Materials and Reagents

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 )
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)
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.
For cell culture medium
1. 1x phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010049 )
2. 0.25% Trypsin (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200072 )
3. DMEM (Thermo Fisher Scientific, Gibco^TM, catalog number: C11965500CP )
4. Fetal bovine serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 10099141 )
5. 100x Nonessential amino acids (Thermo Fisher Scientific, Gibco^TM, catalog number: 11140050 )
6. 100x Penicillin/streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
7. Complete DMEM (see Recipes)
For transcription in vitro
1. MEGAscript^® T7 Transcription Kit (Thermo Fisher Scientific, catalog number: AM1333 )
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)
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)
HCV-neutralizing antibody
1. Antibody AR3A
  Note: AR3A is kindly provided by professor M. Law.
For cell line selecting
1. Puromycin (Sigma-Aldrich, catalog number: P8833-25MG )
2. Blasticidin (Thermo Fisher Scientific, Gibco^TM, catalog number: R21001 )

Equipment

Electroporator (Bio-Rad Laboratories, model: Gene Pulser Xcell^TM )
Electroporation cuvette (Bio-Rad Laboratories, Gene Pulser cuvette, 0.4 cm)
Fluorescence microscope (Olympus, model: IX53 )

Software

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

Procedure

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.
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

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).
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).
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

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)
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.
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.

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