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. Produktion af rekombinant HBV Encoding reporterproteinet <ol><li> Fremstilling af HepG2-celler <ol><li> Forbered celledyrkningsmedium (Dulbeccos modificerede Eagles medium (DMEM) suppleret med 10% føtalt bovint serum (FBS), 100 U / ml penicillin, 100 ug / ml streptomycin, og 100 U / ml ikke-essentielle aminosyrer). </li><li> Plate 4 x 10 6 HepG2 celler i en 10-cm collagen-coatede skål i 10 ml dyrkningsmedium dagen før transfektion. Inkuber HepG2-celler ved 37 ° C i en befugtet 5% <su...

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HBV-genomet har fire primære åbne læserammer (ORF&#39;er), herunder kernen, polymerase, overflade og X ORF&#39;er (figur 1). Transkription af disse fire HBV ORF&#39;er er stramt reguleret af fire promotorer: den prækerne / kerne-promotorholdige enhancer II, S1-promotor, S2 promotoren og X-promotoren indeholdende enhancer I 18. De 3,5 kb og 3,4 kb mRNA oversættes til prækerne og Core proteiner, hhv. Den store kappeproteinet (L) er fremstillet af det største subgenomiske mRN...

Kronisk infektion med hepatitis B virus (HBV) er en stor risikofaktor for kroniske leversygdomme 1. Selv om den nuværende terapeutiske strategier er baseret på nukleotidanaloger, som inhiberer HBV pol funktion og / eller indgivelse af type I interferon, der aktiverer immunresponser i inficerede individer såvel som indirekte undertrykke HBV proliferation gennem interferon-stimuleret genfunktioner 2, kan disse behandlinger ikke fjerne HBV DNA helt 3. Endvidere fremkomsten af HBVs som er resistente over for anti-pol midler er til bekymring 4. Administrationen af ​​kombinerede antivirale midler direkte rettet mod de forskellige trin i human immundefekt virus (HIV) eller hepatitis C-virus (HCV) livscyklussen blev vist at kunne undertrykke eller udrydde virus (es). Svarende til denne idé, udvikling af anti-HBV middel (midler), der virker direkte på de forskellige stadier i HBV livscyklus er vigtig for etablering af fremtidige HBV behandlinger. Generelt etablering af et simpelt in vitro-dyrkningssystem af målet virus fremmer udviklingen af anti-virus midler. Der er dog mindst to hindringer for udviklingen af in vitro dyrkningssystemer til screening anti-HBV midler. Den første er manglen på en bekvem in vitro cellekultursystem for HBV-infektion / proliferation. I modsætning til andre vira, såsom HIV og HCV, som er opformeret i etablerede cellelinjer, er det vanskeligt at dyrke HBV in vitro på grund af eksperimentelle begrænsninger, herunder et snævert værtsområde. Anvendelsen af specifikke cellekultursystemer såsom den humane hepatoma-cellelinie HepaRG, som er følsomt for HBV-infektion, 5, 6, 7, er blevet udviklet for at overvinde disse problemer. Endvidere PxB celler, isoleret fra urokinase-type plasminogenaktivator transgene / SCID mus inokuleret med primære humane hepatocyt (PHH), viste sig at være modtagelige for HBV-infektion og replikation 8. Imidlertid HBV-replikation niveauer i HepaRG er afhængige af cellulær differentiering tilstand efter dyrkning, som kan forårsage inkonsistente og ureproducerbare resultater af HBV infektion / replikering niveauer. PxB er almindeligt anvendt for HBV-infektion eksperimenter, men er begrænset af dets tilgængelighed. En tetracyclin inducerbar HBV ekspression cellelinie HepAD38, er også blevet meget anvendt til at studere HBV-replikation, men dette system tillader kun evaluering efter transkription og ikke på posten trin af HBV-infektion 9. For nylig har identificeringen af natriumtaurocholat cotransporting polypeptid (Ntcp) som en funktionel receptor for HBV muliggjorde udviklingen af en variabel HBV dyrkningssystem 10. Faktisk Ntcp ekspression i ikke-følsomme leverkarcinom celler såsom Huh7 ogHepG2 muliggør HBV-infektion 10 og således har valget af HBV modtagelige cellelinier blevet udvidet, løse mange af de eksperimentelle begrænsninger. Det andet problem er manglen på en simpel assaysystem til evaluering HBV-infektion og replikation. Evaluering af HBV-infektion udføres sædvanligvis ved at analysere HBV-DNA, RNA og proteiner. Imidlertid kvantificering af disse virus markører er tidskrævende, ofte kostbare og ikke altid enkel. Derfor er udviklingen af ​​et simpelt assay-system, såsom anvendelse af et reportergen, kan overvinde problemer i forbindelse med HBV assaysystemer. Men fordi genomet størrelse, der kan pakkes i en HBV capsid er begrænset - mindre end 3,7 kb 11 - størrelsen af et reportergen bør være så kort som muligt. Tilstedeværelsen af ​​multiple cis elementer spredt over hele genomet, som er essentielle for viral replikation, begrænser jobs for i<html> sertion af reportergenet i genomet. Adskillige rapporter har forsøgt at indsætte fremmede gener, herunder HIV-1-Tat, grønt fluorescerende protein, og DsRed, i HBV-genomet 11, 12, 13. Men disse rekombinante HBVs ikke anvendelig til screening af HBV-infektion / replikation, eller for high-throughput screening af faktorer, der påvirker HBV-infektion / replikation,. Dette er primært på grund af den lave produktivitet af rekombinante vira og den reducerede intensiteten af ​​reportergenekspression forårsaget af ineffektiv virusproduktion. For at overvinde disse problemer, vi konstrueret en reporter HBV med et højt udbytte af virus produktion. Denne virus er meget følsom for overvågningen af ​​de tidlige faser af HBV-replikation cyklus, fra post til transkription. For at opnå dette, blev NanoLuc (NL) valgt som markør-gen, fordi det er et lille (171 aminosyrer) manipuleret luminescerende reporterclass = &quot;xref&quot;&gt; 14. Desuden NL er cirka 150 gange lysere end ildflue eller Renilla luciferase, og det luminescerende reaktion er ATP-uafhængig, hvilket antyder, at den falske hit sats vil være lav for high-throughput screening. Produktionen effektivitet rekombinante HBV er ca. 1/5 af moderselskabet HBV, og ligner niveauer indberettet for tidligere HBV rekombinante vira; imidlertid lysstyrke NL overvinder virus produktivitet problemer, så det kan anvendes til massen screening af anti-HBV-midler. Screening af anti-HBV midler ved anvendelse primære hepatocytter, kan HepaRG, HepAD38 og Ntcp-transducerede hepatocytter være anvendelige til screening af anti-HBV-midler ved konventionel fremgangsmåde (r). Men den her beskrevne system har forskellige fordele såsom simpel håndtering, høj følsomhed, og lave omkostninger til screening. Disse fordele er velegnede til analyser med høj kapacitet til at udvikle og identificere nye HBV agenter 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 skematisk afbildning af HBV-genomet, transkriberede RNA&#39;er, virusproteiner og deres kodende område på genomet. Reportergenet og dens placering i genomet er også angivet. Figur 2 viser HBV reporterplasmidet og hjælpe-plasmid indeholdende reportergenet. Den pUC1.2HBV / NL reporter blev konstrueret ved deletion nukleotidpositioner 223-811 fra transkriptionsinitieringsstedet for prækerne-mRNA af 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 nyudviklet hepatitis B virus (HBV) reporter-system til at overvåge de tidlige stadier af HBV livscyklus. Denne forenklede in vitro-system vil støtte i screening af anti-HBV-midler ved anvendelse af en high-throughput strategi.

Udvikling af en hepatitis B virus Reporter system til at overvåge tidlige stadier af 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), ...

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