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

Vi er taknemmelige for Takaji Wakita og Jens Bukh til JFH1 og J6CF isolater, hhv. Derudover har vi takker Charles Ris til Huh-7.5-celler og 9E10 antistof, Steven Foung for E2-specifikt antistof CBH-23, og alle medlemmer af Institut for eksperimentel virologi, Twincore for nyttige forslag og diskussioner.

Cellefusion af PEG 1. Cellekultur <ol><li> Kultur Huh-7.5, HeLa og Hep56.1D naive eller pakkecellelinier 12 på 15 cm celle dyrkningsskåle i DMEM komplet (DMEM cplt) medium, DMEM suppleret med 2 mM L-glutamin, 1x ikke-essentielle aminosyrer, 100 U / ml penicillin, 100 ug / ml streptomycin og 10% føtalt kalveserum. Anvende passende udvælgelse af stabile cellelinier som angivet i tabel 1. </li></ol> 2. Transfektion af HCV-RNA <ol><li> Vas...

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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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Vi præsenterer to metoder til at inducere heterokaryon dannelse i dyrkede celler til analyse af dominante negative begrænsninger, som udelukker HCV-replikation. Under anvendelse af disse fremgangsmåder har vi udelukket nærvær af en dominant konstitutivt udtrykt eller virus-induceret faktor i forskellige humane ikke-leveren og i murine leveren cellelinier. Den første analyse primært analyserer, hvis restriktioner faktorer forebygge HCV samling og frigivelse af infektiøst afkom. Da der i dette tilfælde pakkecelle...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> DMEM </td><td> Invitrogen, Karlsruhe, Tyskland </td><td> 41965-039 </td></tr><tr><td> L-glutamin </td><td> Invitrogen, Karlsruhe, Tyskland </td><td> 25030-024 </td></tr><tr><td> Ikke-essentielle aminosyrer </td><td> Invitrogen, Karlsruhe, Tyskland </td><td> 11140-035 </td></tr><tr><td> Penicillin / streptomycin </td><td> Invitrogen, Karlsruhe, Tyskland </td><td> 15140-122 </td></tr><tr><td> Føtalt kalveserum </td><td> PAA, Cölbe, Tyskland </td><td> A15151 </td></tr><tr><td> α-E2 (CBH23) </td><td> venligst stillet til rådighed af Steven Foung 10 </td><td></td></tr><tr><td> ATP </td><td> Sigma, Steinheim, Tyskland </td><td> A2833-106 </td></tr><tr><td> Glutathion </td><td> Sigma, Steinheim, Tyskland </td><td&gt; 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, Tyskland </td><td> 11811-064 </td></tr><tr><td> Polyethylen-glycol-1500 </td><td> Roche, Mannheim, Tyskland </td><td> 10783641001 </td></tr><tr><td> Paraformaldehyd </td><td> Roth, Karlsruhe, Tyskland </td><td> 0.335,3 </td></tr><tr><td> Triton X-100 </td><td> Roth, Karlsruhe, Tyskland </td><td> 3051,2 </td></tr><tr><td> Gedeserum </td><td> Sigma, Steinheim, Tyskland </td><td> G9023-5 ml </td></tr><tr><td> α-NS5A (9E10) </td><td> Venligst stillet til rådighed af Charles Rice 7 </td><td></td></tr><tr><td> DAPI (4 &#39;, 6&#39;-diamidino-2-phenylindol-dihydrochlorid) </td><td> Invitrogen </td><td> D1306 </td></tr><tr><td> Alexa-Fluor 546 - gede-anti-humant IgG </td><td> Invitrogen, Karlsruhe, Tyskland </td><td> A21089 </td&gt; </tr><tr><td> Alexa-Fluor 488 - gede-anti-muse-IgG </td><td> Invitrogen, Karlsruhe, Tyskland </td><td> A10680 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen, Karlsruhe, Tyskland </td><td> 11668-019 </td></tr><tr><td> CellTracker CMTMR </td><td> Invitrogen, Karlsruhe, Tyskland </td><td> C2927 </td></tr><tr><td> CellTracker CMFDA </td><td> Invitrogen, Karlsruhe, Tyskland </td><td> C2925 </td></tr><tr><td> Fluoromount </td><td> Sigma, Steinheim, Tyskland </td><td> F4680-25ML </td></tr><tr><td> Alle andre kemikalier </td><td> Roth, Karlsruhe, Tyskland </td><td></td></tr><tr><td> Cellekultur materialer </td><td> Sarstedt, Nümbrecht, Tyskland </td><td></td></tr></tbody></table>

two methods of heterokaryon formation to discover hcv restriction factors

Hepatitis C virus (HCV) er en hepatotropisk virus med en værtsområdegenet begrænset til mennesker og chimpanser. Selv HCV-RNA-replikation er blevet observeret i humane ikke-hepatiske og murin cellelinier effektivitet var meget lav, og krævede lange udvælgelsesprocedurer hjælp HCV replicon konstruktioner som udtrykker dominante antibiotikum-selekterbare markører 1-5. HCV in vitro-forskning er derfor begrænset til humane hepatomaceller cellelinier permissive for virus ind og afslutning af den virale livscyklus. Grund HCV&#39;er smalle art tropisme, er der ingen immunokompetente lille dyremodel, som holder hele HCV replikationscyklus 6-8. Ineffektiv replikation af HCV i ikke-humane celler fx af museoprindelse sandsynligvis på grund af manglende genetisk uforenelighed væsentlige vært afhængighed faktorer og / eller ekspression af restriktionssteder faktorer. Vi undersøgte, om HCV formering er undertrykt af dominerende begrænsning FActors i enten humane cellelinjer afledt af ikke-hepatiske væv eller i muselever cellelinier. Til dette formål har vi udviklet to uafhængige betingede trans-komplementering metoder der er afhængige af somatisk celle fusion. I begge tilfælde er færdiggørelse af den virale replikationscyklus kun mulig i heterokaryoner. Følgelig vellykket trans-komplementation, som bestemmes ved at måle de novo produktion af infektiøst virusafkom, indikerer fravær af dominerende begrænsning. Specifikt blev subgenomiske HCV replikoner der bærer et luciferase transgen transficeret ind meget permissive humane hepatomaceller (Huh-7.5-celler). Efterfølgende blev disse celler co-dyrkes og fusioneret til forskellige humane og murine celler, som udtrykker HCV-strukturelle proteiner kerne, envelope 1 og 2 (E1, E2) og accessoriske proteiner P7 og NS2. Forudsat, at cellefusion blev initieret ved behandling med polyethylenglycol (PEG), kulturen frigives infektiøs virussygdom partikel som inficerede naive celler i en receptor-afhængig måde. At vurdere indflydelsen af dominerende begrænsning hele virale livscyklus, herunder celleindtrængen, RNA translation, replikation og viruskonstruktion, vi benyttede en human lever-cellelinie (Huh-7 Lunet N-celler 9), der mangler endogen ekspression af CD81, en væsentlig indgang faktor HCV. I fravær af ektopisk udtrykt CD81, er disse celler i det væsentlige resistente over for HCV-infektion 10. Vigtigt, når de co-dyrkes og fusioneres med celler, der udtrykker human CD81, men manglen på mindst et andet afgørende celleindtastning faktor (dvs. SR-BI, CLDN1, OCLN), kun de resulterende heterokaryoner vist det komplette sæt af HCV indrejse faktorer nødvendige for infektion. Derfor, for at analysere, om dominerende begrænsning faktorer undertrykke afslutningen af ​​HCV-replikation cyklus vi fusioneret Lunet N-celler med forskellige celler fra human-og muse oprindelse, som opfylder ovennævnte critEria. Når co-dyrkede celler blev transficeret med en meget fusogent viralt kappeprotein mutant af prototypen foamy-virus (pFV 11) og derefter udfordret med infektiøse HCV-partikler (HCVcc), blev de novo produktion af infektiøst virus observeret. Dette indikerer, at HCV fuldført sin replikationscyklus i heterokaryoner således udelukke ekspression af dominerende restriktionssteder faktorer i disse cellelinier. Disse hidtil ukendte betingede trans-komplementering fremgangsmåder vil være anvendelige til screening af et stort panel af cellelinier og primære celler til ekspression af HCV-specifikke dominerende restriktionssteder faktorer.

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

Vi beskriver to metoder til betinget<em&gt; Trans</em&gt;-Komplementering af hepatitis C virus (HCV), samling og færdiggørelsen af ​​den fulde virale livscyklus, der er afhængige af heterokaryon dannelse. Disse teknikker er egnede til at screene for cellelinier, der udtrykker dominerende begrænsning faktorer, der regulerer produktionen af ​​infektiøst HCV afkom.

To Metoder til heterokaryon Dannelse til Discover HCV Restriction Faktorer

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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