Name: modeling hepatitis b virus infection in non-hepatic 293t-ne-3nrs cells
Uploaded: 2020-06-05T14:30:00
Description: Watch this Scientific Journal Video about Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells at JoVE.com

This work was supported by the National Natural Science Foundation of China (No. 81870432 and 81570567 to X.L.Z.), (No. 81571994 to P.N.S.), Research Fund for International Young Scientists (No. 81950410640 to W.I.); The Li Ka Shing Shantou University Foundation (No. L1111 2008 to P.N.S.). We would like to thank Prof. Stanley Lin from Shantou University Medical College for useful advice.

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 wi...

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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...

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 virus2,3. 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 4&#945; (HNF4&#945;), retinoid X receptor &#945; (RXR&#945;) and peroxisome proliferator-activated receptor &#945; (PPAR&#945;) 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, HNF4&#945;, RXR&#945; and PPAR&#945; (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 medium6,7. 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.

<table>
	<tbody>
		<tr>
			<td>0.45&#956;m membrane filter</td>
			<td>Millex-HV</td>
			<td>SLHU033RB</td>
			<td>Filter for HepG 2.2.15 supernatant</td>
		</tr>
		<tr>
			<td>293T-NE</td>
			<td>Laboratory construction</td>
			<td>&#8212;&#8212;</td>
			<td>Cell model for HBV infection</td>
		</tr>
		<tr>
			<td>293T-NE-3NRs</td>
			<td>Laboratory construction</td>
			<td>&#8212;&#8212;</td>
			<td>Cell model for HBV infection</td>
		</tr>
		<tr>
			<td>594 labeled goat against rabbit IgG</td>
			<td>ZSGB-BIO</td>
			<td>ZF-0516</td>
			<td>For immunofluorescence assay,second antibody</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>BIOFIL</td>
			<td>TCP010006</td>
			<td>For co-culture</td>
		</tr>
		<tr>
			<td>Amicon Ultra 15 ml</td>
			<td>Millipore</td>
			<td>UFC910008</td>
			<td>For concentration of HepG 2.2.15 supernatant</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Beyotime</td>
			<td>ST023</td>
			<td>For immunofluorescence assay</td>
		</tr>
		<tr>
			<td>Cyclosporin A</td>
			<td>Sangon biotech</td>
			<td>59865-13-3</td>
			<td>inhibitor of HBV infection</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime</td>
			<td>C1006</td>
			<td>For nuclear staining</td>
		</tr>
		<tr>
			<td>Diagnostic kit for Quantification of Hepatitis B Virus DNA(PCR-Fluorescence Probing)</td>
			<td>DAAN GENE</td>
			<td>7265-2013</td>
			<td>For HBV DNA detection</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClong</td>
			<td>SH30243.01</td>
			<td>For culture medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>For improvement of infection efficiency</td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#65288;FBS&#65289;</td>
			<td>CLARK Bioscience</td>
			<td>FB25015</td>
			<td>For culture medium</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>ZEISS</td>
			<td>Axio observer Z1</td>
			<td>For immunofluorescence assay</td>
		</tr>
		<tr>
			<td>HepG2-NE</td>
			<td>Laboratory construction</td>
			<td>&#8212;&#8212;</td>
			<td>Cell model for HBV infection</td>
		</tr>
		<tr>
			<td>HBcAg antibody</td>
			<td>ZSGB-BIO</td>
			<td>ZA-0121</td>
			<td>For immunofluorescence assay, primary antibody</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9062</td>
			<td>For cell wash</td>
		</tr>
		<tr>
			<td>PEG8000</td>
			<td>Merck</td>
			<td>P8260</td>
			<td>For infection medium</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>10378016</td>
			<td>For culture medium</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>CORNING</td>
			<td>3412</td>
			<td>For co-culture</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>sigma-Aldrich</td>
			<td>WXBB7485V</td>
			<td>For PBST</td>
		</tr>
		<tr>
			<td>Virkon</td>
			<td>Douban</td>
			<td>6971728840012</td>
			<td>Viruside</td>
		</tr>
	</tbody>
</table>

modeling hepatitis b virus infection in non-hepatic 293t-ne-3nrs cells

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&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;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.&#160;&#160;

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-HNF4&#945;, pSIN-RXR&#945;, pLV-PPAR&#945;-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...

Watch this Scientific Journal Video about Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells at JoVE.com

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

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

HBV viral replication in extrahepatic plays an important role in the pathogenesis of extrahepatic syndromes. Currently, cell culture models for starting extrahepatic HBV infection are limited. We present an in vitro non-hepatic infection model to help identify novel host factors which affect HBV replication and investigate HBV-related kidney diseases.Compared to traditional HBV infection models, we adopted and engineered 293T cell line, 293T-NE-3NR, and co-cultured it with HepG2.2.15. HBV infection should be performed in a biosafety level two or biosafety level three laboratory. Laboratory safety practices should be followed to ensure the safety of laboratory personnel and all researchers should be vaccinated and detect HBS antibody positive before performing HBV experiments.Begin by culturing the HepG2.2.15 cells keeping in mind that the cell supernatant as well as all the tips, flasks, plates, and tubes that come in contact with HepG2.2.15 should be soaked in 2%virucide overnight prior to disposal. Remove the vial containing HepG2.2.15 cells from liquid nitrogen and thaw it in gentle swirling in a 37 degree Celsius water bath. Transfer the cells to a 25 centimeter square tissue culture flask and add four milliliters of complete culture medium.Culture the HepG2.2.15 cells at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator. Change the medium every three days. When ready, collect the cell supernatant in a 50 milliliter centrifuge tube.Close the lid firmly and wrap it with paraffin film. To remove the cell fragments, filter the HepG2.2.15 supernatant with a 0.45 micrometer membrane into a new 50 milliliter centrifuge tube. Then add 14 milliliters of filtered supernatant to a virus concentrator column and close the lid.Centrifuge the column at 3, 200 times g for 35 minutes in a horizontal spinning centrifuge and collect the HepG2.2.15 supernatant concentrate into a 1.5 milliliter tube. Run real-time PCR to ensure that the concentration if HBV DNA in the HepG2.2.15 supernatant concentrate is within the standard curve range. Dilute the concentrate 20-fold by adding 10 microliters to 190 microliters of DMEM in a 1.5 milliliter microcentrifuge tube.Along with the supernatant concentrate, prepare a negative control, positive control, and four dilutions of the quantitative reference. Add 450 microliters of HBV DNA extraction buffer to each sample and centrifuge them at 12, 000 times g for five minutes at room temperature. Combine 27 microliters of PCR master mix and three microliters of Taq polymerase enzyme per sample in PCR tubes placed on ice.Then add 20 microliters of the centrifuged sample to each tube. Perform real-time PCR according to manuscript directions and export the CT values to obtain a standard curve. Divide the HepG2.2.15 supernatant concentrate into aliquots and store it at minus 80 degrees Celsius until ready to use.Prepare infection medium according to manuscript directions. Then seed HepG2-NE in two wells, 293T-NE-3NR cells in two wells, and 293T-NE in one well. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator for 24 hours.After the incubation, observe the cells and proceed with infection if they're healthy. Add 500 microliters of the infection complex to each well and incubate the cells for another 24 hours. Wash the cells twice with 500 microliters of PBS.Then add one milliliter of medium to each well. Change the medium gently every two days. On day 11, gently wash the cells twice with 500 microliters of PBS and fix the cells with ice cold methanol for immunofluorescence.Seed 100, 000 cells per well of HepG-NE into two wells, 293T-NE-3NRs into two wells, and 293T-NE into one well of the plate. Seed 100, 000 cells per well of HepG2.2.15 into five membrane inserts in a separate six-well plate. Incubate the cells for 24 hours.After the incubation, ensure that the cells are all adherent and in good state. Discard the medium in the six-well plate and the cell membrane inserts. Then place the membrane inserts with HepG2.2.15 into the six-well plate seeded with HepG-NE, 293T-NE-3NRs, and 293T-NE.Incubate the cells at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator, changing the medium every three to four days. After 10 days, remove the membrane inserts, gently wash the cells with PBS twice, and fix the cells with ice cold methanol for immunofluorescence. Incubation with the HBcAg primary antibody and DAPI resulted in a distinct staining when observed with a 10X objective on a fluorescent inverted microscope.Nuclear localization was confirmed by staining with DAPI and NTCP-EGFP expression was identified with green fluorescence. The expression sites of HBcAg were located with red fluorescence. When DAPI, NTCP and HBcAg are in the same cell, the cell has been successfully infected with HBV.The Cyclosporin A group was the negative control because it prevents HBV from entering cells by blocking NTCP. HBcAg was detected in 293T-NE-3NRs, but not in 293T-NE cells indicating NTCP is not the only factor essential for HBV infection in 293T. Good cell status and efficient operation are the key points to getting successful infection results.Gentle washing and changing the media after infection is also important. As an alternative, HBV infection method with hepatoma-based HepG2-NE cells has higher infection efficiency than this method and is suitable for anti-HBV drug screening. Modeling HBV infection in 293T-NE-3NR is a useful compliment to the traditional cell model due to the non-hepatic background of these cells.This method will facilitate the study of HBV infection in non-hepatic cells as well as host factors required for liver of HBV.

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