We are grateful to Takaji Wakita and Jens Bukh for JFH1 and J6CF isolates, respectively. Furthermore we thank Charles Rice for Huh-7.5 cells and the 9E10 antibody, Steven Foung for the E2-specific antibody CBH-23, and all members of the Department of Experimental Virology, Twincore for helpful suggestions and discussions.

Cell Fusion by PEG
1. Cell Culture
<ol>
	<li>Culture Huh-7.5, HeLa and Hep56.1D na&iuml;ve 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 &mu;g/mL streptomycin, and 10% fetal calf serum. Apply appropriate selection to stable cell lines as indicated in Table 1.</li>
</ol>
2. Transfection of HCV RNA
<ol>
	<li>Wash Huh-7.5 cells (one confluent 15 cm dish) twice with Phosphate Buffered Saline (PBS), trypsinize, and resuspend at a concentration of 1.5x107 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.</li>
	<li>Transfect via electroporation 400 &mu;L of the cell suspension (6x106 cells) with 10 &mu;g 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 &mu;F and 270 V as described previously 14.</li>
	<li>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 &deg;C and 5% CO2.</li>
</ol>
3. Induction of Fusion by PEG
<ol>
	<li>Harvest cells as described in 2.1, resuspend in DMEM cplt medium and dilute to 5x105 cells/mL.</li>
	<li>Generate single-cell suspensions of Huh-7.5, HeLa, and Hep56.1D packaging cells, and dilute to 5x105 cells/mL, 2x105 cells/mL, and 2x105 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.</li>
	<li>Prepare 6-well culture plates with 2-3 sterile glass cover slips per well for further analysis of fusion efficiency.</li>
	<li>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 &deg;C and 5% CO2.</li>
	<li>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.</li>
	<li>Aspirate medium from co-cultured cells and wash once with 1 mL PBS, then carefully add 500 &mu;L of pre-warmed 40% PEG-1500 or PBS as control and incubate for 5 min at 37 &deg;C to induce heterokaryon formation.</li>
	<li>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 &deg;C and 5% CO2.</li>
	<li>Forty-eight hours post fusion, collect each supernatant and filter through a 0.45 &mu;m filter.</li>
	<li>Measure production of infectious particles in fused heterokaryons by inoculation of na&iuml;ve Huh-7.5 cells (8x104 cells/well seeded in a 12-well plate, 24 h prior to inoculation) with 500 &mu;L of cell-free culture fluid and subsequent determination of luciferase activity 14 .</li>
</ol>
4. Immunofluorescence to Determine Fusion Efficiency
<ol>
	<li>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.</li>
	<li>Fix cells with 600 &mu;L 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.</li>
	<li>Permeabilize cells with 500 &mu;L of 0.5% Triton X-100 in PBS for 5 min and subsequently wash 3x with PBS.</li>
	<li>Prepare primary antibody solution in PBS supplemented with 5% goat serum. Use anti-NS5A antibody (9E10) at a concentration of 0.5 &mu;g/mL and human monoclonal anti-E2 antibody (CBH-23) at a concentration of 6.4 ng/mL. Apply 250 &mu;L to each well and incubate at RT for 45 min.</li>
	<li>Wash 3x with PBS and detect bound primary antibodies with 250 &mu;L 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 &mu;g/mL in PBS supplemented with 5% goat serum. Incubate for 30 min at RT in the dark.</li>
	<li>Then, wash 1x with PBS and subsequently stain cell nuclei with 250 &mu;L DAPI (4',6'- diamidino-2-phenylindole dihydrochloride) diluted 1:3,000 in PBS, incubate for 1 min at RT in the dark.</li>
	<li>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.</li>
</ol>
Fusion by transient transfection of prototype foamy virus (PFV) glycoprotein
5. Preparation of Viral Inoculum
<ol>
	<li>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.</li>
</ol>
6. Cell Culture, Cell Fusion by Transfection of a Fusogenic PFV Glycoprotein
<ol>
	<li>Culture Lunet N, HeLa, and Hep56.1D hCD81 cells according to the instructions in Table 1 in DMEM cplt medium containing appropriate selections.</li>
	<li>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 1.5x105 cells per 12-well and incubate for 24 h at 37 &deg;C and 5% CO2.</li>
	<li>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.</li>
</ol>
7. Infection Assay
<ol>
	<li>Inoculate heterokaryons 30 h after transfection with 350 &mu;L 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 &deg;C and 5% CO2.</li>
	<li>Harvest supernatant of heterokaryon culture, filter through a 0.45 &mu;m filter and inoculate Huh-7.5 cells, seeded in 96-well plates (1x104 cells/well) the day before, in a limiting dilution assay 13.</li>
	<li>After 72 h, stain infected Huh-7.5 cells with an HCV-specific antibody (NS5A; 9E10) to quantify production of infectious progeny particles.</li>
</ol>
8. Visualization of Cell Fusion
<ol>
	<li>To visualize fusion events, prepare 5 to 10 &mu;M 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).</li>
	<li>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 &deg;C and 5% CO2 (stain Lunet N cells with CellTracker Orange CMTMR and HeLa or Hep56.1D hCD81 cells with CellTracker Green CMFDA, separately).</li>
	<li>Wash cells once with 5 mL of PBS for 1 min and add DMEM cplt medium, incubate for 6 h at 37 &deg;C and 5% CO2.</li>
	<li>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.</li>
	<li>30 h post transfection, wash cells carefully with 1 mL of PBS for 1 min and then fix cells with 250 &mu;L 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.</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Species</td>
			<td>Cell line</td>
			<td>Origin</td>
			<td>Growth medium &amp; selection</td>
		</tr>
		<tr>
			<td>Human</td>
			<td>Huh-7.5</td>
			<td>Subclone of Huh-7 hepatoma cell line 17</td>
			<td>DMEM cplt</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Huh-7.5 [CE1][E2p7NS2]</td>
			<td>Stably expressing viral proteins core, E1, E2, p7, and NS2, transduced by lentiviral gene transfer of two independent gene cassettes</td>
			<td>DMEM cplt + Blasticidin 5 &mu;g/mL</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Huh-7 Lunet N</td>
			<td>Subclone of Huh-7 hepatoma cell line 9</td>
			<td>DMEM cplt</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>HeLa</td>
			<td>Cervical adenocarcinoma cell line (ATCC number: CCL-2)</td>
			<td>DMEM cplt</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>HeLa [CE1][E2p7NS2]</td>
			<td>Stably expressing viral proteins core, E1, E2, p7, and NS2, transduced by lentiviral gene transfer of two independent gene cassettes</td>
			<td>DMEM cplt + G418 750 &mu;g/mL; Blasticidin 5 &mu;g/mL</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>Hep56.1D</td>
			<td>Primary hepatocellular carcinoma (kind gift of J. Encke) 
			Adult C57BL/6J mice</td>
			<td>DMEM cplt</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Hep56.1D [CE1][E2p7NS2]</td>
			<td>Stably expressing viral proteins core, E1, E2, p7, and NS2, transduced by lentiviral gene transfer of two independent gene cassettes</td>
			<td>DMEM cplt + G418 750 &mu;g/mL; Blasticidin 5 &mu;g/mL</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Hep56.1D hCD81</td>
			<td>Stably expressing human CD81 transduced by lentiviral gene transfer</td>
			<td>DMEM cplt + Blasticidin 5 &mu;g/mL</td>
		</tr>
	</tbody>
</table>
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 na&iuml;ve Huh-7.5 cells for subsequent luciferase assays. Notably, when Huh-7.5 replicon cells were fused with either na&iuml;ve 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 na&iuml;ve 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.
<img alt="Figure 1" src="/files/ftp_upload/4029/4029fig1.jpg" /> 
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 na&iuml;ve Huh-7.5 cells by electroporation. The next day, cells were detached and co-cultured with na&iuml;ve 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 na&iuml;ve 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. <a href="https://www.jove.com/files/ftp_upload/4029/4029fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/4029/4029fig2.jpg" /> 
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, na&iuml;ve 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, na&iuml;ve 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 na&iuml;ve Huh-7.5 target cells. Mean values of three independent experiments are given. The horizontal bar represents the detection limit of the assay. <a href="https://www.jove.com/files/ftp_upload/4029/4029fig2large.jpg" target="_blank"> Click here to view larger figure</a>.

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TP received consulting fees from Biotest.

We present two methods to induce heterokaryon formation in cultured cells for the analysis of dominant negative restrictions that preclude HCV replication. Using these procedures we excluded the presence of a dominant constitutively expressed or virus-induced factor in various human non-liver and in murine liver cell lines. The first assay primarily analyzes if restriction factors prevent HCV assembly and release of infectious progeny. Since in this case the packaging cells are fused to HCV replicon cells, possible restr...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>41965-039</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>25030-024</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11140-035</td>
		</tr>
		<tr>
			<td>Penicillin/ streptomycin</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>PAA, C&ouml;lbe, Germany</td>
			<td>A15151</td>
		</tr>
		<tr>
			<td>&alpha;-E2 (CBH23)</td>
			<td>kindly provided by Steven Foung 10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>A2833-106</td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G4251-1G</td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Invivo Gen, San Diego, USA</td>
			<td>Ant-bl-1</td>
		</tr>
		<tr>
			<td>G418 (geneticin)</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11811-064</td>
		</tr>
		<tr>
			<td>Polyethylene-glycol-1500</td>
			<td>Roche, Mannheim, Germany</td>
			<td>10783641001</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Roth, Karlsruhe, Germany</td>
			<td>0335.3</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Roth, Karlsruhe, Germany</td>
			<td>3051.2</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G9023-5mL</td>
		</tr>
		<tr>
			<td>&alpha;-NS5A (9E10)</td>
			<td>Kindly provided by Charles Rice 7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI (4',6'- diamidino-2-phenylindole dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
		</tr>
		<tr>
			<td>Alexa-Fluor 546 - goat anti-human IgG</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>A21089</td>
		</tr>
		<tr>
			<td>Alexa-Fluor 488 - goat anti-mouse IgG</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>A10680</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11668-019</td>
		</tr>
		<tr>
			<td>CellTracker CMTMR</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>C2927</td>
		</tr>
		<tr>
			<td>CellTracker CMFDA</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>C2925</td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>F4680-25ML</td>
		</tr>
		<tr>
			<td>All other chemicals</td>
			<td>Roth, Karlsruhe, Germany</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture materials</td>
			<td>Sarstedt, N&uuml;mbrecht, Germany</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

two methods of heterokaryon formation to discover hcv restriction factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

Watch this Scientific Journal Video about Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors at JoVE.com

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

two-methods-heterokaryon-formation-to-discover-hcv-restriction

Research

JoVE Journal

Immunology and Infection

11.5K Views.  Twincore, Centre for Experimental and Clinical Infection Research. We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Video: Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...