Name: two methods of heterokaryon formation to discover hcv restriction factors
Uploaded: 2012-07-16T12:00:00
Description: Watch this Scientific Journal Video about Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors at JoVE.com

Мы благодарны Такадзи Wakita и Йенс Бух для JFH1 и J6CF штаммов, соответственно. Кроме того, мы благодарим Чарльз Райс Ха-7.5 клеток и антител, 9E10, Стивен Foung для E2-специфических антител CBH-23, и все члены кафедры экспериментальной вирусологии Twincore за полезные советы и обсуждения.

Слияние клеток с помощью ПЭГ 1. Культуре клеток <ol><li> Культура Хух-7.5, HeLa и Hep56.1D наивным или упаковку клеточных линиях 12 на 15 см культуре клеток блюда в полной DMEM (DMEM cplt) среды DMEM с добавлением 2 мМ L-глутамина, 1 без незаменимых аминокислот, 100 U / мл пенициллина, 100 мкг / мл с...

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	<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+permissive+cell+lines+for+subgenomic+and+genomic+hepatitis+C+virus+RNA+replication.">Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.</a> J. Virol. 76, 13001-13014 (2002).</li></ol>

Мы представляем два способа заставить гетерокариона образование в культуре клеток для анализа доминирующих отрицательных ограничений, которые препятствуют репликации ВГС. Использование этих процедур мы исключили наличие доминирующего конститутивно выраженной или вирус-индуциров?...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td></tr><tr><td> DMEM </td><td> Invitrogen, Карлсруэ, Германия </td><td> 41965-039 </td></tr><tr><td> L-глютамин </td><td> Invitrogen, Карлсруэ, Германия </td><td> 25030-024 </td></tr><tr><td> Номера для незаменимых аминокислот </td><td> Invitrogen, Карлсруэ, Германия </td><td> 11140-035 </td></tr><tr><td> Пенициллина / стрептомицина </td><td> Invitrogen, Карлсруэ, Германия </td><td> 15140-122 </td></tr><tr><td> Эмбриональной телячьей сыворотки </td><td> ПАК, Cölbe, Германия </td><td> A15151 </td></tr><tr><td> α-E2 (CBH23) </td><td> любезно предоставленные Стивен Foung 10 </td><td></td></tr><tr><td> ATP </td><td> Sigma, Штайнхайм, Германия </td><td> A2833-106 </td></tr><tr><td> Глутатион </td><td> Sigma, Штайнхайм, Германия </td><td&gt; G4251-1G </td></tr><tr><td> Blasticidin </td><td> Invivo Gen, Сан-Диего, США </td><td> Ant-BL-1 </td></tr><tr><td> G418 (генетицину) </td><td> Invitrogen, Карлсруэ, Германия </td><td> 11811-064 </td></tr><tr><td> Полиэтилен гликоль-1500 </td><td> Roche, Мангейм, Германия </td><td> 10783641001 </td></tr><tr><td> Параформальдегид </td><td> Рот, Карлсруэ, Германия </td><td> 0335,3 </td></tr><tr><td> Тритон Х-100 </td><td> Рот, Карлсруэ, Германия </td><td> 3051,2 </td></tr><tr><td> Коза сыворотке </td><td> Sigma, Штайнхайм, Германия </td><td> G9023-5 мл </td></tr><tr><td> α-NS5A (9E10) </td><td> Пожалуйста, при условии Чарльз Райс 7 </td><td></td></tr><tr><td> DAPI (4 &#39;, 6&#39;-диамидино-2-фенилиндола дигидрохлорид) </td><td> Invitrogen </td><td> D1306 </td></tr><tr><td> Alexa Fluor-546 - козий анти-человеческий IgG </td><td> Invitrogen, Карлсруэ, Германия </td><td> A21089 </tг&gt; </tr><tr><td> Alexa Fluor-488 - антимышиного IgG </td><td> Invitrogen, Карлсруэ, Германия </td><td> A10680 </td></tr><tr><td> Липофектамина 2000 </td><td> Invitrogen, Карлсруэ, Германия </td><td> 11668-019 </td></tr><tr><td> CellTracker CMTMR </td><td> Invitrogen, Карлсруэ, Германия </td><td> C2927 </td></tr><tr><td> CellTracker CMFDA </td><td> Invitrogen, Карлсруэ, Германия </td><td> C2925 </td></tr><tr><td> Fluoromount </td><td> Sigma, Штайнхайм, Германия </td><td> F4680-25МЛ </td></tr><tr><td> Все остальные химические вещества </td><td> Рот, Карлсруэ, Германия </td><td></td></tr><tr><td> Материалы клеточных культур </td><td> Sarstedt, Nümbrecht, Германия </td><td></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

Мы описываем два метода условных<em&gt; Транс-</em&gt;-Дополнения вируса гепатита С (HCV) собраний и завершения полного жизненного цикла вируса, которые опираются на гетерокариона образования. Эти методы предназначены для выявления клеточных линий, которые выражают доминирующие факторы ограничения, которые исключают производство инфекционного гепатита потомства.

Два способа формирования гетерокариона Обнаружение вируса гепатита факторы ограничения

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 ...

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