A Protocol for Analyzing Hepatitis C Virus Replication

Summary

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.

Abstract

Hepatitis C Virus (HCV) affects 3% of the world’s population and causes serious liver ailments including 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.

Introduction

Hepatitis C virus (HCV) causes cirrhosis and liver cancer. It affects 170 million people worldwide with 350,000 people dying annually^1-3. 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 prev....

Protocol

A general outline of the protocol is illustrated in Figure 1.

1. Cells

1. 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.
2. Maintain Huh-7.5.1 cells¹³ in complete growth media containing the above supplements for in vitro analysis of hepatitis C viral replication cycle.
3. Culture viral strains in Huh-7.5.1 cells with the specified supplemented growth media at 37 °C with 5% CO₂.

2.....

Results

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’ end of the HCV genome. A general outline of HCV analysis workflow is presented in Figure 1. To generate HCV genomic RNA with precise 3’ end, the HCV genome containing plasmid is cut with XbaI restriction enzyme and the generated single-stranded overhang was blunted with mung be.......

Discussion

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 previously^11-13,16,17. 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.......

Materials

List of materials used in this article

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