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>

<ol>
	<li>Alter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.+Epidemiology+of+hepatitis+C.">J. Epidemiology of hepatitis C.</a> Hepatology.. 26(3 Suppl 1), (1997).</li><li>Alter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.+Epidemiology+of+hepatitis+C+virus+infection.">J. Epidemiology of hepatitis C virus infection.</a> World J Gastroenterol. 13 (17), 2436-2441 (2007).</li><li>Lavanchy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+hepatitis+C.">The global burden of hepatitis C.</a> Liver Int. 29 (Suppl 1), 74-81 (2009).</li><li>Lindenbach, B. D., Thiel, H. J., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flaviviridae: viruses and their replication in Fields Virology. , 1101-1152 (2007).</li><li>Ciesek, S., Manns, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+in+2010:+the+dawn+of+a+new+era+in+HCV+therapy.">Hepatitis in 2010: the dawn of a new era in HCV therapy.</a> Nat Rev Gastroenterol Hepatol. 8 (2), 69-71 (2011).</li><li>Ghany, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+treatment+of+genotype+1+chronic+hepatitis+C+virus+infection:+2011+practice+guideline+by+the+American+Association+for+the+Study+of+Liver+Diseases.">An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of Liver Diseases.</a> Hepatology. 54 (4), 1433-1444 (2011).</li><li>Choo, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+cDNA+clone+derived+from+a+blood-borne+non-A,+non-B+viral+hepatitis+genome.">Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.</a> Science. 244 (4902), 359-362 (1989).</li><li>Kolykhalov, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+hepatitis+C+by+intrahepatic+inoculation+with+transcribed+RNA.">Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.</a> Science. 277 (5325), 570-574 (1997).</li><li>Lohmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+subgenomic+hepatitis+C+virus+RNAs+in+a+hepatoma+cell+line.">Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.</a> Science. 285 (5424), 110-113 (1999).</li><li>Blight, K. J., Kolykhalov, A. A., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+initiation+of+HCV+RNA+replication+in+cell+culture.">Efficient initiation of HCV RNA replication in cell culture.</a> Science. 290 (5498), 1972-1974 (2000).</li><li>Lindenbach, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+replication+of+hepatitis+C+virus+in+cell+culture.">Complete replication of hepatitis C virus in cell culture.</a> Science. 309 (5734), 623-626 (2005).</li><li>Wakita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+infectious+hepatitis+C+virus+in+tissue+culture+from+a+cloned+viral+genome.">Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.</a> Nat Med. 11 (7), 791-796 (2005).</li><li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+hepatitis+C+virus+infection+in+vitro.">Robust hepatitis C virus infection in vitro.</a> Proc Natl Acad Sci U S A. 102 (26), 9294-9299 (2005).</li><li>Yi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensatory+mutations+in+E1,+p7,+NS2,+and+NS3+enhance+yields+of+cell+culture-infectious+intergenotypic+chimeric+hepatitis+C+virus.">Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus.</a> J Virol. 81 (2), 629-638 (2007).</li><li>Pietschmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+infectious+intragenotypic+and+intergenotypic+hepatitis+C+virus+chimeras.">Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.</a> Proc Natl Acad Sci U S A. 103 (19), 7408-7413 (2006).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+full-length+hepatitis+C+virus+genotype+1+(strain+TN)+infectious+culture+system.">Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system.</a> Proc Natl Acad Sci U S A. 109 (48), 19757-19762 (2012).</li><li>Yi, M., Lemon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+1a+HCV+(H77S)+infection+system.">Genotype 1a HCV (H77S) infection system.</a> Methods Mol Biol. 510, 337-346 (2009).</li><li>Gottwein, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+hepatitis+C+reporter+viruses+with+genotype+1+to+7+core-nonstructural+protein+2+(NS2)+expressing+fluorescent+proteins+or+luciferase+in+modified+JFH1+NS5A.">Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A.</a> J Virol. 85 (17), 8913-8928 (2011).</li><li>Arumugaswami, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+functional+profiling+of+hepatitis+C+virus+genome.">High-resolution functional profiling of hepatitis C virus genome.</a> PLoS Pathog. 4 (10), (2008).</li><li>Chu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+enhancer+and+critical+cis-acting+RNA+elements+in+the+protein-encoding+region+of+the+hepatitis+C+virus+genome.">Systematic analysis of enhancer and critical cis-acting RNA elements in the protein-encoding region of the hepatitis C virus genome.</a> J Virol. 87 (10), 5678-5696 (2013).</li><li>Salloum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab18+binds+to+hepatitis+C+virus+NS5A+and+promotes+interaction+between+sites+of+viral+replication+and+lipid+droplets.">Rab18 binds to hepatitis C virus NS5A and promotes interaction between sites of viral replication and lipid droplets.</a> PLoS Pathog. 9 (8), (2013).</li><li>Gonzalez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+an+optimal+RNA+transfection+reagent+and+comparison+to+electroporation+for+the+delivery+of+viral+RNA.">Selection of an optimal RNA transfection reagent and comparison to electroporation for the delivery of viral RNA.</a> J Virol Methods. 145 (1), 14-21 (2007).</li><li>Phan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+NS2+protein+contributes+to+virus+particle+assembly+via+opposing+epistatic+interactions+with+the+E1-E2+glycoprotein+and+NS3-NS4A+enzyme+complexes.">Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes.</a> J Virol. 83 (17), 8379-8395 (2009).</li><li>Schaller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+hepatitis+C+virus+superinfection+exclusion+by+using+novel+fluorochrome+gene-tagged+viral+genomes.">Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes.</a> J Virol. 81 (9), 4591-4603 (2007).</li><li>Li, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+hepatitis+C+virus+lacking+the+envelope-encoding+genes+for+single-cycle+infection+by+providing+homologous+envelope+proteins+or+vesicular+stomatitis+virus+glycoproteins+in+trans.">Production of hepatitis C virus lacking the envelope-encoding genes for single-cycle infection by providing homologous envelope proteins or vesicular stomatitis virus glycoproteins in trans.</a> J Virol. 85 (5), 2138-2147 (2011).</li><li>Jones, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+p7+and+NS2+proteins+are+essential+for+production+of+infectious+virus.">Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus.</a> J Virol. 81 (16), 8374-8383 (2007).</li><li>Steinmann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+p7+protein+is+crucial+for+assembly+and+release+of+infectious+virions.">Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions.</a> PLoS Pathog. (7), (2007).</li></ol>

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 to be linearized and blunted carefully. The mung-bean nuclease employed to blunt the XbaI overhang is a non-specific nuclease and prolonged incubation will result in damage or degradation of 3&#8217; end of viral genome. The nuclease has to be removed immediately after the specified 30 min incubation by phenol-chloroform extraction or anion exchange column purification. Gel extraction of nuclease treated DNA may not be recommended as the enzyme is active during gel electrophoresis process.
For subsequent in vitro RNA transcription steps, generally, one will also take broad-spectrum precautions for minimizing ribonuclease (RNase) contamination. All the reagents and materials used should be RNase free. To generate high-quality genomic RNA with minimum RNA strand breaks, the RNA need to be subjected to less physical stress by avoiding harsh vortexing and repeated pipetting during purification and downstream handling steps. After completion of RNA transcription, the input plasmid DNA templates have to be removed from transcribed RNA mix by DNase (RNase free) treatment. Incomplete removal can result in carryover of plasmid DNA into transfected cells and affect the quantification of absolute genome copy number of replicated viral RNA. Hence use DNase enzyme at a concentration of 1 unit per microgram of DNA and also having extra 15 min&#160;of DNase incubation at 37 &#186;C can help. The transcribed RNA can be purified by either phenol-chloroform extraction or anion exchange column. Care should be exercised to avoid carryover of phenol or other reagents to the purified RNA sample, which can be cytotoxic and interfere with molecular assays.
For electroporation purposes, the purified RNA has to be dissolved in RNase-free sterile water. If the RNA pellet is difficult to dissolve, heating the sample to 65 &#186;C for 5 min&#160;will help. Once transcription is completed, it is important to store the RNA immediately in 10 &#956;g of working aliquots made in RNase free water at -80 &#186;C. The purpose of the aliquots is to prevent repeated freeze/thaw mediated RNA degradation. For consistent experimental results, it is recommended to transcribe all samples and controls at the same time. High quality RNA is recommended for a higher viral titer yield. For transfecting HCV genomic RNA, polymer and lipid based transfection agents can also be used21,22.
Viral and host factors greatly influence the growth kinetics of HCV strains. Human hepatoma cell line (Huh-7) derivatives Huh-7.5, Huh-7.5.1 and Huh7-Lunet cell lines are commonly used for evaluating viral growth11-13,15. The peak viral production of JFH-1 strain in Huh7-Lunet cells is at 3-4 days post-transfection, whereas in Huh-7.5.1 cells it is at 21 days post-transfection13,15. The intragenotype 2a chimeric virus FNX-HCV having non-structural genes from JFH-1 strain has peak titer at 4 days post-transfection in Huh-7.5.1 cells19,20. For viral production from in vitro transcribed RNA, early passage human hepatoma cell line is preferred. Viral production is significantly reduced while using Huh-7.5.1 cells over passage number 20. To ensure increased cell survivability after electroporation, thawed Huh-7.5.1 cells are allowed a two week recovery period before conducting an experiment and maintained in 12%-15% FBS. Along with this criterion, resuspension of the cells in growth media containing 15% FBS immediately after electroporation increases the cell survival allowing for better viral production. After the first 8 hr time point, the cells are then switched to media containing 10% FBS. To minimize the carry over cellular debris, the viral culture supernatant is subjected to centrifugation and filtration through 4 micron filter. The unconcentrated or concentrated viral supernatants are aliquoted and stored in -80 &#186;C for further in vitro and in vivo experiments. These precautions and suggestions can help researchers for successfully performing the analysis of HCV replication cycle.
Another variation of reporter HCV that harbors a marker gene such as fluorescent protein and a secreted form of Gaussia luciferase (Gluc) are described for HCV research18,23,24. For researchers focusing on investigating HCV genome replication, HCV sub-genomic replicon (lacking the genes core to NS2) is a valuable tool9,10. HCV Replicons are non-infectious, thus require less bio-safety regulations and easier to work with compared to the infectious cell culture HCV and clinical isolates. HCV deficient in NS5B polymerase activity is a critical control for studying HCV replication cycle11,12. HCV lacking envelope glycoproteins (delta E1E2), or p7-NS2 activity can be useful controls for studies involving HCV entry, virion morphogenesis and egress12,19,25-27.
Regarding limitations, currently the infectious cell culture system is available only for HCV genotypes 1 and 2. Authentic viral strains belong to genotypes 3 to 6 that are capable of growing efficiently in cell culture yet to be identified. Intergenotypic recombinant viruses are useful alternatives for studying replication cycle of various genotypes. Replication competent chimeric virus harboring core to NS2 region from genotypes 1 to 7 and rest of the sequence from JFH-1 genotype 2a genome were described15,18. Another limitation is that the viral yield of JFH-1 and its derivative chimeric strains is in the range of 103 to 106 per ml, which is relatively low compared to that of other RNA viruses such as polio and influenza. HCV strain with robust growth needs to be developed. Future investigation includes the use of the reporter HCV for high-throughput library screening to identify potent antivirals and focus on developing vaccine candidates for preventing HCV infection.

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 blunted with mung bean nuclease digestion. The quality of the linearized HCV plasmid was assessed by agarose gel electrophoresis (Figure&#160;2B). The HCV DNA was subjected to T7 RNA polymerase mediated in vitro transcription and the resulting RNA yielded a single product at 9.6 kilobase (Figure&#160;2C).
We tested the growth kinetics of an intragenotype 2a HCV (Figure&#160;3). The Huh-7.5.1 cells were electroporated with in vitro transcribed RNA of wild-type (WT) and polymerase null viruses. We evaluated the viral sense genome replication by RT-qPCR. Results indicated that the WT virus replicated the genome efficiently (Figures&#160;3A and 3B). The wild-type exhibited one to three log higher level of genome replication compared to that of Pol- virus. The WT virus produces the NS3 protein (Figure&#160;3C) that is involved in viral protein cleavage (protease activity), and genome replication (helicase activity). Also the WT virus expressed NS5A protein as assessed by Immunofluorescence assay (Figure&#160;3D). NS5A protein is part of the HCV RNA replicase complex. To visualize viral genome replication, we examined the presence of double- stranded (ds) HCV RNA using an antibody that specifically recognizes dsRNA. During HCV genome amplification, the sense strand RNA is copied to anti-sense genome, thus the double stranded RNA intermediates are present in the infected cell cytoplasm. The NS5A protein and dsRNA co-localizes in the cellular cytoplasm (Figure&#160;3D) of WT transfected cells suggesting the active viral replication. Taken together, the WT virus established active replication in transfected Huh-7.5.1 cells.
Progress in HCV research had been limited due to lack of an infectious cell culture system. The discovery of JFH-1 strain of HCV and the subsequent characterization of chimeric laboratory strains allowed us to investigate the entire HCV replication cycle in cell culture including viral entry, RNA translation, RNA replication and the formation of infectious viral progeny. To assess the HCV titer and de novo infectivity, we inoculated the cell-culture supernatants harvested from HCV RNA transfected cells. The HCV infection was visualized by detecting the HCV NS5A protein and calculated the viral titer by counting the infectious foci (Figure&#160;4). We considered isolated cluster of cells (over 2 cells) positive for NS5A as a single focus. Results indicated that the WT virus is infectious and produces over 104 foci forming units/ml of infectious particle.
We also established a HCV reporter virus harboring Renilla luciferase (Rluc) reporter gene (Figure&#160;5A). A reporter HCV can be used for testing large number of mutant viruses as well as for high-content screening assays. Here we present the assessment of growth phenotypes of WT and Pol- reporter viruses. If the viral genome replicates, the luciferase activity would increase overtime post transfection. The increased genome replication levels can be deduced from increased luciferase activity. Hence, the luciferase activity indirectly provides the measurement of the level of genome replication. The lysates harvested from WT or Pol- viral RNA transfected cells at indicated time points were tested for luciferase activities. At 6 hr post-transfection, both WT and Pol- viruses had similar luciferase activities, indicating similar input level of transfected RNA that had been translated (Figure&#160;5B). However at 48 hr and 96 hr post-transfection, the WT virus exhibited increased genome replication levels compared to that of Pol- virus. Also, the WT virus produced viral NS3 protein as verified by Western blotting analysis (Figure&#160;5C). Subsequently, we tested the infectivity of WT and Pol- reporter viruses by inoculating na&#239;ve Huh-7.5.1 cells with the cell-free supernatants collected from 48 hr and 96 hr post-transfected cell cultures. The Pol- virus had base-level luciferase activity, whereas the WT virus had 2-3 log higher level of replication to that of Pol- reporter virus (Figure&#160;5D). Results demonstrate that we have a robust HCV infectious cell culture system.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51362/51362fig1highres.jpg" width="500" /> 
Figure 1.&#160;General outline of HCV replication analysis workflow. Linearized HCV plasmid construct containing T7 RNA polymerase promoter (T7p) subjected to in vitro transcription. The purified HCV RNA genome is electroporated into Huh-7.5.1 cells and plated in flasks and 48-well plate. At 4 hr, 48 hr, and 96 hr post-transfection, the cellular RNA and culture supernatants are harvested from flasks. The cells from 48-well plate are utilized for harvesting protein lysate and are fixed for Immunofluorescence assay. Genome copy number analysis by RT-qPCR, Western blotting and measuring viral titer are done to assess the HCV replication.
<img alt="Figure 2" fo:content-width="4in" src="/files/ftp_upload/51362/51362fig2highres.jpg" width="400" /> 
Figure 2.&#160;Production of HCV genomic RNAs by in vitro transcription of constructed plasmid DNAs. A) Genomic organization of the J6CF/JFH-1 intragenotype 2a chimeric viruses, FNX-HCV and FNX-HCV Pol null. The J6CF strain region (5&#8217;NTR to part of NS2) is depicted in dark gray and the JFH-1 strain region (part of NS2 to 3&#8217;NTR) is displayed in light gray. NS5B polymerase catalytic domain mutation (GDD to AAG) is indicated with an asterisk. B)&#160;Steps involved in generating linearized HCV plasmid. Gel picture shows the linearized, blunt ended plasmid DNAs produced by XbaI and mung bean nuclease digestion, ready for in vitro transcription. 0.8% agarose gel was used for resolving the DNA. C)&#160;Gel picture depicts the HCV genomic RNAs produced by in vitro transcription using the T7 RNA polymerase system. WT: wild-type; Pol-: Polymerase null; M: marker.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51362/51362fig3highres.jpg" width="500" /> 
Figure 3. Assessing the growth phenotypes of HCV constructs. A)&#160;Evaluating the genome replication kinetics of wild-type (WT) and polymerase null (Pol-) viruses. The genome copy numbers of sense RNA strand assessed by RT-qPCR are presented in the bar graph. The viral genome copies of Pol- virus declined over the period of time indicating replication deficient phenotype. B)&#160;Relative genome replication level of wild-type HCV is compared to that of Pol- virus. C)&#160;Western blot analysis of HCV protein expression. HCV protein NS3 is detected and beta-actin is used as a cellular control. D)&#160;Immunofluorescence assay for investigating viral replication. At 96 hr post-electroporation the cells were fixed and subjected to immunostaining for HCV NS5A protein and double-stranded RNA (ds RNA), a marker for HCV RNA replication intermediates. The nuclei were visualized with Hoechst stain (Scale bar 50 &#956;m). hpt: hours post-transfection. <a href="https://www.jove.com/files/ftp_upload/51362/51362fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51362/51362fig4highres.jpg" width="500" /> 
Figure 4. Examining the infectivity of wild-type HCV and measuring virus titer. A)&#160;Na&#239;ve Huh-7.5.1 cells were used for infection studies. The cell-free supernatant collected at 48 and 96 hr post-transfection (hpt) of HCV RNA were subjected to 10-fold serial dilution and added to the cells in a 96-well plate in triplicate. 72 hr post-infection, the cells were fixed and immunostained for HCV NS5A protein. The cells with NS5A positive staining (red) are infected with virus. For assessing Foci Forming Unit (FFU), the positive foci at the highest dilution were counted. Representative panels of images are shown (Scale bar 100 &#956;m). B)&#160;Mean values and standard deviations of viral titer in FFU per milliliter are shown in the graph. The polymerase null mutant did not produce infectious particles. WT: wild-type; Pol-: Polymerase null. <a href="https://www.jove.com/files/ftp_upload/51362/51362fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51362/51362fig5highres.jpg" width="500" /> 
Figure 5. Evaluating the genome replication and infectivity of reporter HCV. A)&#160;A cartoon of intragenotype 2a chimeric reporter virus is presented. The Renilla luciferase gene is inserted inframe between 5&#8217;NTR and core. B)&#160;The genome replication kinetics of wild-type and Pol- mutant reporter viruses at indicated time points post-transfection is shown in the graph. Protein lysates were harvested at 6 hr, 48 hr and 96 hr time points for measuring Renilla luciferase enzymatic activity. The mean and standard deviation calculated from triplicate Renilla luciferase values (RLV) for each virus are presented in the graph. C)&#160;Western blotting panel shows the HCV NS3 protein expression. A non-specific antigen detected by NS3 primary antibody acts as a loading control. Wild-type (WT) reporter virus produces high level of NS3 protein. D)&#160;Analysis of virus infectivity. Na&#239;ve Huh-7.5.1 cells were inoculated with the cell-free supernatant obtained from transfected culture at 48 hr and 96 hr time points. Renilla luciferase activities of infected cells were measured at 48 hr post-infection. Mean values with standard deviation are shown in the graph. The Pol- reporter virus infected cells had only background level of luciferase activity, whereas WT reporter virus exhibited high level of infectivity.

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

Nanyang Technological University

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. 

Video: A Protocol for Analyzing Hepatitis C Virus Replication

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...