Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

Podsumowanie

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4α, RXRα and PPARα) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

Streszczenie

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T overexpressing human NTCP, HNF4α, RXRα and PPARα) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines. HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.  

Wprowadzenie

Hepatitis B affects the lives of more than 2 billion people and is one of the major threats to public health. Approximately 257 million people are chronically infected with hepatitis B virus (HBV) worldwide, causing a big burden to the society¹. Hepatocytes are not the only cells infected by HBV and other cells in non-hepatic tissue, such as kidney and testis, are also infected by this virus^2,3. Currently, HBV infection cell models are limited to human hepatocytes with only few non-hepatic cell line models. This hampers the study of HBV infection and HBV-related pathology of non-hepatic tissues. Here we present protocols to study HBV infection in non-hepatic 293T cells as well as in hepatoma-based cells.

Sodium taurocholate co-transporting polypeptide (NTCP) is a functional receptor for human hepatitis B and hepatitis D virus⁴. Hepatocyte nuclear factor 4α (HNF4α), retinoid X receptor α (RXRα) and peroxisome proliferator-activated receptor α (PPARα) are liver-enriched transcription factors restricting viral tropism of HBV. They have been verified to promote HBV pregenomic RNA synthesis and support HBV replication in a nonhepatoma cell line⁵. We constructed three different cell lines, HepG2 cell lines expressing NTCP (HepG2-NE), 293T cell line expressing NTCP (293T-NE) and 293T cell line expressing four host genes; NTCP, HNF4α, RXRα and PPARα (293T-NE-3NRs). Two methods for HBV infection were developed based on 293T-NE-3NRs (Figure 1). The first method uses inoculation in 293T-NE-3NRs with high viral genome equivalence (high GEq (150), DMSO and PEG8000) for 24 h. The second method employs co-culturing 293T-NE-3NRs with HepG2.2.15, which can produce HBV particles (low GEq (about 1.83) without DMSO and PEG8000), thus closely emulating natural conditions.

HepG2.2.15 cells are derived from the HepG2 line and chronically secrete infectious HBV, as well as subviral particles into the culture medium^6,7. Cyclosporin A (CsA) is an immunosuppressant clinically used for the suppression of xenograft rejection. Studies have shown that CsA inhibits HBV entry into cultured hepatocytes by inhibiting the transporter activity of NTCP and blocking the binding of NTCP to large envelope proteins⁸.

HepG2-NE cells were used as positive control whereas CsA treated cells were used as negative control. By comparing with the positive and negative control groups, we can find out which host genes play a critical role in HBV infection. Additionally, through this mode of HBV infection, we can also find other novel mechanisms or host genes involved in HBV infection.

Protokół

Culture, collection of supernatants from HepG2.2.15 cells and HBV infection should be performed in biosafety level II (P2) or biosafety level III (P3) laboratory according to the biosafety guidance in the country. Laboratory safety practices should be followed to ensure the safety of laboratory personnel, and all should be vaccinated and detected HBsAb positive before performing HBV experiments. Observe the state of the cells at every step before proceeding to the next step. Human serum samples were used in accordance with the approval obtained by the Institutional Review Board of Shantou University Medical College.

1. HepG2.2.15 cell culture

NOTE: HepG2.2.15 cell supernatant, HepG2.2.15 cell supernatant concentrate and all the tips, flasks, plates, and tubes that come in contact with HepG2.2.15 should be disposed after being soaked in 2% virucide overnight.

1. Remove the vial containing HepG2.2.15 cells from liquid nitrogen and thaw by gently swirling the vial in a 37 °C water bath.
   NOTE: To reduce the possibility of contamination, keep the cap out of the water. Thawing should be rapid (approximately 2 min).
2. Remove the vial from the water bath as soon as the cells are thawed and disinfect it with 70% ethanol.
   NOTE: From this point perform all steps under strict aseptic conditions.
3. Transfer the cells to a 25 cm² tissue culture flask and add 4 mL of complete culture medium (DMEM containing 10% FBS, penicillin 100 U/mL, streptomycin 0.1 mg/mL). Incubate the cells at 37 °C in a humidified incubator with 5% CO₂.

2. Collection of HepG2.2.15 cell culture supernatant

1. Culture HepG2.2.15 cell in T25 flasks at 37 °C, 5% CO₂ in a humidified incubator_. Change the medium every 3 days.
2. Collect the cell supernatant in a 50 mL centrifuge tube. Close the lid firmly and wrap it with a paraffin film.
3. Store the HepG2.2.15 supernatant at -20 °C to keep HBV virus active.

3. Concentrating HepG2.2.15 supernatant

1. Remove HepG2.2.15 supernatant from -20 °C and thaw at 4 °C.
2. To remove the cell fragments, filter the HepG2.2.15 supernatant with a 0.45 µm membrane to a new 50 mL centrifuge tube. First, block the filter by filtering 10 mL of DMEM with 10% FBS before filtering the virus, then filter the cell culture supernatant.
3. Add 14 mL of filtered supernatant to a virus concentrator column and close the lid. Centrifuge the column at 3,200 x g for 35 min in a horizontal spinning centrifuge. Collect the HepG2.2.15 supernatant concentrate (about 200 µL) into a 1.5 mL tube.
4. Wash the column with DMEM once by adding 200 µL of DMEM and collect it into a 1.5 mL tube.

4. Detection of HBV DNA in supernatant concentrate

1. Turn on the dry block heater and set the temperature to 100 °C.
2. To ensure that the concentration of HBV DNA in the HepG2.2.15 supernatant concentrate is within the standard curve range, dilute the concentrate 20-fold by adding 10 µL of HepG2.2.15 supernatant concentrate to 190 µL of DMEM in a 1.5 mL microcentrifuge tube. Add 200 µL of HepG2.2.15 supernatant, negative control (HBV-negative serum from healthy donor), HBV positive control (HBV-positive serum from HBV patient) and four dilutions of the quantitative references provided in the kit (2.0 x 10⁶, 2.0 x 10⁵, 2.0 x 10⁴, 2.0 x 10³ GEq /mL), respectively to separate 1.5 mL microcentrifuge tubes.
3. Add 450 µL of HBV DNA extraction buffer from the kit to all the above eight 1.5 mL tubes, vortex for 15 s and spin down for 10 s. Incubate at 100 °C for 10 min in dry block heater. Centrifuge at 12, 000 x g for 5 min at room temperature.
4. Add 27 µL of PCR master mix and 3 µL of Taq polymerase enzyme to the PCR tubes placed on ice.
   NOTE: PCR master mix provided in the kit contain the primers against the conserved region of HBV DNA.
5. Add 20 µL of centrifuged supernatant liquid to the PCR tubes placed on ice. Cover tightly and spin down for 10 s.
6. Use the following conditions on a real-time PCR machine: 93 °C for 2 min, 10 cycles of 93 °C for 45 s followed by 55 °C for 60 s; then 30 cycles of 93 °C for 30 s followed by 55 °C for 45 s to carry out real-time PCR.
   NOTE: The master mix provided in the commercial kit has primers against the conserved region of HBV DNA.
7. Export the Ct values obtained and generate a standard curve as shown in Table 1 and Figure 2.
8. Based on the standard curve, calculate the HBV DNA concentration of HepG2.2.15 supernatant concentrate diluted 20x as shown in Table 2. Calculate the HBV DNA concentration of HepG2.2.15 supernatant concentrate (Table 2).
9. Divide the HepG2.2.15 supernatant concentrate in aliquots and store at -80 °C until use.

5. In vitro infection of HepG2-NE and 293T-NE-3NRs cells

1. Prepare the following infection medium in DMEM/F-12: 10% FBS, 4 mM glutamine supplement, 0.1 mM NEAA, 1 mM Sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 μg/mL puromycin, 40 ng/mL dexamethasone, 20 μg/mL hydrocortisone and 5 μg/mL insulin. Store at 4 °C.
   NOTE: These NTCP positive cells are puromycin-resistant⁹.
2. Add 0.1% gelatin to 5 wells of 48 well-plate, 200 μL to each well. Incubate the plate at 37 °C for 2 h, discard the supernatant. Incubate the plate at 37 °C for another 2 h.
3. Seed HepG2-NE at a density of 1 x 10⁴ cells in 2 wells each. Seed 293T-NE-3NRs cells at a density of 1 x 10⁴ cells in 2 wells each (all-trans retinoic acid at 1 μmol/L and clofibric acid at 1 mmol/L final concentrations were added to the medium to activate the RXRα and PPARα nuclear hormone receptors, respectively). Seed 293T-NE in 1 well at a density of 1 x 10⁴ cells.
   NOTE: Passage the cells at sub-confluence using 0.25% trypsin. Count the cell number and then seed the cells to an appropriate density.
4. Incubate the cell at 37 °C, 5% CO₂ in a humidified incubator for 24 h. Observe the cells after 24 h and proceed with infection if the cells are healthy.
5. Prepare the infection complex as mentioned in Table 3, add HBV (HepG2.2.15 supernatant concentrate), DMSO, PEG8000 and infection medium to 1.5 mL microcentrifuge tube. Prepare CsA stock by dissolving it in DMSO to a concentration of 5 mM and keep the stock at -20 ºC. The working solution of CsA is 5 μM. Seed 1×10⁴ cells per well of the 48-well plate. Add 100 μL of HepG2.2.15 supernatant concentrate (containing HBV 1.5063 ×10⁷ GEq/mL) to infect cells at 150 GEq/cell.
6. Add 500 μL of the infection complex to each well and incubate the cells at 37 °C, 5% CO₂ in humidified incubator for 24 h.
7. Wash the cells twice with 500 μL of PBS before changing the medium. Add 1 mL of the medium in each well. If the cell state is poor and some cells are floating, change the medium directly. This is day 1 of infection. Change the medium gently every 2 days.
8. On day 11, wash the cells twice with 500 μL of PBS and fix the cells with ice cold methanol for immunofluorescence.

6. HepG2.2.15 co-culture with HepG2-NE / 293T-NE-3NRs / 293T-NE

1. Add 1 mL/well of 0.1% gelatin to 5 wells of 6 well-plate. Incubate the plate at 37 °C for 2 h and discard the supernatant. Incubate the plate again for another 2 h at 37 °C to completely dry the well.
2. Seed 1 x 10⁵ cells/well of HepG-NE into 2 wells, 293T-NE-3NRs into 2 wells, and 293T-NE into 1 well of the plate. Seed 1 x 10⁵ cells/well of HepG2.2.15 onto five membrane inserts (24 mm, 0.4 μm pore diameter, uncoated) in a separate 6 well plate. Incubate the cells at 37 °C, 5% CO₂ in a humidified incubator for 24 h.
3. After 24 h, if the cells are all adherent and in good state, prepare for co-culture. Discard the medium in the 6-well-plate and cell membrane inserts. Place the membrane inserts with HepG2.2.15 into the 6 well plate seeded with HepG-NE, 293T-NE-3NRs and 293T-NE by tweezers. Incubate the cell at 37 °C, 5% CO₂ in a humidified incubator, change the medium every 3-4 days.
   NOTE: Set groups as following: Well 1: 293T-NE co-culture with HepG 2.2.15; Well 2: HepG2-NE co-culture with HepG 2.2.15; Well 3: 293T-NE-3NRs co-culture with HepG2.2.15; Well 4: HepG2-NE co-culture with HepG 2.2.15 (add CsA); Well 5: 293T-NE-3NRs co-culture with HepG2.2.15 (add CsA); Add the complete medium to well number 1, 2 and 3, add the medium with 5 µM CsA to well number 4 and 5 well, about 9 mL for each well, make sure the media are in contact in order for virus particles to migrate from transwells to infect cells.
4. After 10 days, remove the membrane inserts, add PBS to the 6-well-plate and wash gently twice, fix the cells with ice cold methanol for immunofluorescence.

7. Perform immunofluorescence for HBcAg

1. Fix the cells with ice cold methanol at -20 °C for more than 3 h. Discard the methanol. Wash the cells with PBS for 10 min at room temperature on shaker, repeat for 3 times.
2. Add 5% BSA (100 μL/48-well-plate, 500 μL/6-well-plate), shake on horizontal shaker for 1 h at 40 rpm and room temperature.
3. Add the HBcAg primary antibody solution (primary antibody: 5% BSA solution =1:200, 100 μL / 48-well-plate, 500 μL / 6-well-plate), shake overnight at 4 °C.
4. Wash wells with PBST (PBS with 0.1% tween 20), for 10 min at room temperature on shaker, repeat for 3 times.
5. Add the second antibody solution (594 labeled antibody raised in goat against rabbit IgG: PBS = 1:1,000, 100 μL /48-well-plate, 500 μL / 6-well-plate), cover the plate with foil and shake for 2 h at room temperature.
6. Wash again with PBST for three times, shake for 10 min each.
7. Add 1 μg/mL DAPI, shake for 2 min. Wash twice with PBST on a shaker for 5 min each. Discard PBST. Add PBS and observe under immunofluorescence microscope.

Wyniki

We constructed pSIN-NTCP-EGFP plasmid expressing NTCP and EGFP fusion and with puromycin resistance. The plasmid was transfected into HepG2 and 293T cells to construct stable cell lines HepG2-NE and 293T-NE expressing NTCP and EGFP. Plasmids (pSIN-HNF4α, pSIN-RXRα, pLV-PPARα-puro-flag) with puromycin resistance and expression were transfected into 293T-NE cells to construct a stable cell line expressing 4 host genes⁹. The expression of NTCP-EGFP can be observed by green fluorescence...

Dyskusje

Here, we introduce protocols for HBV infection in non-hepatic 293T-NE-3NRs and hepatoma-based HepG2-NE cells. 293T-NE-3NRs were suitable for HBV infection at both high and low GEq. The following critical steps need to be taken into consideration while using our protocol. The cell status is an important factor for a successful infection. The infection medium must be changed timely after the initial period of HBV infection. 293T-NE-3NRs cells are typically fragile following infection with high viral titers. Therefore, thes...

Materiały

Name	Company	Catalog Number	Comments
0.45μm membrane filter	Millex-HV	SLHU033RB	Filter for HepG 2.2.15 supernatant
293T-NE	Laboratory construction	——	Cell model for HBV infection
293T-NE-3NRs	Laboratory construction	——	Cell model for HBV infection
594 labeled goat against rabbit IgG	ZSGB-BIO	ZF-0516	For immunofluorescence assay,second antibody
6-well plate	BIOFIL	TCP010006	For co-culture
Amicon Ultra 15 ml	Millipore	UFC910008	For concentration of HepG 2.2.15 supernatant
BSA	Beyotime	ST023	For immunofluorescence assay
Cyclosporin A	Sangon biotech	59865-13-3	inhibitor of HBV infection
DAPI	Beyotime	C1006	For nuclear staining
Diagnostic kit for Quantification of Hepatitis B Virus DNA(PCR-Fluorescence Probing)	DAAN GENE	7265-2013	For HBV DNA detection
DMEM	HyClong	SH30243.01	For culture medium
DMSO	Sigma-Aldrich	D5879	For improvement of infection efficiency
Fetal bovine serum (FBS)	CLARK Bioscience	FB25015	For culture medium
Fluorescence microscope	ZEISS	Axio observer Z1	For immunofluorescence assay
HepG2-NE	Laboratory construction	——	Cell model for HBV infection
HBcAg antibody	ZSGB-BIO	ZA-0121	For immunofluorescence assay, primary antibody
PBS	ZSGB-BIO	ZLI-9062	For cell wash
PEG8000	Merck	P8260	For infection medium
Penicillin-Streptomycin-Glutamine	Thermo Fisher	10378016	For culture medium
Transwell	CORNING	3412	For co-culture
Tween 20	sigma-Aldrich	WXBB7485V	For PBST
Virkon	Douban	6971728840012	Viruside