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. Produção de recombinante de HBV codifica a proteína repórter <ol><li> Preparação de células HepG2 <ol><li> Prepare meio de cultura celular (meio de Eagle modificado por Dulbecco (DMEM) suplementado com soro fetal bovino a 10% (FBS), 100 U / ml de penicilina, 100 ug / ml de estreptomicina, e 100 U / ml de aminoácidos não essenciais). </li><li> A placa 4 x 10 6 células HepG2 em um prato revestido com colagénio de 10 cm em 10 ml de meio de cultura no dia antes da transfecção. I...

<ol>
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O genoma do VHB tem quatro primários grelhas de leitura abertas (ORFs), incluindo o núcleo, polimerase, de superfície e X ORFs (Figura 1). A transcrição destes quatro HBV ORF é estreitamente regulada por quatro promotores: o promotor pré-core / core contendo potenciador II, promotor S1, S2 promotor e X promotor contendo potenciador I 18. Os mRNAs 3,5 kb e 3,4 kb são traduzidos em proteínas Precore e Core, respectivamente. O grande proteína envelope (L) é produzido a pa...

A infecção crónica pelo vírus da hepatite B (HBV) é um fator de risco para doenças hepáticas crônicas 1. Embora as estratégias terapêuticas correntes baseiam-se em análogos de nucleótidos que inibem a função de HBV pol e / ou a administração de interferão do tipo I que activa as respostas imunitárias em indivíduos infectados, bem como a proliferação de HBV indirectamente supressão através de funções de genes estimulados com interferão 2, estes tratamentos não pode eliminar o HBV DNA completamente 3. Além disso, o aparecimento de HBVs que são resistentes aos agentes anti-Pol é de preocupação 4. A administração de agentes anti-virais que visam directamente combinadas as diferentes etapas do vírus da imunodeficiência humana (HIV) ou ciclo de vida do virus da hepatite C (HCV), foi mostrado para suprimir ou erradicar o vírus (es) com êxito. Semelhante a esta ideia, o desenvolvimento de um agente anti-HBV (s) que actuam directamente sobre as diferentes fases do Hciclo de vida BV é importante para o estabelecimento de futuras terapias HBV. Em geral, o estabelecimento de uma simples no sistema de cultura in vitro do vírus alvo facilita o desenvolvimento de agentes anti-vírus. No entanto, existem pelo menos duas barreiras ao desenvolvimento de sistemas de cultura in vitro para rastrear agentes anti-HBV. O primeiro é a falta de um sistema in vitro de cultura de células conveniente para HBV infecção / proliferação. Ao contrário de outros vírus, tais como HIV e HCV, que são propagadas em linhas celulares estabelecidas, é difícil de cultivar in vitro de HBV devido a limitações experimentais, incluindo uma gama de hospedeiros estreita. A utilização de sistemas de cultura de células específicas, tais como a linha celular de hepatoma humano HepaRG, que é susceptível a infecção por HBV, 5, 6, 7 ter sido desenvolvido para superar estes problemas. Além disso, as células PXB, isoladas de uroquinase-type do ativador do plasminogênio transgênico / SCID inoculados com hepatócitos humanos primários (PHH), mostraram-se suscetíveis à infecção por HBV e replicação 8. No entanto, os níveis de replicação de HBV em HepaRG são dependentes do estado de diferenciação celular após a cultura, o que pode causar resultados inconsistentes e reprodutíveis de níveis de infecção / replicação de HBV. PXB é comumente utilizado nas experiências de infecção por HBV, mas é limitado pela sua disponibilidade. A linha de células de expressão HBV inducible tetraciclina, HepAD38, também tem sido amplamente utilizado para estudar a replicação do VHB, mas este sistema só permite uma avaliação após a transcrição e não na etapa de entrada da infecção por HBV 9. Recentemente, a identificação do polipéptido cotransporting taurocolato de sódio (NTCP) como um receptor funcional para VHB tem permitido o desenvolvimento de um sistema de cultura de HBV variável 10. Na verdade, a expressão PNCT em células de hepatocarcinoma não sensíveis, tais como Huh7 eHepG2 permite a infecção por HBV 10 e assim, a escolha de linhas de células susceptíveis de HBV foi expandido, resolver muitas das limitações experimentais. O segundo problema é a falta de um sistema de ensaio simples para avaliar a infecção por HBV e replicação. A avaliação da infecção por HBV é geralmente realizada por meio da análise HBV DNA, RNA e proteínas. No entanto, a quantificação destes marcadores vírus é demorado, dispendioso e muitas vezes nem sempre simples. Por conseguinte, o desenvolvimento de um sistema de ensaio simples, tais como a utilização de um gene repórter, pode superar os problemas associados com sistemas de ensaio de HBV. No entanto, porque o tamanho do genoma que pode ser empacotado numa cápside de HBV é limitado - menos de 3,7 kb 11 - o tamanho de um gene repórter deverá ser tão curto quanto possível. Além disso, a presença de vários elementos cis espalhados ao longo do genoma, que são essenciais para a replicação virai, limita as posições disponíveis em<html> serção do gene repórter no genoma. Vários relatórios têm tentado inserir genes estranhos, incluindo o HIV-1 Tat, proteína fluorescente verde, e DsRed, no genoma de HBV 11, 12, 13. No entanto, estes HBVs recombinantes não são úteis para o rastreio da infecção por HBV / replicação, ou para o rastreio de alto rendimento de factores que afectam a infecção por HBV / replicação. Isto é principalmente devido à baixa produtividade de vírus recombinantes e a intensidade reduzida da expressão do gene repórter causada pela produção de vírus ineficiente. Para superar estes problemas, foi construída uma VHB repórter com um elevado rendimento de produção de vírus. Este vírus é altamente sensível para monitorizar as fases iniciais do ciclo de replicação de HBV, a partir da entrada para a transcrição. Para alcançar este objectivo, NanoLuc (NL) foi escolhida como um gene marcador, porque ele é um pequeno (171 ácidos aminados) por engenharia repórter luminescenteclass = &quot;xref&quot;&gt; 14. Além disso, a NL é aproximadamente 150 vezes mais brilhante do que de pirilampo ou de Renilla luciferase, e a reacção luminescente é independente de ATP, o que sugere que a taxa de acerto falsa será baixo para rastreio de alto rendimento. A eficiência de produção do HBV recombinante é de aproximadamente 1/5 do progenitor de HBV, e semelhante para os níveis relatados por vírus recombinantes de HBV anteriores; No entanto, o brilho da NL supera os problemas de produtividade de vírus para que ele possa ser utilizado para o rastreio em massa de agentes anti-HBV. Rastreio de agentes anti-HBV, utilizando hepatócitos primários, HepaRG, HepAD38 e hepatócitos NTCP-transduzidas pode ser útil para o rastreio de agentes anti-HBV pelo método convencional (s). No entanto, o sistema descrito aqui tem várias vantagens, tais como manuseio simples, de alta sensibilidade e baixo custo para triagem. Estas vantagens são adequadas para ensaios de alto rendimento para identificar e desenvolver novos agentes de HBV para fins terapêuticos.

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		<col />
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		<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>
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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.

A Figura 1 mostra uma representação esquemática do genoma de HBV, ARN transcritos, proteínas do vírus e a sua região de codificação no genoma. O gene repórter e sua localização no genoma são também indicados. A Figura 2 mostra o plasmídeo repórter e auxiliar plasmídeo VHB que contém o gene repórter. O repórter pUC1.2HBV / NL, foi construído por exclusão posições de nucleótidos 223-811 do local de iniciação da transcrição do AR...

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

Descrevemos aqui um vírus da hepatite B recentemente desenvolvido sistema de repórter (HBV) para monitorar as fases iniciais do ciclo de vida do HBV. Este simplificado sistema in vitro irá ajudar no rastreio de agentes anti-HBV, utilizando uma estratégia de alto rendimento.

Desenvolvimento de um Sistema Reporter vírus da hepatite B de acompanhar as fases iniciais do ciclo de replicação

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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