Name: development of a hepatitis b virus reporter system to monitor the early stages of the replication cycle
Uploaded: 2017-02-01T13:00:00
Description: Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

This work was supported in part by the Research Program on Hepatitis from Japan Agency for Medical Research and Development (AMED) and by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

1. Productie van recombinant HBV coderen de reporterproteïne <ol><li> Bereiding van HepG2-cellen <ol><li> Bereid celkweekmedium (Dulbecco&#39;s gemodificeerd Eagle&#39;s medium (DMEM) aangevuld met 10% foetaal runderserum (FBS), 100 U / ml penicilline, 100 ug / ml streptomycine en 100 U / ml niet-essentiële aminozuren). </li><li> Plaat 4 x 10 6 HepG2 cellen in een 10-cm-collageen gecoate schaal in 10 ml kweekmedium de dag voor transfectie. Incubeer HepG2 cellen bij 37 ° C in een bevochti...

<ol>
	<li>Fattovich, G., Stroffolini, T., Zagni, I., Donato, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma+in+cirrhosis:+incidence+and+risk+factors.">Hepatocellular carcinoma in cirrhosis: incidence and risk factors.</a> Gastroenterology. 127 (5), 35-50 (2004).</li><li>Revill, P., Testoni, B., Locarnini, S., Zoulim, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+strategies+are+required+to+cure+and+eliminate+HBV+infection.">Global strategies are required to cure and eliminate HBV infection.</a> Nat. Rev. Gastroenterol. Hepatol. 13 (4), 239-248 (2016).</li><li>Wursthorn, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peginterferon+alpha-2b+plus+adefovir+induce+strong+cccDNA+decline+and+HBsAg+reduction+in+patients+with+chronic+hepatitis+B.">Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.</a> Hepatology. 44 (3), 675-684 (2006).</li><li>Stuyver, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nomenclature+for+antiviral-resistant+human+hepatitis+B+virus+mutations+in+the+polymerase+region.">Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region.</a> Hepatology. 33 (3), 751-757 (2001).</li><li>Ochiya, T., Tsurimoto, T., Ueda, K., Okubo, K., Shiozawa, M., Matsubara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+system+for+infection+with+hepatitis+B+virus+that+uses+primary+human+fetal+hepatocytes.">An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes.</a> Proc. Natl. Acad. Sci. USA. 86 (6), 1875-1879 (1989).</li><li>Galle, P. R., Hagelstein, J., Kommerell, B., Volkmann, M., Schranz, P., Zentgraf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+experimental+infection+of+primary+human+hepatocytes+with+hepatitis+B+virus.">In vitro experimental infection of primary human hepatocytes with hepatitis B virus.</a> Gastroenterology. 106 (3), 664-673 (1994).</li><li>Gripon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+a+human+hepatoma+cell+line+by+hepatitis+B+virus.">Infection of a human hepatoma cell line by hepatitis B virus.</a> Proc. Natl. Acad. Sci. USA. 99 (24), 15655-15660 (2002).</li><li>Murakami, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+novel+direct+acting+antiviral+agents+for+HBV+using+in+silico+screening.">Discovering novel direct acting antiviral agents for HBV using in silico screening.</a> Biochem. Biophys. Res. Commun. 456 (1), 20-28 (2015).</li><li>Ladner, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+expression+of+human+hepatitis+B+virus+(HBV)+in+stably+transfected+hepatoblastoma+cells:+a+novel+system+for+screening+potential+inhibitors+of+HBV+replication.">Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.</a> Antimicrob. Agents. Chemother. 41 (8), 1715-1720 (1997).</li><li>Yan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+taurocholate+cotransporting+polypeptide+is+a+functional+receptor+for+human+hepatitis+B+and+D+virus.">Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.</a> Elife. 3, (2012).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-competent+infectious+hepatitis+B+virus+vectors+carrying+substantially+sized+transgenes+by+redesigned+viral+polymerase+translation.">Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.</a> PLoS. One. 8, 60306 (2013).</li><li>Chaisomchit, S., Tyrrell, D. L., Chang, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+replicative+and+nonreplicative+hepatitis+B+virus+vectors.">Development of replicative and nonreplicative hepatitis B virus vectors.</a> Gene. Ther. 4 (12), 1330-1340 (1997).</li><li>Hong, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+recombinant+hepatitis+B+virus+vectors+efficiently+deliver+protein+and+RNA+encoding+genes+into+primary+hepatocytes.">Novel recombinant hepatitis B virus vectors efficiently deliver protein and RNA encoding genes into primary hepatocytes.</a> J. Virol. 87 (12), 6615-6624 (2013).</li><li>Hall, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+luciferase+reporter+from+a+deep+sea+shrimp+utilizing+a+novel+imidazopyrazinone+substrate.">Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.</a> ACS. Chem. Biol. 7 (11), 1848-1857 (2012).</li><li>Nishitsuji, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+reporter+system+to+monitor+early+stages+of+the+hepatitis+B+virus+life+cycle.">Novel reporter system to monitor early stages of the hepatitis B virus life cycle.</a> Cancer. Sci. 106 (11), 1616-1624 (2015).</li><li>Sugiyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+hepatitis+B+virus+genotypes+on+the+intra-+and+extracellular+expression+of+viral+DNA+and+antigens.">Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens.</a> Hepatology. 44 (4), 915-924 (2006).</li><li>Seeger, C., Zoulim, F., Mason, W., Knipe, D., Howley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepadnaviruses.">Hepadnaviruses.</a> Field Virology. Volume 1. , 2185-2221 (2013).</li><li>Moolla, N., Kew, M., Arbuthnot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+elements+of+hepatitis+B+virus+transcription.">Regulatory elements of hepatitis B virus transcription.</a> J. Viral. Hepat. 9 (5), 323-331 (2002).</li><li>Cattaneo, R., Will, H., Schaller, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus+transcription+in+the+infected+liver.">Hepatitis B virus transcription in the infected liver.</a> EMBO. J. 3 (9), 2191-2196 (1984).</li><li>Enders, G. H., Ganem, D., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+major+transcripts+of+ground+squirrel+hepatitis+virus:+the+presumptive+template+for+reverse+transcriptase+is+terminally+redundant.">Mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redundant.</a> Cell. 42 (1), 297-308 (1985).</li><li>Haines, K. M., Loeb, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sequence+of+the+RNA+primer+and+the+DNA+template+influence+the+initiation+of+plus-strand+DNA+synthesis+in+hepatitis+B+virus.">The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus.</a> J. Mol. Biol. 370 (3), 471-480 (2007).</li><li>Hirsch, R. C., Lavine, J. E., Chang, L. J., Varmus, H. E., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+gene+products+of+hepatitis+B+viruses+are+required+for+genomic+RNA+packaging+as+well+as+for+reverse+transcription.">Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as well as for reverse transcription.</a> Nature. 344 (6266), 552-555 (1990).</li></ol>

Het HBV genoom vier belangrijke open leesramen (ORF&#39;s), waaronder de kern, polymerase, oppervlak en X ORF (Figuur 1). Transcriptie van deze vier HBV ORF wordt strak gereguleerd door vier initiatiefnemers: de precore / kern promoter met enhancer II, S1 promotor, S2 promotor en X promoter met enhancer I 18. De 3,5 kb en 3,4 kb mRNA&#39;s worden vertaald in PreCore en Core eiwitten, respectievelijk. De grote envelop-eiwit (L) wordt geproduceerd uit de grootste subgenomische mRNA...

Chronische infectie met hepatitis B-virus (HBV) is een belangrijke risicofactor voor chronische leverziekten 1. Hoewel de huidige therapeutische strategieën gebaseerd op nucleotide analogen die HBV pol functie en / of de toediening van type I interferon dat immuunresponsen bij geïnfecteerde individuen geactiveerd als indirect onderdrukt HBV proliferatie doorgaande interferon gestimuleerde genfuncties 2 remmen, kunnen deze behandelingen niet HBV elimineren DNA volledig 3. Bovendien is de opkomst van HBV die resistent zijn tegen anti- pol middelen zijn baart 4. De toediening van gecombineerde antivirale middelen direct gericht de verschillende stappen van humaan immunodeficiëntievirus (HIV) of hepatitis C virus (HCV) levenscyclus bleek succesvol onderdrukken of uitroeien virus (sen). Soortgelijke dit idee, de ontwikkeling van anti-HBV middel (en) die direct aan de verschillende stadia van de H handelenBV life cycle is van belang voor de vaststelling van de toekomstige HBV therapieën. In het algemeen, het opzetten van een eenvoudige in vitro kweeksysteem van het doelwit virus vergemakkelijkt de ontwikkeling van anti-virus middelen. Er zijn echter ten minste twee belemmeringen voor de ontwikkeling van in vitro kweeksystemen voor het screenen van anti-HBV middelen. De eerste is het ontbreken van een geschikt in vitro celkweeksysteem voor HBV-infectie / proliferatie. In tegenstelling tot andere virussen, zoals HIV en HCV, die zijn gekweekt in gevestigde cellijnen, is het moeilijk om HBV kweken in vitro door experimentele beperkingen waaronder een smal gastheerbereik. Het gebruik van specifieke celkweek systemen zoals het humane hepatoma cellijn HepaRG, dat gevoelig is voor HBV-infectie, 5, 6, 7 zijn ontwikkeld om deze problemen te overwinnen. Bovendien PXB cellen, geïsoleerd van urokinase-type plasminogeenactivator transgene / SCID-muizen geïnoculeerd met primaire humane hepatocyten (PHH), bleken gevoelig voor HBV-infectie en replicatie 8 te zijn. Echter, HBV replicatie niveaus HepaRG zijn afhankelijk van de celdifferentiatie toestand na cultuur die inconsistent en reproduceerbare resultaten van HBV-infectie / replicatie niveaus veroorzaken. PXB wordt gebruikt voor HBV-infectie experimenten maar is beperkt door de beschikbaarheid. Een tetracycline induceerbare HBV expressie cellijn HepAD38 is ook op grote schaal gebruikt voor HBV replicatie studie, maar dit systeem staat slechts evaluatie na transcriptie en niet instaphulp HBV infectie 9. Onlangs heeft de identificatie van natriumtaurocholaat (Ntcp) als een functionele receptor voor HBV kon de ontwikkeling van een variabele HBV kweeksysteem 10. Inderdaad, NTCP expressie in niet-gevoelige hepatocarcinoma cellen zoals Huh7 enHepG2 maakt HBV-infectie 10 en derhalve is de keuze van HBV gevoelige cellijnen uitgebreid, oplossen vele experimentele beperkingen. Het tweede probleem is het gebrek aan een eenvoudig testsysteem HBV-infectie en replicatie evalueren. Evaluatie van HBV-infectie wordt gewoonlijk uitgevoerd door het analyseren van HBV DNA, RNA en eiwitten. Echter, kwantificering van deze markers virus is tijdrovend, duur en vaak niet altijd eenvoudig. Daarom is de ontwikkeling van een eenvoudig testsysteem, zoals het gebruik van een reportergen, kunnen problemen met HBV testsystemen overwinnen. Omdat het genoom grootte die kan worden verpakt in een HBV capside beperkt - minder dan 3,7 kb 11 - de grootte van een reportergen moeten zo kort mogelijk zijn. Bovendien is de aanwezigheid van meerdere cis- elementen verspreid door het genoom, die essentieel zijn voor virale replicatie, beperkt de beschikbare functies in<html> insertie van het reportergen in het genoom. Verscheidene rapporten hebben geprobeerd om vreemde genen, waaronder HIV-1 Tat, groen fluorescerend eiwit, en DsRed invoegen in het HBV genoom 11, 12, 13. Echter, deze recombinante HBV niet bruikbaar voor het screenen van HBV-infectie / replicatie of voor high-throughput screening van factoren die HBV-infectie / replicatie. Dit komt voornamelijk door de lage productiviteit van recombinante virussen en de verminderde intensiteit van reportergenexpressie veroorzaakt door inefficiënte virusproductie. Om deze problemen te overwinnen, geconstrueerd we een reporter HBV met een hoge opbrengst van virusproductie. Dit virus is zeer gevoelig voor de controle van de vroege stadia van de replicatiecyclus HBV, from entry transcriptie. Hiervoor NanoLuc (NL) gekozen als een merkergen omdat het een klein (171 aminozuren) ontworpen luminescente reporterclass = &quot;xref&quot;&gt; 14. Bovendien NL is ongeveer 150 maal helderder dan vuurvlieg en Renilla luciferase, en de luminescente reactie ATP-onafhankelijk, wat suggereert dat de valse hit rate laag is voor high-throughput screening zal zijn. De productie-efficiëntie van het recombinante HBV ongeveer 1/5 van de bovenliggende HBV en soortgelijke niveaus over de voorbije HBV recombinante virussen; De helderheid van NL overwint productiviteit virus problemen zodat het kan worden gebruikt voor de massale screening van anti-HBV middelen. Screening van anti-HBV middelen gebruikmakend van primaire hepatocyten kan HepaRG, HepAD38 en NTCP getransduceerde hepatocyten bruikbaar voor het screenen van anti-HBV middelen met gebruikelijke methode (en). De hier beschreven systeem heeft diverse voordelen, zoals eenvoudige bediening, hoge gevoeligheid en lage kosten voor screening. Deze voordelen zijn geschikt voor high throughput assays te ontwikkelen en identificeren van nieuwe HBV middelen voor therapeutische doeleinden.

<table border="0" cellpadding="0" cellspacing="0" width="781">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Nano-Glo Luciferase Assay Regent</td>
			<td style="width:201px;">Promega</td>
			<td style="width:160px;">N1110</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin Mixed Solution</td>
			<td>Nacalai tesque</td>
			<td>09367-34&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:332px;">100 mm/collagen-coated dish</td>
			<td>Iwaki</td>
			<td>4020-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000001&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 6000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>81255</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 8000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Nacalai tesque</td>
			<td>31319-45&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 mol/l-EDTA Solution</td>
			<td>Nacalai tesque</td>
			<td>06894-14&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>Nacalai tesque</td>
			<td>35434-21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millex-HP, 0.45 &#956;m, polyethethersulfone, filter</td>
			<td>Merck Millipore</td>
			<td>SLHP033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagen coated 96-well plate</td>
			<td>Corning</td>
			<td>NO3585</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Passive Lysis 5xBuffer</td>
			<td>Promega</td>
			<td>E1941</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GloMax 96 Microplate Luminometer</td>
			<td>Promega</td>
			<td>E6501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Nacalai tesque</td>
			<td>30403-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luminometer plate</td>
			<td>Greiner bio-one</td>
			<td>655075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HepG2-NTCP1-myc-clone22</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV delta epsilon</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV/NL</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tube</td>
			<td>Violamo</td>
			<td>1-3500-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Myc antibody</td>
			<td>Sigma-Aldrich</td>
			<td>C3956</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBIG</td>
			<td>Japan Blood Products Organization</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IFN-&#946;</td>
			<td>Mochida Pharmaceutical</td>
			<td>14987224005413</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3393</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of a hepatitis b virus reporter system to monitor the early stages of the replication cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Figuur 1 toont een schema van het HBV genoom getranscribeerd RNA, virus eiwitten en hun coderende gebied op het genoom. Het reportergen en de ligging in het genoom zijn ook aangegeven. Figuur 2 toont de HBV reporterplasmide en helper plasmide dat het reporter gen. De pUC1.2HBV / NL reporter werd geconstrueerd door het verwijderen van nucleotideposities 223-811 van de transcriptie initiatie site van de precore mRNA van pUC1.2HBV 16...

Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Hier beschrijven we een nieuw ontwikkelde hepatitis B-virus (HBV) reportersysteem de vroege stadia van de levenscyclus HBV controleren. Deze vereenvoudigde in vitro systeem zal helpen bij het screenen van anti-HBV middelen met een high-throughput strategie.

Ontwikkeling van een Hepatitis B Virus reportersysteem de vroege stadia van de replicatiecyclus Monitor

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

The overall goal of this recombinant HBV reporter system is to make it easy to conduct mass screening for anti-Hepatitis B virus, or HBV agents. This method can help answer key questions in the HBV field, such as screening for anti-HBV agents and identification HBV factors. The main advantage of this technique is that the HBV reporter system can monitor the earlier stages of the HBV replication cycle.After preparing cell culture medium, the day before transvection, place four times 10 to the six HepG2 cells in medium, on a 10 cm collagen coated dish. Incubate the cells at 37 degrees C in a humidifed 5%CO2 incubator. Using a transvectionary agent according to the manufacturer's instructions, transvect the cells with 5 mcg of pUC1 2HBV delta epsilon, and 5 mcg of pUC1 2HBV/NL.The next day, remove the culture medium and add 10 mL of fresh culture medium. Then, one week after transvection, transfer the culture medium containing the recombinant HBV to a 50 mL tube. Next, add 10 mL of fresh culture medium to the plate containing transvected cells.Then collect and centrifuge the culture medium at 2, 300 x g for five minutes to remove the cell debris containing the recombinant HBV. Pass the supernatant through a 45 mcm membrane filter. Then add an equal volume of 25%PEG, 1.5 molar NaCl, to the filtrate, and gently mix.Incubate the sample overnight at four degrees C.The following day, spin the solution at 2, 300 x g and four degrees C, for 20 minutes. Discard the supernatant and dissolve the pellet in 5 mL of TNE buffer. Now centrifuge the sample again to remove cell debris, then load 5 mL of the TNE-containing recombinant HBV onto 8 mL of 20%sucrose in TNE.Centrifuge the two at 100, 000 x g and 15 degrees celsius for three hours. Discard as much of the supernatant as possible and use 1 mL of serum-free DMEM per 40 mL of starting culture to re-suspend the pellet. Incubate the suspension at four degrees celsius overnight.The following morning, filter the suspension through a 45 mcm filter. Then prepare 5 mL aliquots and store the tubes at minus 80 degrees celsius. Plate HepG2 cells, stably expressing sodium taurocholate cotransporting polypeptide, or NTCP, that are susceptible to HBV infection, and incubate at 37 degrees celsius and 5%CO2.One day before infection, plate approximately five times 10 to the fourth HepG2 NTCP cells into the wells of a 96-well collagen coated plate, in 1 mL of culture medium. Thaw recombinant HBV in a 37 degree celsius water bath, until there is a small bit of ice remaining in the vial. Prepare medium for infection by combining 78 mcL of fresh culture medium, 2 mcL of DMSO, 10 mcL of 40%PEG 8000, in 1x PBS, and 10 mcL of recombinant HPV.Add 100 mcL of recombinant HBV solution to each well of a 96-well plate. One day after infection, use 300 mcL of PBS to wash the cell three times to remove the contaminating reporter protein from the virus fraction. Add 200 mcL of culture medium, containing 2%DMSO, to the infected cells, and incubate for one week or less.To carry out analysis of the HBV infection, one week after infection, use PBS to wash the infected cells three times. Add 50 mcL of Lysis Buffer to the infected cells. Rock the culture plate for five minutes then centrifuge at 2, 000 x g for five minutes.Add 50 mcL of the reporter substrate to a Luminometer plate. Then add 20 mcL of the cell lysate to the plate and mix by briefly shaking. Finally, place the plate in the Luminometer and initiate reading.This gel shows digestion of the constructed pUC1 2HBVNL reporter plasmid, and the pUC1 2HBV delta helper plasmid with HindII and EcoRI. Both samples produce the expected band sizes. In this western blot, lysates from HepG2 cells, or HepG2 cells expressing recombinant myc-tagged NTCP and infected with recombinant HBV, were probed with an anti-myc antibody that recognizes the 34 kDa unglycosylated and the 65 kDa glycosolated recombinant proteins.The graphs shown here illustrate the kinetics of the reporter gene. RNA and DNA levels in recombinant HBV infected HepG2 NTCP myc-clone 22, or human primary hepatocyte cells. The levels of HBV RNA in the reporter activity were elevated at three days after infection, in both the infected cells and the primary hepatocytes.In contrast, there was no change in DNA levels at nine days post-infection, because recombinant HBV is deficient for functional core and pole, that play important roles in the intracellular DNA replication pathway. In this figure, inhibition of recombinant HBV by Hepatitis B Immune Globulin, or HBIG, Heparin, and IFN-beta, are illustrated. HBIG and Heparin strongly suppressed the reporter activity to less than 10%at 75 U/mL, and 150 U/mL, respectively.IFN-beta on the other hand, suppressed reporter activity to less than 50%at 1000 U/mL. Once mastered, this technique can be done in one week for preparation of recombinant HBV, in six days for infection recombinant HBV, and in summary, means for measurement of the reporter protein, if it is performed properly. While attempting this procedure, it's important to remember the washing step that removes the contaminating reporter protein from the bias fraction.Following this procedure, other results like infection with wide HBV can be performed in order to answer additional question to that the recombinant HBV did not produce first positive results. After its development, this technique paved the way for researchers in the field of HBV to explore the development of anti-HBV agent in a cell culture model. After watching this video, you should have a good understanding of how to purify and infect cells with recombinant HBV.Don't forget that working with recombinant HBV can be extremely hazardous and precautions such as wearing gloves should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

Development of a Negative Selectable Marker for Entamoeba histolytica

Ontwikkeling van een negatieve selecteerbare marker voor Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

Opheldering van Host-virus interactie in Lytische Replicatie van een Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

A Protocol for Analyzing Hepatitis C Virus Replication

Een protocol voor het analyseren van hepatitis C virus replicatie

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Muis foetale lever Cultuur System Doel Gene functies ontleden in de vroege en late stadia van Terminal Erytropoëse

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analyse van Myd88 gemedieerde cellulaire immuunrespons op West Nile Virus mutantstam Infectie

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescentie te zien op de cellulaire opname van humaan lactoferrine en de Associated antivirale activiteit tegen het hepatitis C virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Een nieuwe Feeder-vrij systeem voor massaproductie van lymfkliertest Natural Killer cellen In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Assembly and Purification of Prototype Foamy Virus Intasomes

Vergadering en zuivering van Prototype schuimend Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

Snelle, veilige en eenvoudige handleiding bed nucleïnezuur extractie voor de opsporing van Virus in monsters van volbloed

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

"Lever-on-a-Chip" culturen van primaire hepatocyten en Kupffer cellen voor Hepatitis B-virusinfectie

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...