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>...

<ol>
	<li>Zhu, Q., Guo, J. T., Seeger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+hepatitis+C+virus+subgenomes+in+nonhepatic+epithelial+and+mouse+hepatoma+cells.">Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells.</a> J. Virol. 77, 9204-9210 (2003).</li><li>Kato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonhepatic+cell+lines+HeLa+and+293+support+efficient+replication+of+the+hepatitis+C+virus+genotype+2a+subgenomic+replicon.">Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon.</a> J. Virol. 79, 592-596 (2005).</li><li>Ali, S., Pellerin, C., Lamarre, D., Kukolj, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+subgenomic+replicons+in+the+human+embryonic+kidney+293+cell+line.">Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line.</a> J. Virol. 78, 491-501 (2004).</li><li>Date, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+2a+hepatitis+C+virus+subgenomic+replicon+can+replicate+in+HepG2+and+IMY-N9+cells.">Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells.</a> J. Biol. Chem. 279, 22371-22376 (2004).</li><li>Chang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+hepatitis+C+virus+(HCV)+RNA+in+mouse+embryonic+fibroblasts:+protein+kinase+R+(PKR)-dependent+and+PKR-independent+mechanisms+for+controlling+HCV+RNA+replication+and+mediating+interferon+activities.">Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities.</a> J. Virol. 80, 7364-7374 (2006).</li><li>Zhong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+hepatitis+C+virus+infection+in+vitro.">Robust hepatitis C virus infection in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 9294-9299 (2005).</li><li>Lindenbach, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+replication+of+hepatitis+C+virus+in+cell+culture.">Complete replication of hepatitis C virus in cell culture.</a> Science. 309, 623-626 (2005).</li><li>Wakita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+infectious+hepatitis+C+virus+in+tissue+culture+from+a+cloned+viral+genome.">Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.</a> Nat. Med. 11, 791-796 (2005).</li><li>Witteveldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD81+is+dispensable+for+hepatitis+C+virus+cell-to-cell+transmission+in+hepatoma+cells.">CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells.</a> J. Gen. Virol. 90, 48-58 (2009).</li><li>Bitzegeio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+hepatitis+C+virus+to+mouse+CD81+permits+infection+of+mouse+cells+in+the+absence+of+human+entry+factors.">Adaptation of hepatitis C virus to mouse CD81 permits infection of mouse cells in the absence of human entry factors.</a> PLoS Pathog. 6, e1000978 (2010).</li><li>Lindemann, D., Goepfert, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+foamy+virus+envelope+glycoproteins.">The foamy virus envelope glycoproteins.</a> Curr Top Microbiol Immunol. 277, 111-129 (2003).</li><li>Brohm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+determinants+important+for+hepatitis+C+virus+p7+function+in+morphogenesis+by+using+trans-complementation.">Characterization of determinants important for hepatitis C virus p7 function in morphogenesis by using trans-complementation.</a> J. Virol. 83, 11682-11693 (2009).</li><li>Steinmann, E., Brohm, C., Kallis, S., Bartenschlager, R., Pietschmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+trans-encapsidation+of+hepatitis+C+virus+RNAs+into+infectious+virus-like+particles.">Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles.</a> J. Virol. 82, 7034-7046 (2008).</li><li>Koutsoudakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+early+steps+of+hepatitis+C+virus+infection+by+using+luciferase+reporter+viruses.">Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses.</a> J. Virol. 80, 5308-5320 (2006).</li><li>Frentzen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Completion+of+hepatitis+C+virus+replication+cycle+in+heterokaryons+excludes+dominant+restrictions+in+human+non-liver+and+mouse+liver+cell+lines.">Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.</a> PLoS Pathog. 7, e1002029 (2011).</li><li>Lindemann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+particle-associated+glycoprotein+signal+peptide+essential+for+virus+maturation+and+infectivity.">A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity.</a> J. Virol. 75, 5762-5771 (2001).</li><li>Blight, K. J., McKeating, J. A., Rice, C. 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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 vues.  Twincore, Centre for Experimental and Clinical Infection Research. Nous décrivons deux méthodes pour conditionnelle Trans-Complémentation de l'hépatite C (VHC) de montage et la réalisation du cycle de vie virale complète, qui reposent sur la formation des hétérocaryons. Ces techniques sont adaptées à l'écran pour les lignées cellulaires qui expriment des facteurs de restriction dominantes, qui empêchent la production de la descendance du VHC infectieux.