Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR
qPCR定量分析感染细胞培养模型中乙型肝炎病毒共价闭合环状DNA   

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Journal of Virology
Nov 2018

 

Abstract

Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and amplification-based assays are currently used for cccDNA quantification. Southern blot, the current gold standard, is time-consuming and not practical for a large number of samples. PCR-based methods show limited specificity when excessive HBV replicative intermediates are present. We have recently developed a real-time quantitative PCR protocol, in which total cellular DNA plus all forms of viral DNA are extracted by silica column. Subsequent incubation with T5 exonuclease efficiently removes cellular DNA and all non-cccDNA forms of viral DNA while cccDNA remains intact and can reliably be quantified by PCR. This method has been used for measuring kinetics of cccDNA accumulation in several in vitro infection models and the effect of antivirals on cccDNA. It allowed detection of cccDNA in non-human cells (primary macaque and swine hepatocytes, etc.) reconstituted with the HBV receptor, human sodium taurocholate cotransporting polypeptide (NTCP). Here we present a detailed protocol of this method, including a work flowchart, schematic diagram and illustrations on how to calculate “cccDNA copies per (infected) cell”.

Keywords: Hepatitis B Virus (乙型肝炎病毒), Covalently closed circular DNA (共价闭合环状DNA), cccDNA (cccDNA), T5 exonuclease (T5外切核酸酶), Quantitative PCR (定量PCR), Copy number per cell (单细胞拷贝数目)

Background

The Hepatitis B virus (HBV), a DNA virus belonging to the family Hepadnaviridae, is a human pathogen persisting in approximately 240 million people globally. HBV infection leads to higher risks of liver cirrhosis and hepatocellular carcinoma (Liang et al., 2015). At present, chronic HBV infection is not curable as current treatments do not eradicate the replicative reservoir, covalently closed circular (ccc)DNA (Levrero et al., 2016). In in vitro infected hepatocytes, cccDNA is formed by cellular repair of relaxed circular duplex (rc)DNA, the genomic form in virions (Guo et al., 2007; Long et al., 2017; Schreiner and Nassal, 2017). cccDNA serves as the viral template for pregenomic RNA which becomes encapsidated into nucleocapsids and is reverse transcribed to generate rcDNA and double-stranded linear (dsl)DNA by the viral polymerase (Bartenschlager and Schaller, 1992; Tu et al., 2017). Of note, albeit the reconstitution of hepatoma cells with the receptor human sodium taurocholate cotransporting polypeptide (NTCP), high excess of inoculated virions with high multiple genome equivalents (mges) (mges > 100) is required to achieve moderate infection rates (e.g., > 20% in HepG2NTCP cells) (Qu et al., 2018). This raises the problem that cccDNA-containing samples taken early after inoculation with HBV contain large amounts of rcDNA from input virions.

Therefore, adequate methodologies for absolute or relative quantification of cccDNA are required. They are divided into two categories: (I) hybridization after separation by gel electrophoresis (Southern blot) and (II) PCR amplification (Li et al., 2017). Southern blot is still a gold standard but not very sensitive, and demands multiple experimental processes and a high-copy load of cccDNA (> 2 x 106 copies using 32P-radioactive isotope/digoxigenin/biotin probe; > 1 x 104 copies using branched DNA technique) (Yu et al., 2015). Therefore, hybridization-based methods are complicated, time-consuming and not practical for a large number of samples (e.g., > 20) to be analyzed in parallel. PCR methods include real-time quantitative (q)PCR, nested qPCR (Xu et al., 2011), digital-droplet PCR (Mu et al., 2015), and rolling circle amplification (Margeridon et al., 2008). Real-time qPCR is the fastest and the most robust method for almost all laboratories. However, unlike Southern blot which gel electrophoretically separates cccDNA, PCR methods are not strictly specific, especially when rcDNA and other HBV replicative intermediates are present in excess, such as in in vitro infection (e.g., MGE > 300), even when cccDNA-specific primer pairs are used (Nassal, 2015; Qu et al., 2018).

To solve this problem, we have developed a qPCR assay using validated cccDNA selective primer pairs and a digestion step by T5 exonuclease, which removes cellular DNA and all HBV intermediates via its exonuclease activity targeting free ends of rcDNA and dslDNA but leaves cccDNA intact (Qu et al., 2018). This assay allowed fast and specific quantification of cccDNA within one working day as shown in Figure 1 (2 h of total DNA extraction; 1.5 h of T5 exonuclease reaction and 2 h of qPCR), accurate calculation of “cccDNA copies/(infected) cell” (Figure 2) and drug efficacy testing on cccDNA levels (Figure 3). The method also provided quantitative judgment on whether cccDNA is formed in new cell models and identified low amount of cccDNA in in vitro infection of non-primate hepatocytes (Lempp et al., 2016 and 2017). This protocol is adapted from Qu et al. (2018) and herein more detailed information on this qPCR quantification after T5 exonuclease digestion is included, and different primer pairs are compared to address the applicability of HBV genotypes. Taken together, this protocol will facilitate studies on cccDNA and help clinicians, technicians and graduate students to analyze cccDNA in samples derived from in vitro infection.

Materials and Reagents

  1. Pipette tips (Neptune, 1,000 μl, 200 μl, 20 μl, 10 μl, DNase-/RNase-free & Biozym, premium tips 1000 μl, 200 μl, 10 μl)
  2. 1.5 ml microcentrifuge tube (Sarstedt AG & Co.KG, SafeSeal tube, catalog number: 72.706)
  3. 0.2 ml microcentrifuge tube (Greiner Bio-one, Sapphire PCR tube, catalog number: 683271)
  4. Hard-Shell PCR plates 96-well, thin-wall (Bio-rad, catalog number: HSP9601)
  5. Primary human hepatocytes (PHH) (obtained from Hannover medical school or prepared in University Hospital Heidelberg) (Possible commercial vendors of PHH are Lonza, Biopredic, BioIVT, ThermoFisher, however susceptibility to HBV may vary), HepaRGNTCP and HepG2NTCP cell lines (Qu et al., 2018)
  6. Plasmid pSHH2.1 (Cattaneo et al., 1983), available upon request from the corresponding author
  7. Primers:
    Primer p1040: 5’-GTGGTTATCCTGCGTTGAT-3’
    Primer p1996: 5’-GAGCTGAGGCGGTATCT-3’
    p1578: 5’-CCGTGTGCACTTCGCTTCA-3’
    p1867: 5’-GCACAGCTTGGAGGCTTGA-3’
    p1583: 5’-TGCACTTCGCTTCACCT-3’
    p2301: 5’-AGGGGCATTTGGTGGTC-3’
  8. Probe
    Probe p1085: 5’-FAM-AGTTGGCGAGAAAGTGAAAGCCTGC-TAMRA-3’
  9. Trypsin [0.25% Trypsin-EDTA (1x)] (Invitrogen, Gibco, catalog number: 25200-056), 4 °C
  10. NucleoSpin Tissue kit (Macherey-Nagel, catalog number: 740952.250)
  11. Buffer T1 (Macherey-Nagel, catalog number: 740940.25)
  12. Proteinase K (Macherey-Nagel, catalog number: 740506), -20 °C
  13. Buffer B3 (Macherey-Nagel, catalog number: 740920)
  14. Ethanol absolute (VWR chemicals, catalog number: 20821.330)
  15. Buffer BW (Macherey-Nagel, catalog number: 740922)
  16. Buffer B5 Concentrate (Macherey-Nagel, catalog number: 740921)
  17. T5 exonuclease (New England Biolabs, catalog number: M0363), -20 °C
  18. NEBuffer 4 (New England Biolabs, catalog number: B7004), -20 °C in aliquots
  19. PerfeCTa qPCR Toughmix (Quanta Biosciences, catalog number: 95112-012), -20 °C in aliquots
  20. SYBR Green Supermix (Bio-Rad, catalog number: 172-5121), -20 °C in aliquots
  21. Myrcludex B (Bachem), available upon request from the corresponding author, -80 °C in aliquots
  22. Recombinant human Interferon-α-2a (PeproTech, catalog number: 300-02AA), -80 °C in aliquots
  23. Human Interferon-α-2a (PBL Assay Science, catalog number: 11100-1), -80 °C in aliquots
  24. Nuclease-free water (B. Braun Melsungen, Aqua ad iniectabilia Braun)
  25. NaCl (Carl Roth GmbH, catalog number: 9265.2)
  26. KCl (Sigma-Aldrich, catalog number: 31248)
  27. Na2HPO4·2H2O (Sigma-Aldrich, catalog number: 04272)
  28. KH2PO4 (Sigma-Aldrich, catalog number: P9791)
  29. Tris-base (Carl Roth GmbH, catalog number: 4855.2)
  30. Dimethyl sulfoxide (Sigma-Aldrich, catalog number: 1.02950)
  31. Fetal bovine serum gold (PAA Laboratories GmbH, catalog number: A15-151), -20 °C
  32. Penicillin/Streptomycin (Thermo Fisher Scientific, catalog number: 15140-122), 4 °C
  33. L-glutamine (Thermo Fisher Scientific, catalog number: 25030-024), 4 °C
  34. MEM non-essential amino acids (Thermo Fisher Scientific, catalog number: 11140-035), 4 °C
  35. Recombinant insulin (Sigma-Aldrich, catalog number: 91077C-1G), -20 °C in aliquots
  36. Hydrocortisone 21-hemisuccinate sodium salt (Sigma-Aldrich, catalog number: H4881), -20 °C in aliquots
  37. Culture medium (see Recipes)
  38. 10x PBS buffer (see Recipes)
  39. 5 mM Tris-HCl (see Recipes)

Equipment

  1. Pipettes (Gilson P1000, P200, P20; Eppendorf P2.5)
  2. Cell counter (Bio-Rad, TC20TM Automated Cell Counter, catalog number: 1450102)
  3. Thermomixer (Eppendorf, ThermoMixer C, catalog number: 5382000015)
  4. Microcentrifuge (Thermo Fisher Scientific, HeraeusTM PicoTM 21 Centrifuge, catalog number: 75002553)
  5. Thermocycler (Labrepco, Biometra T3000 Thermocycler 48, catalog number: 050-723)
  6. qPCR thermocycler (Bio-Rad, C1000 TouchTM Thermal cycler with 96-well Fast Reaction Module, catalog number: 1851196)

Software

  1. CFX96 Real-time System
    (Bio-Rad, http://www.bio-rad.com/en-us/product/cfx96-touch-real-time-pcr-detection-system)
  2. CFX ManagerTM Software
    (Bio-Rad, http://www.bio-rad.com/en-us/sku/1845000-cfx-manager-software?ID=1845000)

Procedure



Figure 1. Working flowchart of this protocol. Susceptible cells (PHH, HepaRGNTCP, HepG2NTCP) infected with high mges (> 300) of HBV are lysed. A. Lysates are incubated at 70 °C in the presence of proteinase K and later loaded on silica column and eluted. B. T5 exonuclease (5 units/1 h) removes host genomic DNA (black) and HBV replicative intermediates (rcDNA, dslDNA, etc.) (green) and preserves cccDNA (red) intact. C. cccDNA is amplified by p1040/p1996 and detected by probe p1085.


  1. HBV DNA extraction from in vitro infected hepatocytes (Figure 1A)
    1. For infection performed in a 24-well plate (culture area: 2 cm2), trypsinize and resuspend infected PHH, differentiated HepaRGNTCP or HepG2NTCP cells (infection procedure see Ni and Urban, 2017) in 1 ml of culture medium. Take 10 μl of the resuspension for cell count by a cell counter (see Equipment). Calculate total cell numbers.
      Notes:
      1. At confluency, cells in one well of a 24-well plate approximately correspond to 180,000 PHH, 200,000 HepaRGNTCP and 500,000 HepG2NTCP cells.
      2. When smaller or larger plates are used, volumes of the respective buffers given in the protocol should be adjusted according to the respective culture area (cm2) of a well.
    2. Spin down the cells (900 x g, 5 min) at room temperature. Wash cells with 1 ml of PBS twice. Spin down the cells and carefully aspirate PBS (Optional: freeze cell pellet at -20 °C for long-term storage). For DNA extraction, manufacturer’s manual of the NucleoSpin Tissue kit (740952.250) is adapted with a minor modification regarding the incubation time in Step A2 (1 h instead of 10~15 min) and the elution volume in Step A6 (50 μl instead of 100 μl). Add 200 μl of lysis buffer T1, pipet up and down and incubate at room temperature for 10 min. Add 25 μl of proteinase K and 200 μl of lysis buffer B3, vortex and incubate the lysate at 70 °C for 1 h with shaking (500 rpm).
      Note: After proteinase K digestion, it is not necessary to re-centrifuge the lysates. Immediately perform the next step.
    3. Add 210 μl of ethanol (96%-100%) to the sample and vortex vigorously for a few seconds. Remove buffer from the lid by a short centrifugation and apply the samples to the column. Centrifuge (11,000 x g, 1 min) at room temperature. Discard flow-through.
    4. Add 500 μl of wash buffer BW. Centrifuge (11,000 x g, 1 min) at room temperature. Discard flow-through.
    5. Add 600 μl of wash buffer B5 (pre-add indicated volume of ethanol to the Buffer B5 Concentrate) to the column. Centrifuge (11,000 x g, 1 min) at room temperature. Discard flow-through. Repeat this step once.
    6. Remove residual ethanol from the column (13,000 x g, 1 min). Place the column into a 1.5 ml microcentrifuge tube. Add 50 μl of pre-warmed (70 °C) elution buffer. Incubate at room temperature for 1 min. Centrifuge (11,000 x g, 1 min). Freeze eluted DNA samples at -20 °C until use or analyze them immediately.
      Notes:
      1. Elution buffers are nuclease-free water or 5 mM Tris-HCl (pH = 8.0). Do not use buffers with EDTA that may inhibit enzymatic activity in the next step.
      2. DNA concentration in eluates is 200-1,000 ng/μl.
      3. Preserve at least 10 μl of the sample without T5 exonuclease digestion for quantification of β-globin (internal standard) for normalization use.

  2. T5 exonuclease hydrolysis of total DNA elutes (Figure 1B)
    1. Assemble the reaction components in a 0.2 ml microcentrifuge tube following the table as shown:


    Note: Do not exceed unit and incubation time of T5 exonuclease since overdigestion leads to partial loss of cccDNA.

    1. Incubate the reaction at 37 °C for 1 h and inactivate the enzyme at 70 °C for 20 min in a thermocycler with heat-lid supply.
    2. Proceed quantification by real-time qPCR or freeze the products at -20 °C until use.
  3. Quantification of cccDNA relative to human β-globin (single-copy gene) by real-time qPCR (Figure 1C)
    1. Quantification of cccDNA
      1. Dilute primers and probe to 10 μM:

        FAM: 6-carboxyfluorescein; TAMRA: 6-carboxytetramethylrhodamine

      2. Prepare the following qPCR reactions in a 96-well Hard-Shell PCR plate:

        #for absolute quantification, load 2 µl of each 1:10 serial-diluted plasmid (pSHH2.1: 109, 108, 107, 106, 105, 104, 103, 102 copies/μl) as templates on the same plate.

      3. Run qPCR program below:


      4. Read absolute cccDNA copy numbers in the CFX ManagerTM Software. Calculate cccDNA copies per well.
    2. Quantification of human β-globin prior to T5 exonuclease digestion
      1. Dilute primers at 10 μM:


      2. Prepare qPCR reactions in a 96-well plate:


      3. Start qPCR program below:


      4. Read levels of human β-globin. Normalize cccDNA copies per well using values of human β-globin. Calculate cccDNA copies per cell (copies per well divided by cell numbers per well). For instance, if cell numbers in a well of 24-well plate are 4.2 x 105 (as determined in Procedure A1) and cccDNA copies per well are 8.52 x 105 (as determined in Procedure C), cccDNA copies per cell are 2.03.

Data analysis

cccDNA copy number per cell or infected cell is calculated according to Figure 2. The cccDNA level in in vitro infected cells can be determined similarly as two examples shown in Figure 3 and Qu et al., 2018. Note that to discriminate signals from the inoculum, we highly recommend using an entry inhibitor (e.g., Myrcludex B) as a control. Routinely Myrcludex B control gives a value below 0.05 copy per infected cell.


Figure 2. Schematic diagram of the calculation of absolute cccDNA copy numbers. A. On the day of experiment, cell number in the well is determined. B. Total DNA samples are extracted as suggested in Procedure. C. Here shows a proper T5 exonuclease digestion as shown in Procedure. D. Absolute cccDNA copies and E level of human β-globin are quantified, respectively as shown in Procedure. “cccDNA copies/cell” is “cccDNA copies/well” divided by “cell numbers”. Optional: F. If calculation of copies per infected cell is required, additional wells have to be arranged in parallel on the same plate during the infection. On the day of DNA extraction, cells in the wells are fixed and subjected to an immunofluorescence assay (HBcAg visualization using DAKO B0586 antibody, etc.) to determine infectivity (%: number of HBcAg positive cells divided by number of total cells) (Qu et al., 2018). “cccDNA copies/infected cell” is the “cccDNA copies/cell” divided by “infectivity (%)”.


Figure 3. cccDNA levels in three in vitro. models and upon IFN-α-2a and Myrcludex B treatment. A. PHH, differentiated HepaRGNTCP and HepG2NTCP cells were infected with HBV at mges/cell of 500. Myrcludex B (1 μM), an entry inhibitor blocking cccDNA formation, was co-administered with HBV inoculum during infection. On Day 7 post infection, cccDNA copies per infected cell were determined. Data shown in triangles were collected from three independent experiments. B. HepG2NTCP cells were infected with HBV at mges/cell of 500 with mock treatment (untreated), co-treated with Myrcludex B (1 μM) during infection, or co- and post-treated with IFN-α-2A at 100 ng/ml purchased from PeproTech and PBL Assay Science. On Day 7 post infection, cccDNA copies per infected cell were analyzed. Statistics: P < 0.01 untreated versus IFN-treated; P = 0.828 PeproTech versus PBL.

Notes

The primer pair (pp1040-1996) is specific for the formed cccDNA in genotype D. Genotype D is the HBV genome in HepAD38 cell line. Users should consider this issue if they try patient-derived serum to perform in vitro infection using this protocol since HBV in patients can be any genotype and pp1040-1996 does not bind the formed cccDNA of other genotypes.
   Genbank accession numbers of other genotypes: A (HE974370.1); B (AB540582.1); C (AB540584.1); E (HE974384.1); F (HE974369.1); G (AP007264.1); H (AB846650.1). pp1578-1867 and pp1583-2301 (Qu et al., 2018) allowing quantification of eight HBV genotypes (A-H) are suggested below. (Table 1)


Table 1. List of primer mismatches to HBV genotypes. The numbers of base-pair mismatch of each forward and reverse primers to eight HBV genotypes are summarized below. Green squares: perfect binding; yellow squares: one base-pair mismatch still allowing template binding; red squares: not binding.


F: forward primer; R: reverse primer. #: The probe of this protocol is not pan-genotypic and has mismatch number as shown: A(1), B(2), C(0), D(0), E(0), F(6), G(1), H(4).

Recipes

  1. Culture medium
    For HepG2NTCP cells: DMEM supplemented with 10% fetal calf serum (heat-inactivated), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 1% MEM non-essential amino acids. For infection, add 2% dimethyl sulfoxide
    For PHH and HepaRGNTCP cells: William’s medium E supplemented with 10% fetal calf serum (heat-inactivated), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 1% MEM non-essential amino acids, 5 μg/ml recombinant insulin, 50 μM hydrocortisone. For differentiation and infection, add 1.5% dimethyl sulfoxide
  2. 10x PBS
    Dissolve 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4·2H2O and 2 g of KH2PO4 in 800 ml of H2O, adjust pH to 7.4, refill H2O to 1 L
  3. Tris-HCl (1 M)
    Dissolve 121.14 g of Tris-base in 800 ml of H2O, adjust pH to 8.0 and refill H2O to 1 L

Acknowledgments

This protocol was adapted from Qu et al., 2018. We herein acknowledge financial supports by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)–Project number 272983813–SFB/TRR179 (B.Q. and S.U.) and German Center for Infection Research (DZIF) TTU Hepatitis projects 05.704 (S.U.). We thank Pascal Mutz (DKFZ, Germany) and Florian WR Vondran (MHH, Germany) for providing PHH. We are grateful to Xue Li for critical reading of this manuscript.

Competing interests

Prof. Dr. Stephan Urban, the corresponding author, holds patents and intellectual property on Myrcludex B.

Ethics

Following written informed consent of the patients, PHH were isolated from liver specimens obtained after partial hepatectomy.

References

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  2. Cattaneo, R., Will, H., Darai, G., Pfaff, E. and Schaller, H. (1983). Detection of an element of the SV40 late promoter in vectors used for expression studies in COS cells. Embo j 2(4): 511-514. 
  3. Guo, H., Jiang, D., Zhou, T., Cuconati, A., Block, T. M. and Guo, J. T. (2007). Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: an intermediate of covalently closed circular DNA formation. J Virol 81(22): 12472-12484. 
  4. Lempp, F. A., Qu, B., Wang, Y. X. and Urban, S. (2016). Hepatitis B virus infection of a mouse hepatic cell line reconstituted with human sodium taurocholate cotransporting polypeptide. J Virol 90(9): 4827-4831. 
  5. Lempp, F. A., Wiedtke, E., Qu, B., Roques, P., Chemin, I., Vondran, F. W. R., Le Grand, R., Grimm, D. and Urban, S. (2017). Sodium taurocholate cotransporting polypeptide is the limiting host factor of hepatitis B virus infection in macaque and pig hepatocytes. Hepatology 66(3): 703-716. 
  6. Levrero, M., Testoni, B. and Zoulim, F. (2016). HBV cure: why, how, when? Curr Opin Virol 18: 135-143. 
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简介

人乙型肝炎病毒(HBV)的持久性需要维持共价闭合环状(ccc)DNA,即感染肝细胞核中的游离基因组储库。 cccDNA消除是目前正在开发的未来治愈疗法的主要目标。在基于细胞培养的体外研究中,基于杂交和扩增的测定目前都用于cccDNA定量。目前的黄金标准Southern印迹耗时且对大量样品不实用。当存在过量的HBV复制中间体时,基于PCR的方法显示出有限的特异性。我们最近开发了一种实时定量PCR方案,其中通过硅胶柱提取总细胞DNA和所有形式的病毒DNA。随后用T5外切核酸酶孵育有效去除细胞DNA和所有非cccDNA形式的病毒DNA,同时cccDNA保持完整并且可通过PCR可靠地定量。该方法已被用于测量几种体外感染模型中cccDNA积累的动力学以及抗病毒剂对cccDNA的影响。它允许检测用人类牛磺胆酸钠协同转运多肽(NTCP)HBV受体重建的非人细胞(原始猕猴和猪肝细胞,等)中的cccDNA。在这里,我们提出了这种方法的详细协议,包括工作流程图,示意图和如何计算“每个(被感染的)细胞的cccDNA拷贝”的插图。
【背景】乙型肝炎病毒(HBV)是一种属于嗜肝DNA病毒科(Hepadnaviridae)家族的DNA病毒,是一种在全球约2.4亿人中持续存在的人类病原体。 HBV感染导致肝硬化和肝细胞癌的风险增加(Liang et al。,2015)。目前,慢性HBV感染是不可治愈的,因为目前的治疗方法不能根除复制性储库,共价闭合环状(ccc)DNA(Levrero et al。,2016)。在体外感染的肝细胞中,cccDNA是通过松弛环状双链(rc)DNA的细胞修复形成的,病毒体中的基因组形式(Guo et al。,2007; Long et al。,2017; Schreiner和Nassal,2017)。 cccDNA作为前基因组RNA的病毒模板,被转录成核衣壳,并通过病毒聚合酶逆转录生成rcDNA和双链线性(dsl)DNA(Bartenschlager和Schaller,1992; Tu 等。,2017)。值得注意的是,尽管用人类牛磺胆酸钠协同转运多肽(NTCP)重建肝癌细胞,但需要高过量的多重基因组当量(mges)(mges> 100)的接种病毒粒子才能达到中度感染率(例如,HepG2 NTCP 细胞中> 20%(Qu et al。,2018)。这引起了以下问题:在接种HBV后早期采集的含有cccDNA的样品含有来自输入病毒体的大量rcDNA。

因此,需要适当的cccDNA绝对或相对定量方法。它们分为两类:(I)通过凝胶电泳分离后的杂交(Southern印迹)和(II)PCR扩增(Li et al。,2017)。 Southern印迹仍然是金标准但不是非常敏感,并且需要多个实验过程和cccDNA的高拷贝负载(> 2 x 10 6 拷贝使用 32 P-放射性同位素/洋地黄毒苷/生物素探针;> 1×10 4 拷贝,使用分支DNA技术)(Yu et al。,2015)。因此,基于杂交的方法是复杂的,耗时的并且对于要并行分析的大量样本(例如,> 20)是不实际的。 PCR方法包括实时定量(q)PCR,嵌套qPCR(Xu et al。,2011),数字液滴PCR(Mu et al。,2015),和滚环扩增(Margeridon et al。,2008)。对于几乎所有实验室而言,实时qPCR是最快且最强大的方法。然而,与通过凝胶电泳分离cccDNA的Southern印迹不同,PCR方法并不严格特异,特别是当rcDNA和其他HBV复制中间体过量存在时,例如体外感染(例如,MGE> 300),即使使用cccDNA特异性引物对(Nassal,2015; Qu et al。,2018)。

为了解决这个问题,我们开发了qPCR分析,使用经验证的cccDNA选择性引物对和T5核酸外切酶消化步骤,通过其核酸外切酶活性去除细胞DNA和所有HBV中间体,靶向rcDNA和dslDNA的游离末端,但使cccDNA保持完整(Qu et al。,2018)。该测定允许在一个工作日内快速和特异性地定量cccDNA,如图1所示(总DNA提取2小时; T5核酸外切酶反应1.5小时和qPCR 2小时),准确计算“cccDNA拷贝/(感染)细胞” “(图2)和cccDNA水平的药物功效测试(图3)。该方法还提供了对新细胞模型中是否形成cccDNA的定量判断,并鉴定了非灵长类动物肝细胞体外感染中的低量cccDNA(Lempp et al。, 2016年和2017年)。该方案改编自Qu 等人(2018),并且在此包括在包括T5核酸外切酶消化后该qPCR定量的更详细信息,并且比较不同的引物对以解决HBV基因型的适用性。总之,该协议将促进对cccDNA的研究,并帮助临床医生,技术人员和研究生分析来自体外感染的样品中的cccDNA。

关键字:乙型肝炎病毒, 共价闭合环状DNA, cccDNA, T5外切核酸酶, 定量PCR, 单细胞拷贝数目

材料和试剂

  1. 移液器吸头(海王星,1,000μl,200μl,20μl,10μl,不含DNase- / RNase&amp; Biozym,优质吸头1000μl,200μl,10μl)
  2. 1.5 ml微量离心管(Sarstedt AG&amp; Co.KG,SafeSeal tube,目录号:72.706)
  3. 0.2 ml微量离心管(Greiner Bio-one,Sapphire PCR tube,目录号:683271)
  4. 硬壳PCR板96孔,薄壁(Bio-rad,目录号:HSP9601)
  5. 原代人肝细胞(PHH)(从汉诺威医学院获得或在海德堡大学医院制备)(PHH的可能商业供应商是Lonza,Biopredic,BioIVT,ThermoFisher,但对HBV的易感性可能不同),HepaRG NTCP 和HepG2 NTCP 细胞系(Qu et al。,2018)
  6. 质粒pSHH2.1(Cattaneo et al。,1983),可根据相应作者的要求提供
  7. 引物:
    引物p1040:5'-GTGGTTATCCTGCGTTGAT-3'
    引物p1996:5'-GAGCTGAGGCGGTATCT-3'
    p1578:5'-CCGTGTGCACTTCGCTTCA-3'
    p1867:5'-GCACAGCTTGGAGGCTTGA-3'
    p1583:5'-TGCACTTCGCTTCACCT-3'
    p2301:5'-AGGGGCATTTGGTGGTC-3'
  8. 探索
    探针p1085:5'-FAM-AGTTGGCGAGAAAGTGAAAGCCTGC-TAMRA-3'
  9. 胰蛋白酶[0.25%胰蛋白酶-EDTA(1x)](Invitrogen,Gibco,目录号:25200-056),4°C
  10. NucleoSpin Tissue kit(Macherey-Nagel,目录号:740952.250)
  11. Buffer T1(Macherey-Nagel,目录号:740940.25)
  12. 蛋白酶K(Macherey-Nagel,目录号:740506),-20℃
  13. Buffer B3(Macherey-Nagel,目录号:740920)
  14. 绝对乙醇(VWR化学品,目录号:20821.330)
  15. Buffer BW(Macherey-Nagel,目录号:740922)
  16. Buffer B5 Concentrate(Macherey-Nagel,目录号:740921)
  17. T5外切核酸酶(New England Biolabs,目录号:M0363),-20℃
  18. NEBuffer 4(New England Biolabs,目录号:B7004),-20℃等分试样
  19. PerfeCTa qPCR Toughmix(Quanta Biosciences,目录号:95112-012),-20°C等分试样
  20. SYBR Green Supermix(Bio-Rad,目录号:172-5121),-20℃等分试样
  21. Myrcludex B(Bachem),可根据相应作者的要求提供,-80°C等分试样
  22. 重组人干扰素-α-2a(PeproTech,目录号:300-02AA), - 80°C等分试样
  23. 人干扰素-α-2a(PBL分析科学,目录号:11100-1),等分试样为-80°C
  24. 无核酸酶水(B. Braun Melsungen,Aqua ad iniectabilia Braun)
  25. NaCl(Carl Roth GmbH,目录号:9265.2)
  26. KCl(Sigma-Aldrich,目录号:31248)
  27. Na 2 HPO 4 •2H 2 O(Sigma-Aldrich,目录号:04272)
  28. KH 2 PO 4 (Sigma-Aldrich,目录号:P9791)
  29. Tris-base(Carl Roth GmbH,目录号:4855.2)
  30. 二甲基亚砜(Sigma-Aldrich,目录号:1.02950)
  31. 胎牛血清金(PAA Laboratories GmbH,目录号:A15-151),-20℃
  32. 青霉素/链霉素(Thermo Fisher Scientific,目录号:15140-122),4℃
  33. L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030-024),4℃
  34. MEM非必需氨基酸(Thermo Fisher Scientific,目录号:11140-035),4℃
  35. 重组胰岛素(Sigma-Aldrich,目录号:91077C-1G),-20℃等分试样
  36. 氢化可的松21-半琥珀酸钠盐(Sigma-Aldrich,目录号:H4881),-20°C等分试样
  37. 培养基(见食谱)
  38. 10x PBS缓冲液(参见食谱)
  39. 5 mM Tris-HCl(见食谱)

设备

  1. 移液器(Gilson P1000,P200,P20; Eppendorf P2.5)
  2. 细胞计数器(Bio-Rad,TC20 TM 自动细胞计数器,目录号:1450102)
  3. Thermomixer(Eppendorf,ThermoMixer C,目录号:5382000015)
  4. 微量离心机(Thermo Fisher Scientific,Heraeus TM Pico TM 21 Centrifuge,目录号:75002553)
  5. 热循环仪(Labrepco,Biometra T3000热循环仪48,目录号:050-723)
  6. qPCR热循环仪(Bio-Rad,C1000 Touch TM 热循环仪,带96孔快速反应模块,目录号:1851196)

软件

  1. CFX96实时系统
    (Bio-Rad, http: //www.bio-rad.com/en-us/product/cfx96-touch-real-time-pcr-detection-system
  2. CFX经理 TM 软件
    (Bio-Rad, http:// www.bio-rad.com/en-us/sku/1845000-cfx-manager-software?ID=1845000

程序



图1.该方案的工作流程图感染高模型(> 300)的易感细胞(PHH,HepaRG NTCP ,HepG2 NTCP )裂解HBV。 A.裂解物在蛋白酶K存在下于70℃温育,然后加载到硅胶柱上并洗脱。 B.T5外切核酸酶(5单位/ 1小时)去除宿主基因组DNA(黑色)和HBV复制中间体(rcDNA,dslDNA,等。)(绿色)并保留完整的cccDNA(红色)。 C.通过p1040 / p1996扩增cccDNA,并通过探针p1085检测。

  1. 从体外感染的肝细胞中提取HBV DNA(图1A)
    1. 对于在24孔板(培养区域:2 cm 2 )中进行的感染,胰蛋白酶消化并重悬感染的PHH,分化的HepaRG NTCP 或HepG2 NTCP 细胞(感染程序见Ni和Urban,2017)在1ml培养基中。通过细胞计数器取10μl重悬浮液进行细胞计数(参见设备)。计算总细胞数。
      注意:
      1. 在汇合时,24孔板的一个孔中的细胞大致对应于180,000 PHH,200,000 HepaRG NTCP 和500,000 HepG2 NTCP 细胞。
      2. 当使用更小或更大的板时,应根据孔的相应培养面积(cm2)调整方案中给出的各缓冲液的体积。
    2. 在室温下旋转细胞(900 x g ,5分钟)。用1ml PBS洗涤细胞两次。旋转细胞并小心吸出PBS(可选:冷冻细胞沉淀在-20°C下长期储存)。对于DNA提取,NucleoSpin Tissue试剂盒(740952.250)的制造商手册适用于对步骤A2中的孵育时间(1小时而不是10~15分钟)和步骤A6中的洗脱体积(50μl而不是100)的微小修改。微升)。加入200μl裂解缓冲液T1,上下吸移,在室温下孵育10分钟。加入25μl蛋白酶K和200μl裂解缓冲液B3,涡旋并在70°C振荡(500 rpm)孵育裂解液1小时。
      注意:蛋白酶K消化后,没有必要重新离心裂解液。立即执行下一步。
    3. 向样品中加入210μl乙醇(96%-100%)并剧烈涡旋几秒钟。通过短暂离心从盖子中取出缓冲液并将样品应用于色谱柱。在室温下离心(11,000 x g ,1分钟)。丢弃流通。
    4. 加入500μl洗涤缓冲液BW。在室温下离心(11,000 x g ,1分钟)。丢弃流通。
    5. 向柱中加入600μl洗涤缓冲液B5(预先将指定体积的乙醇加入缓冲液B5浓缩物中)。在室温下离心(11,000 x g ,1分钟)。丢弃流通。重复此步骤一次。
    6. 从柱中除去残留的乙醇(13,000 x g ,1分钟)。将柱子放入1.5ml微量离心管中。加入50μl预热(70°C)洗脱缓冲液。在室温下孵育1分钟。离心(11,000 x g ,1分钟)。在-20°C冷冻洗脱的DNA样品直至使用或立即分析。
      注意:
      1. 洗脱缓冲液是不含核酸酶的水或5mM Tris-HCl(pH = 8.0)。不要在下一步使用可能抑制酶活性的EDTA缓冲液。
      2. 洗脱液中的DNA浓度为200-1,000 ng /μl。
      3. 保留至少10μl未经T5核酸外切酶消化的样品,用于定量β-珠蛋白(内标)用于标准化使用。

  2. T5核酸外切酶水解总DNA洗脱液(图1B)
    1. 将反应组分装配在0.2ml微量离心管中,如下图所示:


      注意:不要超过T5核酸外切酶的单位和孵育时间,因为过度消化会导致cccDNA部分丢失。
    2. 将反应在37°C孵育1小时,并在带有热盖供应的热循环仪中将酶在70°C下灭活20分钟。
    3. 通过实时qPCR进行定量或将产物冷冻至-20℃直至使用。

  3. 通过实时qPCR定量cccDNA相对于人β-珠蛋白(单拷贝基因)(图1C)
    1. cccDNA的定量分析
      1. 将引物和探针稀释至10μM:

        FAM:6-羧基荧光素; TAMRA:6-羧基四甲基罗丹明

      2. 在96孔硬壳PCR板中准备以下qPCR反应:

        #进行绝对定量,每个1:10连续稀释的质粒加载2μl(pSHH2.1:10 9 ,10 8 ,10 7 ,10 6 ,10 5 ,10 4 ,10 3 ,10 2 拷贝/μl)作为同一平板上的模板。

      3. 运行下面的qPCR程序:


      4. 读取CFX Manager TM 软件中的绝对cccDNA拷贝数。计算每孔的cccDNA拷贝数。
    2. 在T5核酸外切酶消化之前定量人β-珠蛋白
      1. 以10μM稀释引物:


      2. 在96孔板中制备qPCR反应:


      3. 启动以下qPCR程序:


      4. 读取人β-珠蛋白的水平。使用人β-珠蛋白的值使每孔的cccDNA拷贝标准化。计算每个细胞的cccDNA拷贝数(每孔拷贝数除以每孔的细胞数)。例如,如果24孔板孔中的细胞数为4.2×10 5 (如方法A1中所确定),则每孔的cccDNA拷贝数为8.52×10 5 (如方法C中所确定),每个细胞的cccDNA拷贝数是2.03。

数据分析

根据图2计算每个细胞或感染细胞的cccDNA拷贝数。体外感染细胞中的cccDNA水平可以类似于图3和Qu 等所示的两个实例来确定。请注意,为了区分来自接种物的信号,我们强烈建议使用进入抑制剂(例如,Myrcludex B)作为对照。常规Myrcludex B对照给出每个感染细胞低于0.05拷贝的值。


图2.绝对cccDNA拷贝数的计算示意图。 &nbsp; A 。在实验当天,确定孔中的细胞数。 B.按照程序中的建议提取总DNA样品。 C.这里显示了正确的T5核酸外切酶消化,如程序中所示。 D.分别如程序中所示定量绝对cccDNA拷贝和人β-珠蛋白的E水平。 “cccDNA拷贝/细胞”是“cccDNA拷贝/孔”除以“细胞数”。可选:F。如果需要计算每个感染细胞的拷贝数,则在感染期间必须在同一平板上平行排列另外的孔。在DNA提取当天,将孔中的细胞固定并进行免疫荧光测定(使用DAKO B0586抗体进行HBcAg可视化,等)以确定感染性(%:HBcAg阳性细胞数除以总细胞数)(Qu et al。,2018)。 “cccDNA拷贝/感染细胞”是“cccDNA拷贝/细胞”除以“感染性(%)”。


图3.三种体外中的cccDNA水平。模型和IFN-α-2a和Myrcludex B处理。 A.PHH,分化的HepaRG NTCP 和HepG2 NTCP 细胞用HBV感染m /细胞在感染期间,Myrcludex B(1μM)是阻断cccDNA形成的进入抑制剂,与HBV接种物共同施用。在感染后第7天,测定每个感染细胞的cccDNA拷贝。以三个独立实验收集以三角形显示的数据。 B.HPV2 NTCP 细胞用模拟处理(未处理)的mV /细胞500的HBV感染,在感染期间与Myrcludex B(1μM)共同处理,或共同和后处理购自PeproTech和PBL Assay Science的100ng / ml的IFN-α-2A。在感染后第7天,分析每个感染细胞的cccDNA拷贝。统计: P &lt;未处理0.01对IFN处理; P = 0.828 PeproTech与PBL。

笔记

引物对(pp1040-1996)对基因型D中形成的cccDNA具有特异性。基因型D是HepAD38细胞系中的HBV基因组。如果患者使用该方案尝试使用患者来源的血清进行体外感染,则应考虑此问题,因为患者中的HBV可以是任何基因型,并且pp1040-1996不结合其他基因型的形成的cccDNA。
其他基因型的Genbank登录号:A(HE974370.1); B(AB540582.1); C(AB540584.1); E(HE974384.1); F(HE974369.1); G(AP007264.1); H(AB846650.1)。 pp1578-1867和pp1583-2301(Qu et al。,2018)允许定量八种HBV基因型(A-H),如下所述。 (表格1)

表1. HBV基因型的引物错配列表。下面总结了每种正向和反向引物与8种HBV基因型的碱基对错配数。绿色方块:完美装订;黄色方块:一个碱基对错配仍然允许模板结合;红色方块:没有约束力。


F:正向引物; R:反向引物。 #:该协议的探针不是泛基因型,并且具有错配数,如下所示:A(1),B(2),C(0),D(0),E(0),F(6),G (1),H(4)。

食谱

  1. 文化媒体
    对于HepG2 NTCP 细胞:补充有10%胎牛血清(热灭活),100U / ml青霉素,100μg/ ml链霉素,2mM L-谷氨酰胺,1%MEM非必需的DMEM氨基酸。感染时,加入2%二甲基亚砜
    对于PHH和HepaRG NTCP 细胞:William's培养基E补充10%胎牛血清(热灭活),100 U / ml青霉素,100μg/ ml链霉素,2 mM L-谷氨酰胺,1% MEM非必需氨基酸,5μg/ ml重组胰岛素,50μM氢化可的松。为了分化和感染,加入1.5%二甲基亚砜
  2. 10x PBS
    溶解80克NaCl,2克KCl,11.5克Na 2 HPO 4 •2H 2 O和2克KH 2 PO 4 ,将pH调节至7.4,再填充H 2 O至1L
  3. Tris-HCl(1M)
    将121.14克Tris碱溶于800毫升H 2 O中,调节pH至8.0,再填充H 2 O至1L

致谢

该协议改编自Qu et al。,2018。我们在此承认Deutsche Forschungsgemeinschaft(DFG,德国研究基金会) - 项目编号272983813-SFB / TRR179(BQ和SU)和德国中心的财政支持感染研究(DZIF)TTU肝炎项目05.704(SU)。我们感谢Pascal Mutz(DKFZ,德国)和Florian WR Vondran(德国MHH)提供PHH。我们感谢薛莉对这份手稿的批判性阅读。

利益争夺

相应作者Stephan Urban教授拥有Myrcludex B的专利和知识产权。

伦理

根据患者的书面知情同意,从部分肝切除术后获得的肝脏标本中分离出PHH。

参考

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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Qu, B. and Urban, S. (2019). Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR. Bio-protocol 9(7): e3202. DOI: 10.21769/BioProtoc.3202.
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