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

हम Takaji Wakita और JFH1 और J6CF आइसोलेट्स के लिए जेन्स Bukh, क्रमशः के लिए आभारी हैं. इसके अलावा हम चार्ल्स चावल हं 7.5 कोशिकाओं के लिए और 9E10 प्रतिरक्षी, E2 विशिष्ट एंटीबॉडी CBH-23 के लिए स्टीवन Foung, और उपयोगी सुझाव और विचार विमर्श के लिए Twincore प्रायोगिक विषाणु विज्ञान विभाग के सभी सदस्यों का धन्यवाद.

खूंटी द्वारा सेल फ्यूजन 1. सेल संस्कृति <ol><li> हं 7.5 HELA और पैकेजिंग भोले या सेल लाइनों 15 सेमी DMEM पूरा (DMEM cplt) मध्यम में सेल संस्कृति बर्तन पर 12 Hep56.1D संस्कृति, DMEM की 2 मिमी एल glutamine, है 1x गैर आवश्यक अमीनो एसि?...

<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. 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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. 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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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हम दो तरीकों प्रमुख नकारात्मक प्रतिबंध है कि HCV प्रतिकृति छीन के विश्लेषण के लिए संवर्धित कोशिकाओं heterokaryon गठन प्रेरित उपस्थित थे. इन प्रक्रियाओं का प्रयोग हम विभिन्न मानव गैर - जिगर और murine जिगर सेल लाइनों ...

<table border="1"><tbody><tr><td> अभिकर्मक का नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> DMEM </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> 41965-039 </td></tr><tr><td> एल-glutamine </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> 25030-024 </td></tr><tr><td> गैर आवश्यक अमीनो एसिड </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> 11140-035 </td></tr><tr><td> पेनिसिलिन स्ट्रेप्टोमाइसिन / </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> 15140-122 </td></tr><tr><td> भ्रूण बछड़ा सीरम </td><td> पा, Cölbe जर्मनी </td><td> A15151 </td></tr><tr><td> α E2 (CBH23) </td><td> कृपया स्टीवन Foung 10 के द्वारा प्रदान की </td><td></td></tr><tr><td> एटीपी </td><td> सिग्मा, Steinheim, जर्मनी </td><td> A2833-106 </td></tr><tr><td> Glutathione </td><td> सिग्मा, Steinheim, जर्मनी </td><td&gt; G4251-1G </td></tr><tr><td> Blasticidin </td><td> InVivo जनरल, सैन डिएगो, संयुक्त राज्य अमरीका </td><td> चींटी - बीएल-1 </td></tr><tr><td> G418 (geneticin) </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> 11811-064 </td></tr><tr><td> Polyethylene - glycol-1500 </td><td> Roche, मैनहेम, जर्मनी </td><td> 10783641001 </td></tr><tr><td> Paraformaldehyde </td><td> Roth, कार्लज़ूए, जर्मनी </td><td> 0,335.3 </td></tr><tr><td> एक्स-100 ट्राइटन </td><td> Roth, कार्लज़ूए, जर्मनी </td><td> 3051.2 </td></tr><tr><td> बकरी सीरम </td><td> सिग्मा, Steinheim, जर्मनी </td><td> G9023 - 5ml </td></tr><tr><td> α NS5A (9E10) </td><td> कृपया चार्ल्स 7 राइस द्वारा प्रदान की </td><td></td></tr><tr><td> DAPI (4, 6&#39;-diamidino-2-phenylindole dihydrochloride) </td><td> Invitrogen </td><td> D1306 </td></tr><tr><td> 546 एलेक्सा Fluor - बकरी आईजीजी विरोधी मानव </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> A21089 </tघ&gt; </tr><tr><td> एलेक्सा Fluor 488 - बकरी विरोधी माउस आईजीजी </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> A10680 </td></tr><tr><td> 2000 lipofectamine </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> 11668-019 </td></tr><tr><td> CellTracker CMTMR </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> C2927 </td></tr><tr><td> CellTracker CMFDA </td><td> Invitrogen, कार्लज़ूए, जर्मनी </td><td> C2925 </td></tr><tr><td> Fluoromount </td><td> सिग्मा, Steinheim, जर्मनी </td><td> F4680-25ml </td></tr><tr><td> अन्य सभी रसायन </td><td> Roth, कार्लज़ूए, जर्मनी </td><td></td></tr><tr><td> सेल संस्कृति सामग्री </td><td> Sarstedt, Nümbrecht, जर्मनी </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

हम सशर्त के लिए दो विधियों का वर्णन<em&gt; ट्रांस</emहेपेटाइटिस सी वायरस (HCV) विधानसभा और पूर्ण वायरल जीवन चक्र है, जो heterokaryon गठन पर निर्भर के पूरा होने के complementation. इन तकनीकों में सेल लाइनों है कि प्रमुख प्रतिबंध कारक है, जो संक्रामक HCV के संतान का उत्पादन रोकना व्यक्त के लिए स्क्रीन के लिए उपयुक्त हैं.

HCV के प्रतिबंध कारक डिस्कवर Heterokaryon गठन के दो तरीके

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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