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 av rekombinant HBV Kodning av Reporter Protein <ol><li> Framställning av HepG2-celler <ol><li> Förbereda cellodlingsmedium (Dulbeccos modifierade Eagles medium (DMEM) kompletterat med 10% fetalt bovint serum (FBS), 100 U / ml penicillin, 100 | ig / ml streptomycin och 100 U / ml icke-essentiella aminosyror). </li><li> Plate 4 x 10 6 HepG2-celler i en 10 cm kollagenbelagda skålen i 10 ml odlingsmedium dagen före transfektion. Inkubera HepG2-celler vid 37 ° C i en fuktad 5%...

<ol>
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HBV-genomet har fyra primära öppna läsramar (ORF) inklusive kärnan, polymeras, yta och X ORF (Figur 1). Transkription av dessa fyra HBV ORF är hårt reglerad av fyra promotorer: den förkärna / core promotor som innehåller förstärkare II, S1-promotor, S2 promotorn och X-promotorn innehåller förstärkare I 18. De 3,5 kb och 3,4 kb mRNA översätts till förkärna och kärnproteiner, respektive. Den stora kuvert protein (L) framställs av den största subgenomiska mRNA (...

Kronisk infektion med hepatit B-virus (HBV) är en stor riskfaktor för kroniska leversjukdomar 1. Även om nuvarande terapeutiska strategier är baserade på nukleotidanaloger som hämmar HBV pol funktion och / eller administrering av typ I interferon som aktiverar immunsvar i infekterade individer samt indirekt undertrycka HBV spridning genom interferon-stimulerade genfunktioner 2, kan dessa behandlingar inte eliminera HBV DNA helt 3. Dessutom är uppkomsten av HBVs som är resistenta mot anti-pol medel till oro 4. Administrationen av kombinerade antivirala medel direkt inriktade på olika stegen i humant immunbristvirus (HIV) eller hepatit C-virus (HCV) livscykel visades framgångsrikt undertrycka eller utrota viruset (er). Liknar denna idé, utveckling av anti-HBV-agens (er) som verkar direkt på de olika stegen i HBV livscykel är viktigt för att etablera framtida HBV terapier. I allmänhet, inrättandet av en enkel in vitro-kultursystem av målet viruset underlättar utvecklingen av antivirusmedel. Men det finns åtminstone två hinder för utvecklingen av kultur in vitro-system för att screena anti-HBV-medel. Den första är bristen på en praktisk in vitro cellkultursystem för HBV-infektion / proliferation. Till skillnad från andra virus, såsom HIV och HCV, som fortplantas i etablerade cellinjer, är det svårt att odla HBV in vitro på grund av experimentella begränsningar inklusive ett smalt värdområde. Användning av specifika cellkultursystem, såsom den humana hepatomcellinjen HepaRG, som är känslig för HBV-infektion, 5, 6, har 7 utvecklats för att övervinna dessa problem. Dessutom PxB celler, isolerade från urokinas-type plasminogenaktivator transgena / SCID-möss ympade med primär human hepatocyte (PHH), visade sig vara mottagliga för HBV-infektion och replikering 8. Men HBV-replikation nivåer i HepaRG är beroende av celldifferentiering tillståndet efter kultur, vilket kan orsaka inkonsekventa och reproducerbara resultat av HBV-infektion / replikering nivåer. PxB används vanligen för HBV-infektion experiment, men begränsas av dess tillgänglighet. En tetracyklin inducerbar HBV uttryck cellinje HepAD38 har också använts i stor utsträckning för att studera HBV-replikation, men detta system endast tillåter utvärdering efter transkription och inte på ingångssteget av HBV-infektion 9. Nyligen har identifieringen av natriumtaurokolat cotransporting polypeptid (NTCP) som en funktionell receptor för HBV möjliggjort utvecklingen av en variabel HBV kultursystem 10. I själva verket NTCP uttryck i icke-mottagliga hepatokarcinom celler såsom Huh7 ochHepG2 möjliggör HBV-infektion 10 och därmed har valet av HBV mottagliga cellinjer utökats, lösa många av de experimentella begränsningar. Det andra problemet är bristen på ett enkelt testsystem för att utvärdera HBV-infektion och replikation. Utvärdering av HBV-infektion utförs vanligen genom att analysera HBV-DNA, RNA och proteiner. Men är kvantifiering av dessa virusmarkörer tidskrävande, ofta kostsamma och inte alltid enkelt. Därför är utvecklingen av ett enkelt analyssystem, såsom användning av en reportergen, kan övervinna problem som är associerade med HBV analyssystem. Emellertid, därför att genomstorleken som kan förpackas i en HBV-kapsid är begränsad - mindre än 3,7 kb 11 - storleken av en reportergen bör vara så kort som möjligt. Vidare, närvaron av multipla cis-element utspridda i genomet, som är väsentliga för viral replikation, begränsas de lediga tjänster för i<html> sertion av reportergenen i genomet. Flera rapporter har försökt att sätta in främmande gener, inklusive HIV-1 Tat, grönt fluorescerande protein, och DsRed, in i HBV-genomet 11, 12, 13. Emellertid är dessa rekombinanta HBVs är inte användbara för screening av HBV-infektion / replikation, eller för high-throughput screening av faktorer som påverkar HBV-infektion / replikation. Detta är främst på grund av den låga produktiviteten av rekombinanta virus och minskad intensitet av reporter genuttryck orsakas av ineffektiv virusproduktionen. För att övervinna dessa problem, konstruerade vi en reporter HBV med ett högt utbyte av virusproduktion. Detta virus är mycket känslig för att övervaka de tidiga stadierna av HBV-replikationscykeln, från inträde till transkription. För att uppnå detta, var NanoLuc (NL) valdes som en markörgen för att det är en liten (171 aminosyror) engineered luminescent reporterclass = &quot;xref&quot;&gt; 14. Dessutom är NL cirka 150 gånger starkare än eldfluga eller Renilla luciferas, och ljusreaktionen är ATP-oberoende, vilket tyder på att den falska träffsäkerhet kommer att vara låg för high-throughput screening. Produktionseffektiviteten av den rekombinanta HBV är ungefär 1/5 av moder HBV, och liknande till nivåer som rapporterats för tidigare HBV rekombinanta virus; emellertid ljusstyrkan i NL övervinner virusproduktivitetsfrågor så att den kan användas för mass screening av anti-HBV-medel. Screening av anti-HBV-medel med användning av primära hepatocyter, kanske HepaRG, HepAD38 och NTCP-omvandlade leverceller vara användbara för screening av anti-HBV-medel genom konventionell metod (er). Det system som beskrivs här har olika fördelar, såsom enkel hantering, hög känslighet, och låg kostnad för screening. Dessa fördelar är lämpliga för hög genomströmning analyser för att utveckla och identifiera nya HBV-medel för terapeutiska ändamål.

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		<col />
		<col />
		<col />
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	<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 visar en schematisk bild av HBV-genomet, transkriberade RNA, virusproteiner och deras kodande regionen på genomet. Reportergenen och dess läge i genomet är också indikerade. Figur 2 visar den HBV-reporter-plasmid och hjälparfag plasmid innehållande reportergenen. Den pUC1.2HBV / NL reporter konstruerades genom att deletera nukleotidpositioner 223-811 från transkriptionsinitieringsstället av den förkärna mRNA från pUC1.2HBV <sup class="...

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

Här beskriver vi en nyutvecklad hepatit B-virus (HBV) reportersystem för att övervaka de tidiga stadierna av HBV-livscykeln. Denna förenklade in vitro-system kommer att hjälpa till vid screening av anti-HBV-medel med användning av en hög genomströmning strategin.

Utveckling av en hepatit B-virus Reporter system för att övervaka de tidiga stadierna av replikationscykeln

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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