We thank F. Chisari for providing Huh-7.5.1 cell line. We would like to thank Justine Ho for editing the manuscript. This work was supported by Cedars-Sinai Medical Center Institutional Programmatic Research Award and National Center for Advancing Translational Sciences, Grant UL1TR000124 to V.A.

A general outline of the protocol is illustrated in Figure&#160;1.
1. Cells
<ol>
	<li>Prepare complete growth media that contains 10-15% fetal bovine serum (FBS), 10 mM nonessential amino acids, 10 mM Hepes, penicillin (100 units/ml), streptomycin (100 mg/ml), and 2 mM L-glutamine.</li>
	<li>Maintain Huh-7.5.1 cells13 in complete growth media containing the above supplements for in vitro analysis of hepatitis C viral replication cycle.</li>
	<li>Culture viral strains in Huh-7.5.1 cells with the specified supplemented growth media at 37 &#176;C with 5% CO2.</li>
</ol>
2. Virus and Plasmid Constructs
<ol>
	<li>Generate the complementary DNA (cDNA) form of HCV RNA and clone it into a plasmid vector for ease of genetic manipulation. Note: The discovery of the Japanese fulminant hepatitis 1, JFH-1 (genotype 2a HCV), isolate has been critical for HCV research and is primarily used to study the HCV replication cycle11-13. Inter- and intragenotypic chimeric viruses based on JFH-1 HCV are commonly used in research14-18. Construction of synthetic intragenotype 2a chimeric virus, FNX-HCV, and a monocistronic chimeric reporter strain was described previously19 and construction details are beyond the scope of this protocol.</li>
	<li>Use the pFNX-HCV intragenotype 2a virus as it contains a 5&#8217;NTR, structural regions and p7, and part of the NS2 non-structural regions (nucleotides 1 to 2878) of the J6CF parental strain (NCBI accession no. AF177036), as well as the non-structural components of the JFH-1 viral strain (NCBI accession no. AB047639). Note: FNX-HCV was synthesized based on the sequence of Jc1 intragenotype 2a chimeric HCV reported previously15.</li>
	<li>Generate a monocistronic chimeric reporter viral construct, the pFNX-Rluc, based on the pFNX-HCV strain (above in step 2.2) by inserting a Renilla luciferase gene between the 5&#8217; NTR and core gene (between nt 388 and 389). Subsequently, connect the luciferase gene and core gene of this construct using a foot and mouth disease virus 2A (F2A) peptide sequence, which would work as a cleavage signal.</li>
	<li>Engineer the RNA polymerase-null (Pol-) viral construct using either the pFNX-HCV or the pFNX-Rluc viral background. Replace the NS5B polymerase catalytic residues, GDD (aa 2759-2761; nt 8615-8623), of either of these viral backbones with AAG amino acid residues.</li>
</ol>
3. In Vitro HCV RNA Transcription
<ol>
	<li>Use an intragenotype 2a chimeric virus FNX-HCV and FNX-HCV Pol null (Pol-) HCV for evaluation of viral replication (Figure&#160;2A).</li>
	<li>Linearize the viral plasmids with XbaI restriction enzyme and then treat with mung bean nuclease to generate blunt ends. Purify the digested plasmids by anion-exchange chromatography. Verify the integrity of the linearized plasmid by subjecting DNA to agarose gel electrophoresis (Figure&#160;2B).</li>
	<li>Transcribe the linearized viral plasmids using the T7- RNA polymerase.</li>
	<li>Purify the newly synthesized DNase-treated RNA using an RNA purification kit.</li>
	<li>Verify the RNA production by agarose gel electrophoresis (Figure&#160;2C).</li>
	<li>Quantify the RNA by spectrophotometry.</li>
	<li>Store the generated RNA in -80 &#176;C in 10 &#956;g working aliquots. Note: To minimize variation of RNA quality within each experimental design by transcribing all RNA for each viral sample and control at the same time.</li>
</ol>
4. HCV RNA Transfection and Sample Collection
<ol>
	<li>Harvest the Huh-7.5.1 cells using trypsin.</li>
	<li>To rinse cells, centrifuge and resuspend the suspension twice with cold low serum media. Resuspend the cells in low serum media at 1 x 107cells per ml.</li>
	<li>Mix a total of 10 &#956;g of transcribed viral RNA with 400 &#956;l of resuspended cells (4 x 106 cells) in a 0.4-cm electroporation cuvette. Transfect the cells via electroporation at 270 V, 100 &#937;, and 950 &#956;F.</li>
	<li>Resuspend the electroporated cells in 10 ml complete growth media with 15% FBS. Note: Increased survivability of electroporated cells is seen when the Huh-7.5.1 cells were cultured in 15% Fetal Bovine Serum.</li>
	<li>Plate the cells in both T-25 flasks (~1.2 x 106 cells per flask) and 48-well plates (1 x 104 cells per well) for each of the following time points: 4, 48 and 96 hr.</li>
	<li>Replace the media at 4-8 hr post-transfection with fresh supplemented growth media with 10% FBS to remove dead cell debris from the cultured flasks and plates.</li>
	<li>Harvest cell culture supernatants at the 48, 96 hr time points and remove cellular debris from collected samples by centrifugation of cells at 1,500 rpm for 10 min at 4 &#186;C.</li>
	<li>Store the cell-free supernatants at -80&#160;&#186;C. Lyse the cells for protein and RNA analysis by Western blot and reverse transcription-quantitative PCR at the indicated time points.</li>
</ol>
5. Reverse Transcription-quantitative PCR (RT-qPCR) for Assessing the HCV Genome Copies
<ol>
	<li>Perform a two-step RT-qPCR to determine the HCV genomic RNA copy number.</li>
	<li>Reverse transcribe 1 &#956;g of total cellular RNA using reverse transcriptase and a primer specific for HCV sense strand that binds to 5&#8217;NTR (JFH RTQ R: 5&#8217;CCTATCAGGCAGTACCACA-3&#8217;) or a primer specific for housekeeping gene peptidylprolyl isomerase G (PPIG R: 5&#8217;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#8217;). Also reverse transcribe FNX-HCV RNA (generated in Step 3) of known genome copies (101 to 109 standard) using HCV sense strand primer.</li>
	<li>Carry out qPCR by using 50 ng of the resulting transcribed cDNA using specific HCV primers (JFH RTQ F: 5&#8217;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R: 5&#8217;CCTATCAGGCAGTACCACA-3&#8217;) and DNA binding green dye containing qPCR super mix. Perform qPCR for housekeeping gene PPIG as well (primers PPIG F: 5&#8217;-GAAGAGTGCGATCAAGAACCCATGAC-3&#8217;; PPIG R: 5&#8217;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#8217;) Note: For calculating accurate intracellular HCV RNA level across samples, use cellular housekeeping gene, PPIG, expression level (Ct cycle) for normalization. Use the copy number of the FNX-HCV genome as the standard for the copy number determination.</li>
	<li>Use the following conditions when running qPCR to determine HCV RNA copy number: 95&#160;&#186;C for 15 sec and 60&#160;&#186;C for 30 sec (40 cycles) using real-time PCR system.</li>
	<li>See Figures 3A and 3B for genome replication results.</li>
</ol>
6. Western Blotting Analysis for Detecting HCV Protein Expression (Figure 3C)
<ol>
	<li>Use cell lysates from viral RNA transfected at 96 hr post transfection for protein western blotting analysis.</li>
	<li>Resolve the cell lysate using SDS-PAGE and transfer to polyvinylidene difluoride (PVDF) membrane.</li>
	<li>Block the membrane using a blocking solution containing 5% skim milk, 0.2% Tween 20 in phosphate buffered saline (PBS).</li>
	<li>Incubate the membrane with primary mouse monoclonal antibody NS3 (1 in 1,000 dilution) and beta-actin (1 in 5,000 dilution).</li>
	<li>Add goat anti-mouse IgG conjugated to horseradish peroxidase (1 in 5,000 dilution) and detect by chemiluminescence.</li>
</ol>
7. Immunofluorescence Assay (IFA)
<ol>
	<li>Fix the HCV-infected and transfected cells using methanol for 30 min at -20&#160;&#186;C for immunofluorescence assay.</li>
	<li>Wash the cell with PBS three times and block with IFA blocking buffer.</li>
	<li>Use rabbit polyclonal anti-NS5A primarily antibody (kindly provided by Dr. Dasgupta, UCLA) or mouse monoclonal anti-dsRNA antibody J2 at a dilution of 1:200 (1 &#956;g/ml) and incubate for 5 hr to overnight at 4 &#186;C.</li>
	<li>Wash the cells with PBS three times after the primary antibody.</li>
	<li>Add goat anti-rabbit IgG-488 polyclonal secondary antibody or goat anti-mouse IgG-594 polyclonal secondary antibody at a 1:1,000 dilution (1 &#956;g/ml) and incubate for 1 hr at room temperature.</li>
	<li>Wash cells with PBS three times and stain nuclei using Hoechst dye and view using fluorescent microscope (Figure&#160;3D).</li>
</ol>
8. Measuring Virus Titer
<ol>
	<li>Plate na&#239;ve Huh-7.5.1 cells at approximately 3 x 103 cells/well using a 96-well plate. The next day, perform 10-fold serial dilutions of cell-free culture supernatant harvested from HCV RNA transfected cells using growth media and inoculate in triplicate onto Huh-7.5.1 cells.</li>
	<li>Fix cells at 72&#160;hr post-infection using methanol (for 30 min at -20 &#186;C) and immunostain for HCV NS5A protein as stated in the section 7.</li>
	<li>Use the highest dilution to count for the NS5A positive foci and calculate the average number of focus forming unit (FFU) per milliliter. See Figure&#160;4 for HCV titer.</li>
</ol>
9. Renilla Luciferase Reporter Assay for Viral Genome Replication and Infectivity
<ol>
	<li>For evaluating viral genome replication, plate HCV RNA-electroporated Huh-7.5.1 cells in triplicate in 48-well plates (1 x 104 cells/well).</li>
	<li>Lyse the cells with 75 &#956;l of passive lysis buffer at the 6 hr, 48 hr, and 96 hr post-transfection.</li>
	<li>Rock the plates gently for 15 min at room temperature and store at -80&#160;&#186;C.</li>
	<li>To measure the Renilla luciferase enzymatic activity, thaw the protein lysate and Renilla luciferase assay reagents to room temperature.</li>
	<li>Add 20 &#956;l of protein lysate per well of a 96-well white luminescence plate and place the plate in a luminometer.</li>
	<li>Dispense 100 &#956;l of Renilla luciferase assay reagent (coelenterazine substrate + buffer) per well and after a 2 sec pre-read delay; integrate the emitted light for 10 sec.</li>
	<li>Calculate the mean and standard deviation for each sample from the luciferase values of biological triplicates and subject the data to statistical analysis (Figure&#160;5).</li>
	<li>For assessing infectivity, inoculate 500 &#956;l of cell-free supernatant obtained from HCV RNA-transfected cells for the indicated time points (48 hr and 96 hr) in triplicate onto na&#239;ve Huh-7.5.1 cells in 48-well plates.</li>
	<li>Replace the viral inoculum with 500 &#956;l of fresh medium per well after 6&#160;hr post-infection.</li>
	<li>Lyse the cells at 48 hr post-infection and subject to Renilla luciferase assay as described in steps 8.2 to 8.7 (Figure&#160;5).</li>
</ol>
10. Statistical Analysis
<ol>
	<li>The error bars in the graphs indicate standard deviations (SD). The P values were calculated by the unpaired t test.</li>
</ol>

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The authors have nothing to disclose.

This illustration describes a method for analyzing the hepatitis C Virus replication cycle. HCV is a human pathogen and the prescribed biosafety protocol will have to be strictly followed. Infectious HCV cell culture systems have been described previously11-13,16,17. There are few crucial points we implement when following the illustrated protocol. First, it is of high importance to have good quality of intact full length viral genomic RNA for downstream studies. The input plasmid carrying the viral cDNA has t...

Hepatitis C virus (HCV) causes cirrhosis and liver cancer. It affects 170 million people worldwide with 350,000 people dying annually1-3. HCV is a positive strand RNA virus with a genome size of 9.6 kb. The HCV genome is translated as a single polyprotein of ~3,000 amino acid residues that is proteolytically cleaved by various cellular and viral proteases into 10 polypeptides. HCV is the prototype virus in the genus Hepacivirus and belongs to family Flaviviridae4. Upon exposure, HCV establishes chronic infection in 80% of the individuals. The infection is mostly asymptomatic and timely diagnosis can allow therapeutic intervention to prevent liver deterioration. Current treatment is suboptimal and no vaccine is available5,6.
The etiology of hepatitis C was first described in 1989 7. Studying HCV replication is important for hepatitis C vaccine and treatment research, but it had been long hampered by the lack of an efficient viral culture system. A molecular clone of HCV was shown to be infectious in chimpanzees upon intrahepatic inoculation8. Subsequently, HCV sub-genomic replicons were described, which allowed to dissect the viral genome replication stage in a cell culture system9,10. Discovery of a genotype 2a HCV isolate JFH-1 (Japanese Fulminant hepatitis-1), capable of infecting cell culture opened new avenues for HCV replication research11-13. Genotype 2a strain JFH-1 based inter- and intra-genotypic chimeric viruses and genotype 1 HCV based infectious culture systems are available as well14-18.
We have successfully used JFH-1 strain and HCV intragenotype 2a chimeric virus to obtain the high-resolution functional profiling map of protein domains and cis-acting RNA elements19,20. According to this, here we describe an effective culture system routinely used that allows studying various stages of the HCV replication cycle and host-pathogen interaction. We present virological assays to assess viral genome replication and de novo infectivity of intragenotype 2a HCV and a Renilla luciferase based reporter HCV.

<table border="0" cellpadding="0" cellspacing="0" width="1121">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Name of Material/ Equipment</td>
			<td style="width:267px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">10-017-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Non essential amino acid</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">MT25025CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">HEPES</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Glutamax</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Opti-MEM Reduced Serum Medium,no Phenol Red</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11058-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Huh-7.5.1</td>
			<td style="width:267px;">The Scripps Research Institute</td>
			<td style="width:119px;"></td>
			<td>The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:569px;">Plasmids (pFNX-HCV, pFNX-HCV Pol null, pFNX-Rluc, and pFNX-Rluc Pol null)&#160;</td>
			<td style="width:267px;">Cedars-Sinai Medical Center</td>
			<td style="width:119px;"></td>
			<td>The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">XbaI</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">R0145S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Mung Bean Nuclease</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">M0250S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">T7 RiboMAX Express Large Scale RNA Production System</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">P1320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Rneasy Mini Kit</td>
			<td style="width:267px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Nanodrop 2000</td>
			<td style="width:267px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Electroporation Cuvette (4 mm)</td>
			<td style="width:267px;">Bioexpress</td>
			<td>E-5010-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Gene Pulser Xcell Total System</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">165-2660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-dsRNA antibody J2&#160;</td>
			<td>English &#38; Scientific Consulting Kft.</td>
			<td style="width:119px;">10010200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 488</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 594</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVDF membrane package</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">162-0263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Blotting Grade Blocker Non Fat Dry Milk</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6404XTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Tween-20</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6531XTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [8 G-2]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab65407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [H23]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab13830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td style="width:119px;">115-035-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham ECL Prime Western Blotting Detection Reagents&#160;</td>
			<td style="width:267px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SUPERSCRIPT III RT&#160;</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">18080085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SYBR QPCR SUPERMIX W/ROX</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11744500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ViiA 7 real-time PCR system</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Renilla Luciferase Assay System kit</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">E2810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">RNase-Free DNase</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">M6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">GloMax-Multi Detection System (Luminometer)</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for analyzing hepatitis c virus replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C virus is a RNA virus. Thus for genetic manipulation purpose, the HCV genomic cDNA has been cloned into a bacterial plasmid vector. A T7 RNA polymerase promoter sequence was introduced immediately before the 5&#8217; end of the HCV genome. A general outline of HCV analysis workflow is presented in Figure&#160;1. To generate HCV genomic RNA with precise 3&#8217; end, the HCV genome containing plasmid is cut with XbaI restriction enzyme and the generated single-stranded overhang was blu...

Watch this Scientific Journal Video about A Protocol for Analyzing Hepatitis C Virus Replication at JoVE.com

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

a-protocol-for-analyzing-hepatitis-c-virus-replication

Research

JoVE Journal

Immunology and Infection

23.9K Views.  Cedars-Sinai Medical Center. Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle. 