/mL, respectively. Note that packaging cells ectopically express HCV proteins core, E1, E2, p7 and NS2 after transduction with two independent lentiviral vectors transducing a C-E1 and E2-p7-NS2 expression cassette, respectively ^{13, 15}.
3. Prepare 6-well culture plates with 2-3 sterile glass cover slips per well for further analysis of fusion efficiency.
4. Combine 1 mL of transfected Huh-7.5 cells harboring the subgenomic replicon with 1 mL of packaging cells, seed into one well of a 6-well plate, and incubate for 24 h at 37 °C and 5% CO₂.
5. At this time point, cell density should range between 60-80% confluency before induction of cell fusion in order to allow cells to be in close proximity for cell membranes to fuse.
6. Aspirate medium from co-cultured cells and wash once with 1 mL PBS, then carefully add 500 μL of pre-warmed 40% PEG-1500 or PBS as control and incubate for 5 min at 37 °C to induce heterokaryon formation.
7. Aspirate PEG and carefully wash 3-5 times for 1 minute (min) per wash with 2 mL PBS to remove excess PEG and finally add 2 mL of DMEM cplt medium to each well and incubate at 37 °C and 5% CO₂.
8. Forty-eight hours post fusion, collect each supernatant and filter through a 0.45 μm filter.
9. Measure production of infectious particles in fused heterokaryons by inoculation of naïve Huh-7.5 cells (8×10⁴ cells/well seeded in a 12-well plate, 24 h prior to inoculation) with 500 μL of cell-free culture fluid and subsequent determination of luciferase activity ¹⁴ .

4. Immunofluorescence to Determine Fusion Efficiency

1. After collecting cell culture supernatants (section 3.8), wash wells containing glass cover slips once with 1 mL of PBS for 1 min. All following steps should be performed at room temperature (RT) and washing should be done with 1 mL PBS for 1 min per wash.
2. Fix cells with 600 μL of 3% paraformaldehyde (PFA) in PBS for 15 min and then wash 3x with PBS. Carefully transfer cover slips into a 24-well plate using forceps.
3. Permeabilize cells with 500 μL of 0.5% Triton X-100 in PBS for 5 min and subsequently wash 3x with PBS.
4. Prepare primary antibody solution in PBS supplemented with 5% goat serum. Use anti-NS5A antibody (9E10) at a concentration of 0.5 μg/mL and human monoclonal anti-E2 antibody (CBH-23) at a concentration of 6.4 ng/mL. Apply 250 μL to each well and incubate at RT for 45 min.
5. Wash 3x with PBS and detect bound primary antibodies with 250 μL secondary goat anti-mouse or goat anti-human IgG-specific antibodies conjugated to Alexa-Fluor 546 or Alexa-Fluor 488, at a concentration of 2 μg/mL in PBS supplemented with 5% goat serum. Incubate for 30 min at RT in the dark.
6. Then, wash 1x with PBS and subsequently stain cell nuclei with 250 μL DAPI (4′,6′- diamidino-2-phenylindole dihydrochloride) diluted 1:3,000 in PBS, incubate for 1 min at RT in the dark.
7. Wash 4x with PBS and 1x with H₂O, then mount cover slips upside down onto a drop of Fluoromount on a microscope slide, let it dry in the dark and evaluate sample by indirect fluorescence microscopy.

Fusion by transient transfection of prototype foamy virus (PFV) glycoprotein

5. Preparation of Viral Inoculum

1. Produce a high titer virus stock by transfecting in vitro transcribed HCV RNA into Huh-7.5 cells and harvesting cell culture supernatant after 48 h and 72 h as described previously ¹⁴. Determine viral titer by limiting dilution assay (TCID₅₀) ¹³.

6. Cell Culture, Cell Fusion by Transfection of a Fusogenic PFV Glycoprotein

1. Culture Lunet N, HeLa, and Hep56.1D hCD81 cells according to the instructions in Table 1 in DMEM cplt medium containing appropriate selections.
2. Detach cells as described in 2.1, prepare single cell suspensions in DMEM cplt medium, and co-cultivate Lunet N cells with HeLa or Hep56.1D hCD81 cell lines at appropriate ratios (e.g. 1:2 for Hep56.1D hCD81 or 1:1 for HeLa cells) yielding a total cell density of 1.5×10⁵ cells per 12-well and incubate for 24 h at 37 °C and 5% CO₂.
3. The next day, transiently transfect cells with the highly fusogenic PFV envelope protein (pczHFVenvEM066 ¹⁶) by using Lipofectamine 2000 according to the manufacturer’s instructions.

7. Infection Assay

1. Inoculate heterokaryons 30 h after transfection with 350 μL of virus stock (MOI of ~2.3) for 12 h overnight, wash once with PBS and add 1 mL of DMEM cplt medium per well. Incubate for 48 h at 37 °C and 5% CO₂.
2. Harvest supernatant of heterokaryon culture, filter through a 0.45 μm filter and inoculate Huh-7.5 cells, seeded in 96-well plates (1×10⁴ cells/well) the day before, in a limiting dilution assay ¹³.
3. After 72 h, stain infected Huh-7.5 cells with an HCV-specific antibody (NS5A; 9E10) to quantify production of infectious progeny particles.

8. Visualization of Cell Fusion

1. To visualize fusion events, prepare 5 to 10 μM solutions of CellTracker dyes in DMEM medium without additives and complete the steps below 6 h before co-culture (described in sections 6.2 and 6.3).
2. Wash adherent cells once with 5 mL of PBS and then incubate cells with 4 mL of staining solution per 10 cm dish for 45 min at 37 °C and 5% CO₂ (stain Lunet N cells with CellTracker Orange CMTMR and HeLa or Hep56.1D hCD81 cells with CellTracker Green CMFDA, separately).
3. Wash cells once with 5 mL of PBS for 1 min and add DMEM cplt medium, incubate for 6 h at 37 °C and 5% CO₂.
4. Prepare seeding and transfection of stained cells as described above in sections 6.2 and However, add a cover slip to the cell culture dish before seeding.
5. 30 h post transfection, wash cells carefully with 1 mL of PBS for 1 min and then fix cells with 250 μL of 3% PFA for 15 min at RT. Wash cells once with 1 mL PBS for 1 min, stain with DAPI for 1 min as described in section 4.6, wash once with 1 mL PBS for 1 min, repeat wash with H₂O and mount cover slips on glass slides (see section 4.7). Analyze cells with a fluorescence microscope.

Table 1. Specifications about origin and culture condition of used cell lines Cplt: complete, G418: geneticin, h: human.

9. Representative Results

In this study, we applied two different methods of somatic cell fusion that allow us to specifically investigate the impact of restriction factors on completion of the HCV replication cycle in heterokaryons. Of note, the conditional design of trans-complementation upon fusion of the two different cell types assures that only in true heterokaryons between human liver cells and non-permissive cell lines, virus assembly (approach 1) or virus entry and the full HCV replication cycle (approach 2) are accomplished.

In the first approach Huh-7.5 cells, highly permissive for HCV, were transiently transfected with a subgenomic replicon expressing a luciferase transgene and HCV non-structural proteins supporting subgenomic HCV RNA replication (Huh-7.5 replicon cells). These cells were co-cultured and fused to cell lines expressing HCV structural proteins core, E1, E2, as well as accessory proteins p7, and NS2. Figure1A shows an overview of the experimental procedure. Importantly, following PEG fusion, all proteins required for particle assembly should be present in the formed heterokaryon which can be confirmed by immunofluorescence against individual viral proteins. Figure 1B depicts an exemplary fusion event in which signals for both NS5A expressed in Huh-7.5 replicon cells and E2 expressed in Huh-7.5[CE1][E2p7NS2] packaging cells were detected in the same cytoplasm upon PEG-induced heterokaryon formation. When co-cultured cells were instead treated with PBS, no fusion was induced thus only single-positive cells and no co-localization of signals was observed. Furthermore, to control transgene expression, we performed ELISAs specific for core and E2 (previously reported in detail ¹⁵). In addition to the control packaging cell line Huh 7.5[CE1][E2p7NS2], we generated HeLa and Hep56.1D packaging cells as representatives for human non-liver and mouse liver cells, respectively. HCV RNA replication efficiency in these latter cells is low and HCV assembly and release has not been shown. Figure 1C illustrates the results of the fusion assay. Supernatants were collected from the co-culture at 48 h post fusion induction and used to inoculate naïve Huh-7.5 cells for subsequent luciferase assays. Notably, when Huh-7.5 replicon cells were fused with either naïve cell lines or treated with PBS as control, no infectivity was detected in the culture fluids. However, when cell fusion between Huh-7.5 replicon and packaging cell lines was induced by PEG, trans-complementation between replicon and constitutively expressed structural proteins rescued virus production in the resulting heterokaryons and infectious viral particles were released into the culture fluids. We therefore concluded that virus production required cell fusion and expression of viral proteins in the packaging cell lines. Strikingly, not only the heterokaryons of Huh-7.5 human hepatoma cells, but also heterokaryons with human non-liver (HeLa[CE1][E2p7NS2]) and mouse liver (Hep56.1D[CE1][E2p7NS2]) cells allowed virus release, indicating that assembly and release are not dominantly inhibited by possible restriction factors in these cell types. Expression of structural proteins in packaging cell lines and authentic entry into target cells was shown in Frentzen et al. ¹⁵.

An alternative method of cell fusion was utilized to analyze possible restrictions affecting cell entry, RNA translation and RNA replication stages of the replication cycle and to exclude that a high viral burden in transfected Huh 7.5 replicon cells would preclude detection of restriction factors by saturation. To this end, we developed an independent assay based on inoculation of heterokaryons with HCVcc and detection of de novo virus production in these cells. As outlined in Figure 2A, somatic cell fusion was induced by the expression of a fusogenic glycoprotein after co-cultivation of the cells. This method was chosen because sensitivity was increased likely due to increased fusion efficiency. Heterokaryons were visualized by staining separate cell lines with CellTracker dyes prior to co-cultivation. A heterokaryon with homogenously distributed dyes within the cytoplasm indicates fusion of two cell types as indicated in Figure 2B. Only in heterokaryons of Lunet N cells with HeLa cells or Hep56.1D hCD81 cells are all HCV entry factors expressed thus selectively rendering heterokaryons susceptible to HCV cell entry. Challenge with HCVcc resulted in complete replication of HCV in heterokaryons which is evident from de novo production of infectious viral progeny approximately 10-fold above the background of the assay (Figure 2C). As a control, Lunet N cells were fused with Lunet N cells. Importantly only very low levels of infectious HCV close to the detection limit were observed. Similar results were obtained when Lunet N cells were fused with naïve Hep56.1D cells. In both cases, human CD81 was absent so that HCV could not productively infect the heterokaryons. This very low infectivity detected in these two latter cases is likely attributable to low level of infection of Lunet N cells and/or low levels of residual infectious virus input from the inoculum. In contrast HeLa cells and Hep56.1D hCD81 cells express human CD81, thus complementing the lacking cell entry factor in the heterokaryons and permitting HCV cell entry. The 10-fold increased level of infectious HCV detected in the medium of heterokaryons involving these two cell lines therefore likely reflects de novo production of infectious particles in heterokaryons. Thus, we concluded that completion of the HCV replication cycle is not restricted excluding the expression of dominant HCV restriction factors in these cell lines.

Figure 1. Trans-complementation of HCV assembly and release in heterokaryons. (A) Schematic outline 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 by electroporation. The next day, cells were detached and co-cultured with naïve cells or packaging cells constitutively expressing HCV core, E1, E2, p7 and NS2. 24 h post co-cultivation, heterokaryon formation was induced by treatment with 40% PEG using PBS as negative control. After 48 h, cell-free supernatant was harvested to inoculate naïve Huh-7.5 cells. Infectivity was determined by luciferase activity. (B) For detection of heterokaryons, cells were immunostained using monoclonal antibodies against E2 and NS5A. Simultaneous expression of both proteins within cells is indicative of cell fusion. (C) Luciferase measurements were performed to quantify viral infectivity produced from heterokaryons. Mean values of three independent experiments and the standard deviations of the means are given. The horizontal bar represents the background RLU determined in uninfected Huh-7.5 cells. Click here to view larger figure.

Figure 2. Completion of HCV replication cycle after inoculation of heterokaryons. (A) Schematic overview of the experimental procedure. Huh-7 Lunet N cells lacking CD81 were co-cultured with the indicated cell lines lacking or expressing human CD81. Importantly, these latter cells lack at least one HCV entry factor and therefore cannot be productively infected. The next day, fusion was initiated by transfection of a fusogenic viral envelope protein from PFV. Thirty hours later, cells were challenged with infectious HCV particles. To measure cell entry, RNA replication and virus production sustained by these heterokaryons, released infectivity in the cell-free culture fluids was determined 48 h post inoculation. To this end, naïve Huh-7.5 cells were used as target cells in a limiting dilution assay (TCID₅₀). (B) For detection of heterokaryons, cells were stained with CellTracker Green or CellTracker Orange 6 h prior to co-cultivation and transfection. Thirty hours later, cells were fixed and stained with DAPI. (C) Lunet N cells were fused to HeLa cells, naïve Hep56.1D or Hep56.1D cells expressing human CD81. These cultures were inoculated with HCVcc (MOI of 2.3). Culture fluids were collected 48 h later and de novo particle release from heterokaryons was determined by TCID₅₀ using naïve Huh-7.5 target cells. Mean values of three independent experiments are given. The horizontal bar represents the detection limit of the assay. Click here to view larger figure.