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Jan 2017

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HCV Reporter System (Viral Infection-Activated Split-Intein-Mediated Reporter System) for Testing Virus Cell-to-cell Transmission ex-vivo
用于测试病毒细胞间传递的HCV报告系统(病毒感染-激活的断裂内含肽介导的报告系统)   

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Abstract

Hepatitis C virus (HCV) spread involves two distinct entry pathways: cell-free transmission and cell-to-cell transmission. Cell-to-cell transmission is not only an efficient way for viruses to spread but also an effective method for escaping neutralizing antibodies. We adapted the viral infection-activated split-intein-mediated reporter system (VISI) and developed a straightforward model for Live-cell monitoring of HCV cell-to-cell transmission ex-vivo: co-culture of HCV infected donor cells (red signal) with uninfected recipient cells (green signal) and elimination of the cell-free transmission by adding potent neutralizing antibody AR3A in the supernatant. With this model, the efficiency of cell-to-cell transmission can be evaluated by counting the number of foci designated by the green signal of recipient cells.

Keywords: HCV (HCV), Reporter system (报告系统), Fluorescence signal (荧光信号), Virus spread (病毒传播), Cell-to-cell transmission (细胞间传递)

Background

Accumulating evidence support that viruses can use different routes of spread in infected tissues (Sattentau, 2008; Zhong et al., 2013). For HCV transmission, both cell-free transmission and cell-to-cell transmission can mediate virus transfer between hepatocytes. While cell-free transmission initiates HCV infection, cell-cell transmission is thought to transfer HCV to adjacent hepatocytes directly. It provides an excellent way to resist the neutralizing antibodies and contribute to the viral persistence (Brimacombe et al., 2011; Xiao et al., 2014). Previous articles also proved some host factors which contributed to cell-cell transmission, such as scavenger receptor BI (SR-BI), CD81, tight junction proteins claudin-1 (CLDN1), Occludin (OCLN), epidermal growth factor receptor (EGFR) (Witteveldt et al., 2009; Catanese et al., 2013; Zona et al., 2013). But the exact mechanisms of this process still need to explore. We optimized a viral infection-activated split-intein-mediated reporter system (VISI) for live-cell visualization of HCV infection (Zhao et al., 2017). Based on the study in Huh7.5.1 cell line using a technique of split GFP/RFP reconstitution by intein protein splicing (Figure 1A), it showed that VISI system is a very sensitive and low background system. With this system, we can clearly visualize the HCV infected cell by its nuclear fluorescence signal. In addition, combining VISI-GFP and VISI-mCherry cells, we can further monitor HCV cell-to-cell transmission in the presence of potent neutralizing antibody AR3A.

Materials and Reagents

  1. For cell culture materials
    1. Sterile 100 mm polystyrene Petri dish (Thermo Fisher Scientific, catalog number: 172931 )
    2. Sterile flat-bottom 96-well plate (Thermo Fisher Scientific, catalog number: 167008 )
    3. Sterile 60 mm polystyrene Petri dish (Thermo Fisher Scientific, catalog number: 150288 )
    4. Sterile 6-well plate (Thermo Fisher Scientific, catalog number: 140675 )
    5. Sterile 50 ml Conical Centrifuge Tube (Thermo Fisher Scientific, catalog number: 339652 )

  2. Plasmids and Cell lines
    1. Lentiviral vector:
      pWPI-blasticidin-NLS-GFPn-INTEINn (Genbank: KY067203)
      pWPI-puromycin-INTEINc-GFPc-NLS-IPS (Genbank: KY067204) (for construction of Huh7.5.1-VISI-GFP cell line)
      pWPI-blasticidin-NLS-mCherry(n)-INTEINn (Genbank: KY067205)
      pWPI-puromycin-INTEINc-mCherry(c)-NLS-IPS (Genbank: KY067206) (for construction of Huh7.5.1-VISI-mCherry cell line)
    2. Two auxiliary plasmids:
      psPAX2 (the HIV-1 packaging plasmid)
      pMD2.G (a vesicular stomatitis virus glycoprotein [SV-G] expression vector)
    Note: Lentiviral vector and two auxiliary plasmids were kindly provided by professor R. Bartenschlager.
    1. 293T cell (ATCC, catalog number: CRL-3216 )
    2. Huh7.5.1 cell (Human hepatocyte-derived cell line Huh7.5.1 is kindly provided by professor F. Chisari)

  3. HCV virus strains
    1. HCV JC1 (GenBank: JF343782.1)
    2. Subgenomic JFH1 (sgJFH1) (GenBank: AB114136.1)
    Note: They were kindly provided by professor T. Wakita, professor C. M. Rice, professor J. Bukh, and professor R. Bartenschlager.

  4. For cell culture medium
    1. 1x phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010049 )
    2. 0.25% Trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 25200072 )
    3. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: C11965500CP )
    4. Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 10099141 )
    5. 100x Nonessential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
    6. 100x Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    7. Complete DMEM (see Recipes)

  5. For transcription in vitro
    1. MEGAscript® T7 Transcription Kit (Thermo Fisher Scientific, catalog number: AM1333 )

  6. For electroporation buffer
    1. ATP (Thermo Fisher Scientific, catalog number: R0441 )
    2. L-Glutathione (Sigma-Aldrich, catalog number: V900456-25G )
    3. Potassium chloride (KCl) (Sinopharm Chemical Reagent, catalog number: 10016318 )
    4. Calcium chloride (CaCl2) (Shanghai Experiment Reagent, catalog number: 117600 )
    5. Dipotassium hydrogen phosphate (K2HPO4) (Shanghai Experiment Reagent, catalog number: 168120 )
    6. Potassium phosphate monobasic (KH2PO4) (Shanghai Experiment Reagent, catalog number: 175650 )
    7. HEPES (Sigma-Aldrich, catalog number: H4034-25G )
    8. EGTA (Sangon Biotech, catalog number: E0732-50G )
    9. Magnesium chloride (MgCl2) (Sinopharm Chemical Reagent, catalog number: 10012818 )
    10. Cytomix buffer (see Recipes)

  7. For Calcium Phosphate (CaPO4) transfection buffer
    1. HEPES (Sigma-Aldrich, catalog number: H4034-25G )
    2. Sodium chloride (NaCl) (Sinopharm Chemical Reagent, catalog number: 10019318 )
    3. Disodium hydrogen phosphate (Na2HPO4·12H2O) (Shanghai Experiment Reagent, catalog number: 174710 )
    4. Calcium chloride (CaCl2) (Shanghai Experiment Reagent, catalog number: 117600 )
    5. Calcium Phosphate (CaPO4) transfection buffer (see Recipes)

  8. HCV-neutralizing antibody
    1. Antibody AR3A
      Note: AR3A is kindly provided by professor M. Law.

  9. For cell line selecting
    1. Puromycin (Sigma-Aldrich, catalog number: P8833-25MG )
    2. Blasticidin (Thermo Fisher Scientific, GibcoTM, catalog number: R21001 )

Equipment

  1. Electroporator (Bio-Rad Laboratories, model: Gene Pulser XcellTM )
  2. Electroporation cuvette (Bio-Rad Laboratories, Gene Pulser cuvette, 0.4 cm)
  3. Fluorescence microscope (Olympus, model: IX53 )

Software

  1. GraphPad Prism (GraphPad Software, https://www.graphpad.com/)

Procedure

  1. Construction of HCV reporter system (VISI system) cell line
    The mechanism and construction of VISI system have been described in detail in our research paper (Zhao et al., 2017). The materials (plasmids and cell lines used in VISI system) are available upon request from our lab.
    1. Plasmid construction
      Technically, most reporters are applicable to VISI system, such as green fluorescent protein (GFP), red fluorescent protein (RFP), luciferases, antibiotic-resistant proteins. Here, we chose two different fluorescent proteins: green fluorescent protein (GFP, green signal) and mCherry (red signal), for HCV reporter system construction (VISI-GFP/VISI-mCherry). N/C-terminal sequences of VISI are inserted into lentiviral vectors pWPI-blasticidin or pWPI-puromycin, respectively.
      Note: These four DNA sequences have been uploaded (Genbank: KY067203 [N-terminal of VISI-GFP], KY067204 [C-terminal of VISI-GFP], KY067205 [N-terminal of VISI-mCherry], KY067206 [C-terminal of VISI-mCherry]).
    2. Generation of Huh7.5.1-VISI by lentiviral transduction
      1. Lentiviral production
        Day 1:
        Plate 1.2 x 106 293T cells in complete DMEM into a 6 cm dish.
        Day 2:
        Replace the medium with 4 ml fresh complete DMEM and warm up the solutions of calcium phosphate (CaPO4) transfection (2x HEPES solution, 2.5 M calcium chloride solution, sterile H2O). After 1-2 h, dilute 15 μg DNA (pWPI vector:psPAX2:pMD2.G = 6.4 μg:6.4 μg:2.1 μg) in a final volume of 270 μl sterile H2O, and add 30 μl calcium chloride solution (2.5 M). Then vortex the mixed liquid gently and add 2x HEPES solution (300 μl) drop by drop simultaneously. Finally transfer the mixture into a 6 cm dish by dropwise, gently shake to distribute the precipitate evenly.
        Day 3:
        Change the medium with 4 ml fresh complete DMEM at 16 h post calcium phosphate transfection.
        Day 4:
        Harvest the supernatant (lentivirus) at 48 h post calcium phosphate transfection, after filtration with 0.45 μm filter, store in aliquots at -80 °C.
      2. These lentiviral preparations are used to transduce the targeted cell line Huh7.5.1 of which 5 x 105 cells are seeded into a 6-well plate. Eight hours after transduction, discard the lentiviral preparation and replace with fresh complete medium. Two days later, resuspend the transduced cells and transfer them into a 10 cm dish. Then add antibiotics after cell adhesion.
      3. After selection by both puromycin (1 μg/ml) and blasticidin (4 μg/ml) for days, Huh7.5.1-VISI-GFP and Huh7.5.1-VISI-mCherry cell lines will be successfully obtained, respectively.

  2. Construction of cell model for testing cell-to-cell transmission
    We choose Huh7.5.1-VISI-mCherry as donor cells that were electroporated with purified HCV RNA. Huh7.5.1-VISI-GFP is used as recipient cells which could receive virus genome from donor cells.
    1. First, Huh7.5.1-VISI-mCherry is electroporated with in vitro transcripts of HCV (Jc1) or subgenomic JFH1 (sgJFH1) which in vitro transcripted from HCV infectious clone pFK-JC1 or subgenomic construct pFK-sgJFH1 (Long et al., 2011). These two HCV genomes could be the positive and negative control of this experiment.
      1. pFK-JC1 and pFK-sgJFH1 are linearized by restriction enzyme Mlu1 or Xba1 respectively.
      2. Assemble transcription reaction at room temperature (1 μg linearized template, 2 μl Enzyme Mix, 2 μl ATP solution, 2 μl CTP solution, 2 μl GTP solution, 2 μl UTP solution, 2 μl 10x Reaction buffer, Add proper amount of Nuclease-free water to 20 μl), Mix thoroughly and incubate for 3 h at 37 °C.
      3. Then add 1 μl DNase to digest the template and incubate for 15 min at 37 °C.
      4. Lithium Chloride (LiCl) precipitation can be used to purified transcriptional RNA. Add 30 μl LiCl precipitation solution and chill for 1 h at -20 °C, then centrifuge for 15 min at maximum speed to pellet the RNA.
      5. After wash twice with 70% ethanol, resuspend the RNA pellet with 20 μl RNase-free water. And store in 10 μg aliquots at -80 °C.
      6. Add 10 μg of in vitro transcripts (RNA) with the cell suspension–5 x 106 Huh7.5.1-VISI-mCherry cells are resuspended in 400 μl cytomix buffer (Recipe 2). Then the cells are transfected by electroporation at 960 mF and 270 V using Bio-Rad Gene Pulser system and a cuvette with a gap width of 0.4 cm (Bio-Rad). Immediately after electroporation, resuspend the cells in complete DMEM and seed as required.
    2. Electroporated Huh7.5.1-VISI-mCherry are individualized and mixed with Huh7.5.1-VISI-GFP cells at a ratio of 1:30.
      Note: For single well of 96-well plates, it needs 1,000 Huh7.5.1-VISI-mCherry cells plus 30,000 Huh7.5.1-VISI-GFP cells.
    3. Culture the cells in the presence of HCV-neutralizing antibody AR3A at 50 μg/ml to block cell-free transmission (Figure 1B). Replace antibody-containing medium every 24 h.
    4. After co-culture for 72-96 h, HCV cell-to-cell transmission efficiency can be evaluated by capturing the fluorescent images and examining the number of GFP or mCherry glowing cells.


      Figure 1. Schematic diagram of VISI system and HCV cell-to-cell transmission system. A. Fusion proteins of N-terminal and C-terminal halves of VISI are located in nucleus and mitochondria by different location signal (nuclear localization signal [NLS] or IPS), respectively; upon virus infection, HCV NS3-4A cleavage activates transportation of the C-terminal piece from mitochondria to the nucleus where intact GFP is reconstituted through split-intein splicing. B. Huh7.5.1-VISI-mCherry cells electroporated with the HCV genome are used as donor cells; and Huh7.5.1-VISI-GFP cells are used as recipient cells. Donor cells are co-cultured with recipient cells at a cell number ratio of 1:30. To block cell-free transmission, we added HCV-neutralizing antibody AR3A into the supernatant. After incubation for 96 h, images are taken under a fluorescence microscope (Figure 2A).

Data analysis

  1. For experimental design, there are three replicates for each experiment. For each replicate, we capture 10-15 fluorescent images so as to count a minimum of 40 virus foci (Figure 2A).
  2. For data analysis, to evaluate the efficiency of HCV cell-to-cell transmission, we randomly take the fluorescent images of virus foci that several red donor cells are surrounded by some green recipient cells or not (Figure 2A). The numbers of red donor cells and green recipient cells should be counted, respectively (Figure 2B).
  3. For statistical analysis, we use GraphPad Prism to analyze the difference between samples by using the two-tailed, unpaired Student’s t-test.


    Figure 2. Data processing and analysis of HCV cell-to-cell transmission system. A. HCV cell-to-cell transmission phenomena is observed by fluorescence microscopy after co-culture for 96 h. Donor cells Huh7.5.1-VISI-mCherry electroporated with the HCV genome JC1 or subgenomic JFH1 (sgJFH1), and Huh7.5.1-VISI-GFP cells are used as recipient cells. So red nuclear fluorescence represent HCV positive donor cells and green nuclear fluorescence represent recipient cells which infected with HCV in cell-to-cell transmission. B. Numbers of HCV-positive donor cells (Huh7.5.1-VISI-mCherry cells) or HCV-positive recipient cells (Huh7.5.1-VISI-GFP cells) per focus after co-culture for 96 h are counted. In the scatter plot, red circles represent the number of HCV-positive donor cells per focus; green squares represent the number of HCV-positive recipient cells per virus foci; horizontal lines represent the median for 40 randomly selected foci.

Recipes

  1. Complete DMEM
    DMEM medium mix with 10% FBS (440 ml DMEM + 50 ml FBS)
    1x Non-essential amino acids (5 ml)
    1x Penicillin/streptomycin (5 ml Penicillin/streptomycin)
  2. Cytomix buffer, pH 7.6 (electroporation buffer)

    Notes:
    1. The pH value of the mixture solution is adjusted to 7.6 with 3 M potassium hydrate (KOH). Filtrate through a 0.22 μm filter, and store in 10 ml aliquots at -20 °C.
    2. The above solutions are prepared with double distilled water unless otherwise indicated.
  3. Calcium Phosphate (CaPO4) transfection buffer
    1. 2.5 M CaCl2 (in ddH2O), filtrate through a 0.22 μm filter, and store in 10 ml aliquots at -20 °C
    2. 2x HEPES buffer, pH 7.1

      Note: The pH value of the mixture solution is adjusted to 7.1. Filtrate through a 0.22 μm filter, and store in 10 ml aliquots at -20 °C. For single calcium phosphate (CaPO4) transfection in a 6-cm dish, it needs 270 μl ddH2O (contain 15 μg DNA plasmids), 30 μl 2.5 M Cacl2 and 300 μl 2x HEPES buffer.

Acknowledgments

This protocol was adapted from procedures published in Zhao et al. (2017). We thank F. Chisari for kindly providing Huh7.5.1 cell line; M. Law for the gift of HCV-neutralizing antibody AR3A; and T. Wakita, C. M. Rice, J. Bukh, and R. Bartenschlager for providing HCV strains.
This work was supported by the National Key R&D program of China (2016YFC1200400), the “100 talents program” from the Chinese Academy of Sciences, and the National Science and Technology Major Project of the Ministry of Science and Technology of China (2014ZX10002002-001-004 and 2015CB554300). The authors declare that there are no conflicts of interest.

References

  1. Brimacombe, C. L., Grove, J., Meredith, L. W., Hu, K., Syder, A. J., Flores, M. V., Timpe, J. M., Krieger, S. E., Baumert, T. F., Tellinghuisen, T. L., Wong-Staal, F., Balfe, P. and McKeating, J. A. (2011). Neutralizing antibody-resistant hepatitis C virus cell-to-cell transmission. J Virol 85(1): 596-605.
  2. Catanese, M. T., Loureiro, J., Jones, C. T., Dorner, M., von Hahn, T. and Rice, C. M. (2013). Different requirements for scavenger receptor class B type I in hepatitis C virus cell-free versus cell-to-cell transmission. J Virol 87(15): 8282-8293.
  3. Long, G., Hiet, M. S., Windisch, M. P., Lee, J. Y., Lohmann, V. and Bartenschlager, R. (2011). Mouse hepatic cells support assembly of infectious hepatitis C virus particles. Gastroenterology 141(3): 1057-1066.
  4. Sattentau, Q. (2008). Avoiding the void: cell-to-cell spread of human viruses. Nat Rev Microbiol 6(11): 815-826.
  5. Witteveldt, J., Evans, M. J., Bitzegeio, J., Koutsoudakis, G., Owsianka, A. M., Angus, A. G., Keck, Z. Y., Foung, S. K., Pietschmann, T., Rice, C. M. and Patel, A. H. (2009). CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells. J Gen Virol 90: 48-58.
  6. Xiao, F., Fofana, I., Heydmann, L., Barth, H., Soulier, E., Habersetzer, F., Doffoel, M., Bukh, J., Patel, A. H., Zeisel, M. B. and Baumert, T. F. (2014). Hepatitis C virus cell-cell transmission and resistance to direct-acting antiviral agents. PLoS Pathog 10(5): e1004128.
  7. Zhao, F., Zhao, T., Deng, L., Lv, D., Zhang, X., Pan, X., Xu, J. and Long, G. (2017). Visualizing the essential role of complete virion assembly machinery in efficient hepatitis C virus cell-to-cell transmission by a viral infection-activated split-intein-mediated reporter system. J Virol 91(2): e01720-16.
  8. Zhong, P., Agosto, L. M., Munro, J. B. and Mothes, W. (2013). Cell-to-cell transmission of viruses. Curr Opin Virol 3(1): 44-50.
  9. Zona, L., Lupberger, J., Sidahmed-Adrar, N., Thumann, C., Harris, H. J., Barnes, A., Florentin, J., Tawar, R. G., Xiao, F., Turek, M., Durand, S. C., Duong, F. H., Heim, M. H., Cosset, F. L., Hirsch, I., Samuel, D., Brino, L., Zeisel, M. B., Le Naour, F., McKeating, J. A. and Baumert, T. F. (2013). HRas signal transduction promotes hepatitis C virus cell entry by triggering assembly of the host tetraspanin receptor complex. Cell Host Microbe 13(3): 302-313.

简介

丙型肝炎病毒(HCV)传播涉及两种不同的进入途径:无细胞传播和细胞间传播。 细胞间传播不仅是病毒传播的有效方式,也是逃避中和抗体的有效方法。 我们采用了病毒感染激活的分裂 - 内含肽介导的报告系统(VISI),并开发了一种直接模型,用于活细胞监测HCV细胞间传递离体:共培养 HCV感染的供体细胞(红色信号)与未感染的受体细胞(绿色信号)和通过在上清液中加入有效的中和抗体AR3A消除无细胞的传递。 利用该模型,可以通过计数受体细胞的绿色信号指定的病灶数来评估细胞间传递的效率。

【背景】越来越多的证据证明病毒可以在受感染的组织中使用不同的传播途径(Sattentau,2008; Zhong et al。,2013)。对于HCV传播,无细胞传播和细胞间传播均可介导肝细胞之间的病毒转移。虽然无细胞传播引发HCV感染,但认为细胞 - 细胞传递直接将HCV转移至相邻的肝细胞。它提供了抵抗中和抗体并有助于病毒持久性的极好方法(Brimacombe et al。,2011; Xiao et al。,2014)。之前的文章也证明了一些促进细胞传递的宿主因子,如清道夫受体BI(SR-BI),CD81,紧密连接蛋白claudin-1(CLDN1),Occludin(OCLN),表皮生长因子受体(EGFR)。 (Witteveldt et al。,2009; Catanese et al。,2013; Zona et al。,2013)。但是这个过程的确切机制仍然需要探索。我们优化了病毒感染激活的分裂 - 内含肽介导的报告系统(VISI),用于HCV感染的活细胞可视化(Zhao et al。,2017)。基于Huh7.5.1细胞系的研究,使用内切蛋白剪接分裂GFP / RFP重建技术(图1A),它表明VISI系统是一个非常敏感和低背景系统。通过该系统,我们可以通过其核荧光信号清楚地显现HCV感染的细胞。此外,结合VISI-GFP和VISI-mCherry细胞,我们可以在有效的中和抗体AR3A存在下进一步监测HCV细胞间传递。

关键字:HCV, 报告系统, 荧光信号, 病毒传播, 细胞间传递

材料和试剂

  1. 用于细胞培养材料
    1. 无菌100毫米聚苯乙烯培养皿(Thermo Fisher Scientific,目录号:172931)
    2. 无菌平底96孔板(Thermo Fisher Scientific,目录号:167008)
    3. 无菌60毫米聚苯乙烯培养皿(Thermo Fisher Scientific,目录号:150288)
    4. 无菌6孔板(Thermo Fisher Scientific,目录号:140675)
    5. 无菌50 ml锥形离心管(Thermo Fisher Scientific,目录号:339652)

  2. 质粒和细胞系
    1. 慢病毒载体:
      pWPI-blasticidin-NLS-GFPn-INTEINn(Genbank:KY067203)
      pWPI-嘌呤霉素-INTEINc-GFPc-NLS-IPS(Genbank:KY067204)(用于构建Huh7.5.1-VISI-GFP细胞系)
      pWPI-blasticidin-NLS-mCherry(n)-INTEINn(Genbank:KY067205)
      pWPI-嘌呤霉素-INTEINc-mCherry(c)-NLS-IPS(Genbank:KY067206)(用于构建Huh7.5.1-VISI-mCherry细胞系)
    2. 两种辅助质粒:
      psPAX2(HIV-1包装质粒)
      pMD2.G(水疱性口炎病毒糖蛋白[SV-G]表达载体)
    注:慢病毒载体和两种辅助质粒由R. Bartenschlager教授友情提供。
    1. 293T细胞(ATCC,目录号:CRL-3216)
    2. Huh7.5.1细胞(人肝细胞衍生细胞系Huh7.5.1由F. Chisari教授友情提供)

  3. HCV病毒株
    1. HCV JC1(GenBank:JF343782.1)
    2. 亚基因组JFH1(sgJFH1)(GenBank:AB114136.1)
    注:由T. Wakita教授,C. M. Rice教授,J. Bukh教授和R. Bartenschlager教授友情提供。

  4. 用于细胞培养基
    1. 1x磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10010049)
    2. 0.25%胰蛋白酶(Thermo Fisher Scientific,Gibco TM ,目录号:25200072)
    3. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:C11965500CP)
    4. 胎牛血清(Thermo Fisher Scientific,Gibco TM ,目录号:10099141)
    5. 100x非必需氨基酸(Thermo Fisher Scientific,Gibco TM ,目录号:11140050)
    6. 100x青霉素/链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
    7. 完整的DMEM(见食谱)

  5. 转录体外
    1. MEGAscript ® T7转录试剂盒(赛默飞世尔科技,目录号:AM1333)

  6. 用于电穿孔缓冲液
    1. ATP(赛默飞世尔科技,目录号:R0441)
    2. L-谷胱甘肽(Sigma-Aldrich,目录号:V900456-25G)
    3. 氯化钾(KCl)(国药化学试剂,目录号:10016318)
    4. 氯化钙(CaCl 2 )(上海实验试剂,目录号:117600)
    5. 磷酸氢二钾(K 2 HPO 4 )(上海实验试剂,目录号:168120)
    6. 磷酸二氢钾(KH 2 PO 4 )(上海实验试剂,目录号:175650)
    7. HEPES(Sigma-Aldrich,目录号:H4034-25G)
    8. EGTA(Sangon Biotech,目录号:E0732-50G)
    9. 氯化镁(MgCl 2 )(国药化学试剂,目录号:10012818)
    10. Cytomix缓冲液(见食谱)

  7. 对于磷酸钙(CaPO 4 )转染缓冲液
    1. HEPES(Sigma-Aldrich,目录号:H4034-25G)
    2. 氯化钠(NaCl)(国药化学试剂,目录号:10019318)
    3. 磷酸氢二钠(Na 2 HPO 4 ·12H 2 O)(上海实验试剂,目录号:174710)
    4. 氯化钙(CaCl 2 )(上海实验试剂,目录号:117600)
    5. 磷酸钙(CaPO 4 )转染缓冲液(见食谱)

  8. HCV中和抗体
    1. 抗体AR3A
      注意:AR3A由M. Law教授提供。

  9. 用于细胞系选择
    1. 嘌呤霉素(Sigma-Aldrich,目录号:P8833-25MG)
    2. 杀稻瘟素(Thermo Fisher Scientific,Gibco TM ,目录号:R21001)

设备

  1. Electroporator(Bio-Rad Laboratories,型号:Gene Pulser Xcell TM )
  2. 电穿孔比色皿(Bio-Rad Laboratories,Gene Pulser比色杯,0.4 cm)
  3. 荧光显微镜(奥林巴斯,型号:IX53)

软件

  1. GraphPad Prism(GraphPad软件, https://www.graphpad.com/ )

程序

  1. 构建HCV报告系统(VISI系统)细胞系
    VISI系统的机制和结构已在我们的研究论文中详细描述(Zhao et al。,2017)。我们的实验室可根据要求提供材料(VISI系统中使用的质粒和细胞系)。
    1. 质粒构建
      从技术上讲,大多数记者适用于VISI系统,如绿色荧光蛋白(GFP),红色荧光蛋白(RFP),萤光素酶,抗生素抗性蛋白。在这里,我们选择了两种不同的荧光蛋白:绿色荧光蛋白(GFP,绿色信号)和mCherry(红色信号),用于HCV报告系统构建(VISI-GFP / VISI-mCherry)。将VISI的N / C末端序列分别插入慢病毒载体pWPI-杀稻瘟素或pWPI-嘌呤霉素中。
      注意:上传了这四个DNA序列(Genbank:KY067203 [VISI-GFP的N-末端],KY067204 [VISI-GFP的C-末端],KY067205 [VISI-mCherry的N-末端],KY067206 [ VISI-mCherry的C-terminal])。
    2. 通过慢病毒转导产生Huh7.5.1-VISI
      1. 慢病毒生产
        第1天:
        将1.2×10 6个 293T细胞在完全DMEM中加入6cm培养皿中。
        第2天:
        用4ml新鲜完全DMEM替换培养基并加热磷酸钙(CaPO 4 )转染溶液(2x HEPES溶液,2.5M氯化钙溶液,无菌H 2 O)。 1-2小时后,稀释15μgDNA(pWPI载体:psPAX2:pMD2.G =6.4μg:6.4μg:2.1μg),终体积为270μl无菌H 2 O,并加入30μl氯化钙溶液(2.5M)。然后轻轻涡旋混合液体并同时逐滴加入2x HEPES溶液(300μl)。最后,将混合物逐滴转移到6厘米的培养皿中,轻轻摇动,使沉淀物均匀分布。
        第3天:
        在磷酸钙转染后16小时,用4ml新鲜的完全DMEM更换培养基。
        第4天:
        在磷酸钙转染后48小时收集上清液(慢病毒),用0.45μm过滤器过滤后,以等分试样储存在-80°C。
      2. 这些慢病毒制剂用于转导靶细胞系Huh7.5.1,其中将5×10 5个细胞接种到6孔板中。转导后8小时,丢弃慢病毒制剂并用新鲜的完全培养基替换。两天后,重悬转导细胞并将其转移到10cm培养皿中。然后在细胞粘附后加入抗生素。
      3. 在用嘌呤霉素(1μg/ ml)和杀稻瘟素(4μg/ ml)选择数天后,将分别成功获得Huh7.5.1-VISI-GFP和Huh7.5.1-VISI-mCherry细胞系。

  2. 用于测试细胞间传递的细胞模型的构建
    我们选择Huh7.5.1-VISI-mCherry作为用纯化的HCV RNA电穿孔的供体细胞。 Huh7.5.1-VISI-GFP用作可从供体细胞接收病毒基因组的受体细胞。
    1. 首先,Huh7.5.1-VISI-mCherry用HCV的体外转录物(Jc1)或亚基因组JFH1(sgJFH1)进行电穿孔,其体外转录自HCV感染性克隆pFK- JC1或亚基因组构建体pFK-sgJFH1(Long et al。,2011)。这两个HCV基因组可以是该实验的阳性和阴性对照。
      1. pFK-JC1和pFK-sgJFH1分别通过限制酶Mlu1或Xba1线性化。
      2. 在室温下组装转录反应(1μg线性化模板,2μl酶混合物,2μlATP溶液,2μlCTP溶液,2μlGTP溶液,2μlUTP溶液,2μl10x反应缓冲液,加入适量无核酸酶加水至20μl),充分混合并在37°C下孵育3小时。
      3. 然后加入1μlDNA酶消化模板,37°C孵育15分钟。
      4. 氯化锂(LiCl)沉淀可用于纯化的转录RNA。加入30μlLiCl沉淀溶液,在-20°C下冷却1小时,然后以最大速度离心15分钟以沉淀RNA。
      5. 用70%乙醇洗涤两次后,用20μl不含RNase的水重悬RNA沉淀。并在-80°C下以10μg等分试样储存。
      6. 添加10μg体外转录物(RNA)与细胞悬液-5 x 10 6 Huh7.5.1-VISI-mCherry细胞重悬浮于400μlcytomix缓冲液中(配方2)。然后使用Bio-Rad Gene Pulser系统和间隙宽度为0.4cm的比色皿(Bio-Rad)通过电穿孔在960mF和270V下转染细胞。电穿孔后立即将细胞重悬于完全DMEM中并根据需要接种。
    2. 将电穿孔的Huh7.5.1-VISI-mCherry个体化并与Huh7.5.1-VISI-GFP细胞以1:30的比例混合。
      注意:对于单孔96孔板,需要1,000个Huh7.5.1-VISI-mCherry细胞和30,000个Huh7.5.1-VISI-GFP细胞。
    3. 在HCV中和抗体AR3A存在下以50μg/ ml培养细胞以阻断无细胞传播(图1B)。每24小时更换含抗体的培养基。
    4. 共培养72-96小时后,可通过捕获荧光图像并检查GFP或mCherry发光细胞的数量来评估HCV细胞间传递效率。


      图1. VISI系统和HCV细胞间传播系统的示意图。 A. VISI的N端和C端半部的融合蛋白通过不同的位置信号位于细胞核和线粒体中(核定位信号[NLS]或IPS),分别;在病毒感染后,HCV NS3-4A切割激活C-末端片段从线粒体到细胞核的转运,其中完整的GFP通过分裂内含肽剪接重建。 B.用Huh7.5.1-VISI-mCherry用HCV基因组电穿孔的细胞用作供体细胞;和Huh7.5.1-VISI-GFP细胞用作受体细胞。供体细胞与受体细胞共培养,细胞数比为1:30。为了阻断无细胞传播,我们将HCV中和抗体AR3A添加到上清液中。孵育96小时后,在荧光显微镜下拍摄图像(图2A)。

数据分析

  1. 对于实验设计,每个实验有三个重复。对于每个重复,我们捕获10-15个荧光图像,以便计算至少40个病毒灶(图2A)。
  2. 对于数据分析,为了评估HCV细胞间传播的效率,我们随机拍摄病毒灶的荧光图像,其中几个红色供体细胞被一些绿色受体细胞包围或不被包围(图2A)。应分别计数红色供体细胞和绿色受体细胞的数量(图2B)。
  3. 对于统计分析,我们使用GraphPad Prism通过使用双尾,未配对的学生 t - 测试来分析样本之间的差异。


    图2. HCV细胞 - 细胞传递系统的数据处理和分析。 A.共培养96小时后,通过荧光显微镜观察HCV细胞 - 细胞传播现象。供体细胞Huh7.5.1-VISI-mCherry用HCV基因组JC1或亚基因组JFH1(sgJFH1)电穿孔,Huh7.5.1-VISI-GFP细胞用作受体细胞。因此,红色核荧光代表HCV阳性供体细胞,绿色核荧光代表在细胞间传递中感染HCV的受体细胞。 B.计数共培养96小时后每个焦点的HCV阳性供体细胞(Huh7.5.1-VISI-mCherry细胞)或HCV阳性受体细胞(Huh7.5.1-VISI-GFP细胞)的数目。在散点图中,红色圆圈表示每个焦点的HCV阳性供体细胞的数量;绿色方块表示每个病毒灶的HCV阳性受体细胞数;水平线代表40个随机选择的焦点的中位数。

食谱

  1. 完成DMEM
    DMEM培养基与10%FBS(440ml DMEM + 50ml FBS)混合 1x非必需氨基酸(5毫升)
    1x青霉素/链霉素(5 ml青霉素/链霉素)
  2. Cytomix缓冲液,pH 7.6(电穿孔缓冲液)

    注意:
    1. 用3M水合钾(KOH)将混合物溶液的pH值调节至7.6。通过0.22μm过滤器过滤,并在-20°C下以10 ml等分试样储存。
    2. 除非另有说明,否则上述溶液用双蒸水制备。
  3. 磷酸钙(CaPO 4 )转染缓冲液
    1. 2.5 M CaCl 2 (在ddH 2 O中),通过0.22μm过滤器过滤,并在-20°C下以10 ml等分试样储存
    2. 2x HEPES缓冲液,pH 7.1


      注意:将混合溶液的pH值调节至7.1。通过0.22μm过滤器过滤,并在-20°C下以10 ml等分试样储存。
      对于6 cm培养皿中的单磷酸钙(CaPO 4 )转染,需要270μlddH < sub> 2 O(含有15μgDNA质粒),30μl2.5M Cacl 2 和300μl2xHEPES缓冲液。

致谢

该方案改编自Zhao et al。(2017)中公布的程序。我们感谢F. Chisari提供Huh7.5.1细胞系; M.用于HCV中和抗体AR3A的礼物的法律;和T. Wakita,C。M. Rice,J。Bukh和R. Bartenschlager提供HCV菌株。
中国(2016YFC1200400)的d计划,“100个人才计划”由中国科学院和国家科学与中国的科技部科技重大专项(2014ZX10002002,这项工作是由国家重点的R&amp支持-001-004和2015CB554300)。作者声明没有利益冲突。

参考

  1. Brimacombe,CL,树丛,J.,梅雷迪思,LW,胡,K.,赛德,AJ,布鲁姆,MV,Timpe,JM,克里格,SE,BAUMERT,TF,Tellinghuisen,TL,皇斯塔尔,F.,Balfe ,P。和McKeating,JA(2011)。 中和抗体抗性丙型肝炎病毒细胞间传播。 J Virol 85(1):596-605。
  2. Catanese,M.T.,Loureiro,J.,Jones,C.T。,Dorner,M.,von Hahn,T。和Rice,C.M。(2013)。 对丙型肝炎病毒无细胞清除受体B类I的不同要求与细胞对细胞的关系细胞传播。 J Virol 87(15):8282-8293。
  3. Long,G.,Hiet,M。S.,Windisch,M。P.,Lee,J.Y.,Lohmann,V。和Bartenschlager,R。(2011)。 小鼠肝细胞支持传染性丙型肝炎病毒颗粒的组装。 Gastroenterology 141(3):1057-1066。
  4. Sattentau,Q。(2008)。 避免空白:人类病毒的细胞间传播。 Nat Rev Microbiol 6(11):815-826。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhao, F., Zhao, T., Deng, L., Lv, D., Zhang, X., Pan, X., Xu, J. and Long, G. (2018). HCV Reporter System (Viral Infection-Activated Split-Intein-Mediated Reporter System) for Testing Virus Cell-to-cell Transmission ex-vivo. Bio-protocol 8(15): e2949. DOI: 10.21769/BioProtoc.2949.
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