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. Produksjon av rekombinant HBV Koding Reporter Protein <ol><li> Utarbeidelse av HepG2 celler <ol><li> Fremstille cellekulturmedium (Dulbeccos modifiserte Eagles medium (DMEM) supplementert med 10% føtalt bovint serum (FBS), 100 U / ml penicillin, 100 ug / ml streptomycin, og 100 U / ml ikke-essensielle aminosyrer). </li><li> Plate 4 x 10 6 HepG2-celler i en 10-cm kollagen-belagte fatet i 10 ml kulturmedium dagen før transfeksjon. Inkuber HepG2 celler ved 37 ° C i en fuktig 5%</sub...

<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>

HBV-genomet har fire primære åpne leserammer (ORF) inkludert i kjernen, polymerase, overflate og X-ORF (figur 1). Transkripsjon av disse fire HBV ORF er strengt regulert av fire arrangører: den Precore / core promoter inneholder Enhancer II, S1 promoter, S2 promoter og X promoter inneholder enhancer jeg 18. 3,5 kb og 3,4 kb mRNA er oversatt til Precore og Core proteiner, henholdsvis. Den store innhylle protein (L) er produsert fra den største subgenomt mRNA (2,5 kb), og den m...

Kronisk infeksjon med hepatitt B-virus (HBV) er en viktig risikofaktor for kroniske leversykdommer 1. Selv om dagens terapeutiske strategier er basert på nukleotid-analoger som hemmer HBV pol funksjon og / eller administrering av type I interferon som aktiverer immunresponser hos infiserte individer, så vel som indirekte å undertrykke HBV spredning gjennom interferon-stimulerte genfunksjoner 2, kan disse behandlingene ikke eliminere HBV DNA helt tre. Videre fremveksten av HBVs som er resistente mot anti-pol midler er av interesse 4. Administrasjonen av kombinerte antivirale midler direkte rettet mot de ulike trinnene i humant immunsviktvirus (HIV) eller hepatitt C virus (HCV) livssyklus ble vist å kunne undertrykke eller utrydde viruset (es). I likhet med denne ideen, utvikling av anti-HBV middel (e) som virker direkte på de ulike stadier av HBV livssyklus er viktig for å etablere fremtidige HBV-terapier. Generelt, etablering av en enkel in vitro kultursystem av målet viruset forenkler utviklingen av antivirusmidler. Det er imidlertid minst to barrierer for utvikling av in vitro kultursystemer for å screene anti-HBV-midler. Den første er mangelen på en enkel in vitro-cellekultursystem for HBV-infeksjon / proliferasjon. I motsetning til andre virus, slik som HIV og HCV, som forplanter seg i etablerte cellelinjer, er det vanskelig å dyrke HBV in vitro på grunn av eksperimentelle begrensninger, inkludert et snevert vertsområde. Bruken av spesifikke cellekultursystemer som for eksempel den humane hepatom-cellelinje HepaRG, som er utsatt for HBV-infeksjon, 5, 6, 7 er blitt utviklet for å overvinne disse problemene. Videre PXB celler, isolert fra urokinase-type plasminogen aktivator transgene / SCID mus inokulert med primære humane hepatocytter (PHH), viste seg å være utsatt for HBV-infeksjon og replikasjon 8. Men HBV replikasjonsnivået i HepaRG er avhengig av cellulær differensiering tilstand etter kultur, noe som kan føre til inkonsistente og uforklarlige resultatene av HBV infeksjon / replikering nivåer. PXB er ofte brukt for HBV smitteforsøk, men er begrenset av sin tilgjengelighet. En tetracyklin-induserbar ekspresjon av HBV-cellelinje, HepAD38, har også blitt mye brukt til å studere HBV replikasjon, men dette systemet tillater bare evaluering etter transkripsjon, og ikke på inngangstrinnet av HBV-infeksjon 9. Nylig har identifikasjon av natriumtaurocholat cotransporting polypeptid (NTCP) som en funksjonell reseptor for HBV er tillatt utviklingen av en variabel HBV kultursystem 10. Faktisk NTCP uttrykk i ikke-følsomme hepatokarsinom celler som Huh7 ogHepG2 gjør det mulig for HBV-infeksjon 10 og dermed har valget av HBV mottagelig cellelinjer blitt utvidet, løse mange av de eksperimentelle begrensninger. Det andre problem er mangelen på en enkel bestemmelsessystem for å evaluere HBV-infeksjon og replikasjon. Evaluering av HBV-infeksjon blir vanligvis utført ved å analysere HBV DNA, RNA og proteiner. Imidlertid kvantifisering av disse virus markørene er tidkrevende, kostbart og ofte ikke alltid er enkelt. Derfor er utviklingen av et enkelt assay-system, for eksempel ved hjelp av et reporter-gen, kan overvinne problemene forbundet med HBV analysesystemer. Imidlertid, fordi den genomstørrelse som kan pakkes inn i en HBV kapsid er begrenset - mindre enn 3,7 kb 11 - størrelsen på et reportergen bør være så kort som mulig. Videre, tilstedeværelsen av flere cis-elementer er spredt over hele genomet, som er essensielle for virusreplikasjon, begrenser de stillinger som er tilgjengelige for i<html> sertion av reportergenet i genomet. Flere rapporter har forsøkt å sette inn fremmede gener, inkludert HIV-1 Tat, grønt fluorescerende protein, og DsRed, inn i HBV-genomet 11, 12, 13. Men disse rekombinante HBVs ikke er nyttige for screening av HBV-infeksjon / replikasjon, eller for high-throughput screening av faktorer som påvirker HBV-infeksjon / replikasjon. Dette er hovedsakelig på grunn av den lave produktivitet av rekombinante virus og den reduserte intensitet av reportergenekspresjon skyldes ineffektiv virusproduksjon. For å overvinne disse problemene, konstruerte vi et reporter HBV med et høyt utbytte av virusproduksjon. Dette viruset er svært følsom for overvåking av de tidlige stadiene av den HBV-replikasjonssyklus, fra inngang til transkripsjon. For å oppnå dette, ble NanoLuc (NL) valgt som et markør-gen, fordi det er en liten (171 aminosyrer) konstruert luminescerende reporterclass = &quot;xref&quot;&gt; 14. Videre er NL ca 150 ganger lysere enn ildflue eller Renilla luciferase, og den selvlysende reaksjonen er ATP-uavhengig, noe som tyder på at den falske hit rate vil være lav for høy gjennomstrømming screening. Den produksjonseffektivitet av det rekombinante HBV er omtrent 1/5 av den overordnede HBV, og tilsvarende nivå som er rapportert for tidligere HBV rekombinante virus; imidlertid, vil lysstyrken NL overvinner virus produktivitets problemer slik at den kan brukes for masse screening av anti-HBV-midler. Screening av anti-HBV-midler ved hjelp av primære hepatocytter kan HepaRG, HepAD38 og NTCP-transduserte hepatocytter være nyttige for screening av anti-HBV-midler ved konvensjonell metode (r). Imidlertid er systemet som er beskrevet her har en rekke fordeler som enkel håndtering, høy følsomhet og lav kostnad for screening. Disse fordelene er egnet for høy gjennomstrømning analyser for å utvikle og identifisere nye HBV midler til terapeutiske formål.

<table border="0" cellpadding="0" cellspacing="0" width="781">
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		<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.

Figur 1 viser en skjematisk fremstilling av HBV-genomet, transkriberte RNA, virusproteiner og deres kodende område i genomet. Reporteren genet og dens plassering i genomet er også indikert. Figur 2 viser HBV reporter plasmid og hjelper-plasmid som inneholder reportergenet. Den pUC1.2HBV / NL reporter ble konstruert ved å slette nukleotidposisjoner 223-811 fra transkripsjons-initierings-setet av den Precore mRNA av 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

Her beskriver vi en nyutviklet hepatitt B-virus (HBV) reporter system for å overvåke de tidlige stadier av HBV-livssyklusen. Denne forenklede in vitro-system skal hjelpe til screening av anti-HBV-midler ved hjelp av en high-throughput-strategi.

Utvikling av en hepatitt B virus Reporter system for å overvåke tidlige stadier av 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), ...

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.

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