1 Development of an intergenotypic HCV cell culture method to assess antiviral susceptibilities and

53 Pages·2010·1.49 MB·English

JVI Accepts, published online ahead of print on 17 February 2010
J. Virol. doi:10.1128/JVI.02698-09
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
JVI02698-09 (Revised)
Development of an intergenotypic HCV cell culture method to assess antiviral susceptibilities
and resistance development of HCV NS3 protease genes from HCV genotypes 1-6

Ingrid Imhof D
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Peter Simmonds* n
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Centre for Infectious Diseases, University of Edinburgh, Summerhall, Edinburgh, EH9 1QH :/
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*Correspondence: Tel: [phone] e
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Fax: [phone] m
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Email: [email protected] r
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Document totals: Word count for summary 245 8

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Word count for main text 8435 g
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ABSTRACT

Protease inhibitors (PIs) of hepatitis C virus (HCV) provide an additional or alternative
therapy for chronic infection. However, assessment of their efficacy and ability to inhibit
replication of different genotypes is hampered by the lack of a convenient animal model or a
method for in vitro culture of HCV other than the type 1/2-based replicons and the infectious
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genotype 2a clone, JFH1. To address this problem, we constructed a panel of replication
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competent chimaeric Jc1 (pFK JFH1/J6/C-846) clones containing protease and NS4A coding a
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sequences from all six major genotypes, enabling the determination of replication and the
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susceptibility to PIs. Chimaeras showed substantial variability in replication kinetics, attributable

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in part to naturally occurring polymorphisms and differing requirements for adaptive mutations
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in NS3 and NS4A. Through calculation of inhibitory concentrations (IC50) of BILN-2061, i.a
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measuring Foci Forming Units/ml (FFU/ml) reductions and replication inhibition, consistent,
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genotype-associated differences in antiviral susceptibilities were observed. IC values for /
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genotype 1b, 4a and 6a-derived chimaeras (1-3 nM) were approximately 100-fold lower than for D
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genotypes 2a, 3a and 5a (range 80-720 nM), implying major differences in response to therapy. e
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In vitro passage in increasing concentrations of BILN-2061 rapidly induced resistance-associated e
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mutations at position 168 in chimaeras of all 6 genotypes and at position 156 in genotypes 1b ,

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and 4a, each with substantial variability in the identity of substituted amino acids. The system
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will allow future comprehensive phenotypic characterisation of naturally occurring and treatment
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induced mutations for PIs in trial or entering clinical use. s
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JVI02698-09 (Revised)
INTRODUCTION

Worldwide about 170 million individuals are estimated to be infected with hepatitis C
virus (HCV) (1, 48). Chronic HCV infection is a leading cause of chronic liver diseases, such as
cirrhosis and hepatocellular carcinoma (6). HCV has a positive-sense, single-stranded RNA
genome of approximately 9,600 nucleotides, belonging to the family of Flaviviridae (7). A single D
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polyprotein of around 3000 amino acids (53) is translated and processed by cellular and viral n
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proteases to generate 10 different structural and non-structural proteins (16, 18, 19). d
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The error-prone RNA-dependent RNA polymerase (RdRp) NS5B, and the resulting high fr
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mutation frequencies during replication, contributes to the substantial genetic and antigenic h
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heterogeneity of HCV, with seven major genotypes showing >30% nucleotide sequence :/
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divergence from each other and numerous subtypes identified to date (5, 50-52). The distribution a
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of genotypes varies by geographical location and risk groups for infection; the predominant .o
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genotypes within the USA, Europe, Australia and East Asia (Japan, Taiwan, Thailand and China) o
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are 1, 2 and 3. Genotype 4 is largely confined to the Middle East, Egypt and Central Africa, e
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whereas genotypes 5 and 6 are found predominantly in South Africa and South East Asia, m
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respectively (49). r
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The current treatment of pegylated interferon and Ribavirin has limited efficacy and 2
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serious side effects; infections with genotype 1 in particular respond poorly even to prolonged 8

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treatment, with 48% failing to clear infections after 48 weeks of combined therapy (33, 39). To g
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address this problem, several direct antiviral inhibitors of the NS3/4A serine protease and the t
RNA-dependent RNA polymerase have been developed. Among the former are the non-covalent
inhibitor BILN 2061 (24) and the covalent inhibitors SCH 503034 (30) and VX-950 (37). In
ongoing trials encouraging results have been reported for the covalent inhibitors (12, 17, 42, 44),
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whereas the non-covalent inhibitor BILN 2061 development has been halted due to
cardiotoxicity in laboratory animals ((58) and reviewed recently by de Bruijne et al (9)).
Research into antiviral drugs and vaccines has been hampered by the lack of a full viral
life cycle cell culture system. Only recently a full length HCV cell culture system in which
infectious virus can be generated in Huh7 cells from transfection of complete HCV genomic
RNA sequences has been described (26, 59). Viable JFH1-based intergenotypic recombinants D
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containing genotype specific structural proteins (core, E1, E2), p7 and NS2 have been developed n
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for all seven genotypes (14, 15, 21, 38, 45, 65), which allow the study of vaccines and entry d
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inhibitors for all genotypes. However, full length HCV cell culture systems allowing the study of fr
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the NS3 protease are currently only available for genotype 2a (JFH1 and HC-J6) (26, 34, 59) and h
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1a (H77), which needs adaptive mutations to replicate efficiently (64). The limited number of :/
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replication competent full length reference sequences limits the assessment of how genetic a
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variation between the different genotypes and within subtypes influence susceptibility to .o
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antiviral therapy and development of resistance. o
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The aim of the current study was to develop effective cell culture systems for the six e
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major genotypes of HCV, to compare the susceptibility of each to the protease inhibitor (PI), m
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BILN 2061, and through passaging in sub-inhibitory concentrations of the drug, to compare the r
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ability and mechanism of antiviral resistance development between genotypes. The use of 2
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replicon vectors, where protease genes of different genotypes were inserted into the genotype 1b 8

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replicon, has been demonstrated in several reports (3, 40). Other approaches involve the release g
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of reporter molecules upon NS3/4A cleavage (29). None of these methods however construct t
infectious virus that would allow the whole replication cycle of HCV to be analysed.
Recently, the full-length replication competent clone Jc1 (pFK JFH1/J6/C-846) has been
developed. This clone comprises the HCJ6 core and envelope coding sequences and a portion of
the NS2 gene, with the remainder of the polyprotein derived from JFH-1 (26, 38). It replicates
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autonomously and yields high infectious titres in the Huh7.5 cells. It was therefore chosen as a
backbone for the construction of the intra- and intergenotypic recombinants in the current study,
and enabled the activities of HCV protease inhibitors against different genotypes and diverse
natural isolates to be directly assessed. The development of these replication competent
intergenotype chimaeras will improve the ability to predict clinical doses, efficacy and
development of drug resistance mutations in a diverse range of HCV variants circulating D
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worldwide. n
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MATERIALS AND METHODS fr
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HCV plasmids, sequences and clinical specimens. pJFH1 and pJFH1-GND (AB047639) used :/
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in the construction of the intra- and intergenotypic recombinants were provided by T. Wakita a
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(Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan) (59) and pFK JFH1/J6/C-846_dg .o
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(Jc1) by R. Bartenschlager (Department of Molecular Virology, University of Heidelberg, o
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Heidelberg, Germany) (38). pJ6CF (AF177036) and pH77* (differs to pH77 (AF011751) at e
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M1205T) were provided by J. Bukh (NIH, Hepatitis Viruses Section, National Institute of m
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Health, Bethesda, Maryland) (62, 63) and HCV3a-Gla (p3a) by E.A. McCruden (Division of r
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Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK) (47). 2
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HC-J4 (p1b, D10750) was described by Okamoto et al. (35). ED43* (p4a), differs to ED43 8

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(NC_009852) in 4 amino acids (T1048A, T1064I, I1160T, R1176A); EUH1480* (p5a) differs to g
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EUH1480 (NC_009826) by 9 amino acids (L1045V, F1061V, I1072T, L1081V, K1117T, t
G1118R, R1122P, I1694V, T1695I) and EUHK2* (p6a) differs to EUHK2 (Y12083) in 6 amino
acids (D1065A, V1070L, P1085A, F1087S, K1094R, I1196V). Plasmids p4a, p5a and p6a were
provided by Richard Elliot (Centre for Biomolecular Sciences, University of St Andrews, St
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Andrews, UK). Differences in clones to prototype sequences were present in received clones and
likely arose during cloning.
Sequences of HCV isolates used for sequence diversity analysis were retrieved from the
HCV sequence database (22) and the NCBI GenBank. 570 NS3 and NS4A sequences of
genotype 1a; 459 NS3 sequences of genotype 1b; 242 NS3 and 180 NS4A sequences of
genotype 3a: 39 NS3 sequences of genotype 4a and 15 NS3 sequences of genotype 6a were D
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analysed. Anonymised research samples of residual plasma from previous investigations of HCV n
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epidemiology and treatment response were used for estimation of sequence diversity in NS3. d
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Cell Culture. Huh-7.5 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, h
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Invitrogen) supplemented with 4500 mg/l glucose, 2mM L-glutamine, 10% heat-inactivated fetal :/
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calf serum (Harlan Sera-Lab), non-essential amino acids, 20 mM Hepes, 100 U/ml penicillin and a
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100 µg/ml streptomycin and incubated at 37˚C, 5% CO and 100% relative humidity. Cells were .o
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split every second to third day at a ratio of 1:2 to 1:3. o
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Construction of pJFH1- and Jc1-Based Intra- and Intergenotypic Recombinants m
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a) Replacement of NS3 Protease Gene (Fx/Jx). All nucleotide positions are referred to r
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according to their position in the H77 reference position, AF009606. To construct F1a, F1b, F2a, 2
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F3a, F4a, F5a and F6a a BstBI restriction site was generated at the junction of NS2 and NS3 of 8

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the pJFH1 plasmid and a BglII restriction enzyme site at the junction of the NS3 protease and g
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NS3 helicase domain. To reduce the possibility of PCR errors, the NS3 gene was subcloned into t
Zero Blunt TOPO using the naturally occurring restriction sites NotI and SpeI. QuickChange Site
Directed Mutagenesis kit (Stratagene) was used to introduce two silent point mutations to
generate a BstBI (C3398T and C3401G) and one to create a BglII restriction site (G3993A). Each
of the introduced base changes was verified by DNA sequence analysis. The NS3 protease
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region was amplified of pH77* with primers 1aBstBI and 1aBglII, of pJ6CF with primers
2aBstBI and 2aBglII and so on. A list of all the primers used can be found in the Supplementary
Material. Primers were designed to include JFH1 sequence of the NS2 region and the
corresponding intergenotypic sequence of the NS3 protease region. Products were digested with
BstBI and BglII (all restriction enzymes are from New England Biolabs), gel purified (Gel
extraction kit, QIAGEN) and ligated into pJFH1. pJFH1 has similarly been digested with BstBI D
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and BglII, dephosphorylated using alkaline phosphatase (New England Biolabs) and gel purified. n
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Each introduced NS3 protease gene was verified by DNA sequence analysis. To generate the d
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corresponding Jc1 based chimaeras (Jx), recombinant plasmids were digested with NotI and SfiI, fr
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gel purified and ligated into Jc1 which was digested with NotI and SfiI and dephosphorylated. h
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b) Replacement of NS3 Protease and NS4A Cofactor Gene (Fxx/Jxx) a
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To generate F1a1a, F1b1b, F2a2a, F3a3a, F4a4a, F5a5a and F6a6a two new restriction sites were .o
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introduced into pJFH1. To reduce the possibility of PCR errors, the NS4A was subcloned into o
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Zero Blunt TOPO using the naturally occurring restriction sites NsiI and XbaI. A BlpI restriction e
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site was generated by introducing a silent point mutation (C5297G) at the NS3 helicase and m
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NS4A junction and a MluI site at the NS4A and NS4B junction by introducing 4 point mutations r
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(T5478A, T5480G, A5481C, G5483T), were one was non-silent. The NS4A region was 2
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amplified of pH77* with primer 1aSapI and 1aMluI, introducing a SapI (5′-end) and a MluI (3′- 8

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end) restriction site. The same strategy was used to amplify NS4A from genotype 1a-6a g
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prototype plasmids using the corresponding genotype specific primers (primer sequences can be t
found in the Supplementary Material). Products were digested with SapI and MluI, gel purified
and ligated into the TOPO vector containing the JFH1 insert digested with BlpI and MluI and
dephosphorylated. A5478 was mutated back to T by site directed mutagenesis to recreate native
JFH1 amino acid sequence (Ala to Thr) outside of the NS4A region. To generate Fxx and Jxx,
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TOPO vector including the modified insert was digested with NotI and SfiI, gel purified and
ligated into pJHF1 and Jc1 respectively digested with NotI and SfiI and dephosphorylated.

Amplification of NS3 Protease Gene from Study Subject Plasma. HCV RNA was isolated
from 150 µl study subject plasma using the QIAGEN RNeasy Kit according to the manufactures
guidelines. Extracted RNA was eluted into 30 µl of RNase-free water. To generate cDNA and D
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primary PCR product, 0.75 µM of genotype specific outer primers (NS3p1a/1b/3a/4a/6aOS, n
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NS3p1a/1b/3a/4a/6aOA, sequences see Supplementary Material) and 10 µl of extracted RNA d
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were used in a 50 µl reaction using SuperScript III One-Step RT-PCR System with Platinum Taq fr
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DNA Polymerase (Invitrogen) as recommended by the manufacturer. The PCR protocol h
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consisted of an RT step at 43˚C for 1 h, followed by 20 cycles of, 53˚C for 1 min; 55˚C for 1 :/
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min; and final extension at 70˚C for 15 min. Subsequent to a denaturation step at 94˚C for 2 min, a
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35 cycles of, 94˚C for 30 sec; 54˚C for 30 sec; 68˚C for 2 min; and a final extension at 68˚C for 5 .o
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min were performed. The first round PCR products were used as templates in a nested secondary o
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PCR reaction using genotype specific primers (1a/1b/3a/4a/6aBstBIs and 1a/1b/3a/4a/6aBglIIas) e
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introducing BstBI (5’end) and BglII (3’end) restriction sites. Nested PCR was performed using m
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the high fidelity KOD hot start polymerase (Novagen) as recommended by the manufacturer. r
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Each PCR product was sequenced to obtain a consensus sequence of the corresponding NS3 2
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protease gene. The nested PCR products were treated with BstBI and BglII and gel purified. To 8

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remove the NS3 protease gene, Jxx’s including prototype NS3 protease genes were likewise g
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digested with BstBI and BglII, dephosphorylated and gel purified. Purified study subject-derived t
NS3 protease genes were then ligated into the corresponding Jxx, creating intergenotypic
chimaeras where the NS3 protease gene is study subject-derived and the NS4A cofactor is of
prototype sequence.

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Construction of Adapted Genomes. RNA was extracted from infectious supernatant using the
QIAmp viral RNA mini kit (QIAGEN) according to the manufactures protocol. If no infectious
supernatant was generated RNA was alternatively extracted from cell pellets using QIAGEN
shredder columns followed by a QIAGEN RNeasy Kit as described above. A PCR product was
generated using Superscript III as described above. JFH-NotI and JFH-SpeI were used to amplify
a 1.2-kb-long fragment encompassing the HCV NS3 protease, JFH-5230 and JFH-5536 for a D
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300-bp-long fragment encompassing the NS4A cofactor. The PCR-product was then subjected to n
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bulk sequence determination. To generate J2a2a- (J2a2a- ), J5a5a- d
C3538G T1066S C3416G, T3968C, A4081T e
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(J5a5a-Q1247L) and J6a6a-A3458G, G3459T (J6a6a-V1040L) the fragment encompassing the HCV NS3 fro
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protease was digested with SpeI and NotI and cloned into the corresponding (J2a2a, J5a5a or h
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J6a6a) recombinant plasmids. To generate J3a3a-3- (J3a3a-3- ) and J3a3a-8- :/
C5328G, T5329C L1663A /
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(J3a3a-8- ) point mutations were introduced by site-directed mutagenesis and a
C5328G, T5329C L1663A s
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cloning. Modified fragments were verified by sequencing. .o
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RNA Synthesis and Transfection. Plasmid templates were linearized by XbaI (for pJFH1 and e
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pJFH1 chimaeras) or MluI (for Jc1 and Jc1 chimaeras) digestion and treated with Mung Bean m
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Nuclease (New England Biolabs) to remove 5′ end overhangs. The linearized DNA template was r
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cleaned by phenol/chloroform extraction following ethanol precipitation. RNA was synthesised 2
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from 1 µg DNA template with T7 RNA Polymerase (Promega) for 1 h at 37˚C. Following 8

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treatment with RNase-free DNase, RNA was cleaned up using the RNeasy Kit (QIAGEN) and g
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the integrity of the RNA analyzed by non-denaturing agarose gel electrophoresis. RNA t
concentrations were determined using spectrophotometry and 10 µg aliquots were stored at
-80˚C.
RNA was transfected into Huh7.5 cells by electroporation. Huh7.5 cells were washed
with phosphate-buffered saline (PBS) and detached with trypsin. Cells were pelleted by
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centrifugation (1,600 rpm for 7 min at 4˚C), then resuspended in 10 ml chilled DEPC-treated
PBS, counted and washed 3 times with chilled DEPC-treated PBS (1,600 rpm for 4 min at 4˚C),
then chilled on ice for at least 5 min. 10 µg of RNA was mixed with 5 x 106 cells suspended in
400 µl of PBS and transferred to an electroporation cuvette (0.4-cm gap width, Bio-Rad, Munich,
Germany). Electroporation consisted of one square wave pulse for 25 ms of current delivered by
the Bio-Rad Gene Pulser Xcell electroporation device, set at 150V. Transfected cells were D
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immediately resuspended in 4.5 ml 50:50 mix of conditioned and fresh media (containing 10% n
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FCS), then transferred into T25 flasks containing 10 ml complete growth medium or seeded into d
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24-well plates for NS5A immunostaining and incubated at 37˚C, 5% CO2 and 100% relative fro
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humidity. Cells were passaged every 3 to 4 days by trypsinisation and reseeding with a 1:3 to 1:4 h
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split ratio into fresh culture vessels or 24-well plates for NS5A immunostaining. Virus :/
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containing supernatant was collected, cleared of cell debris by centrifugation and stored at 4˚C a
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overnight or at -80˚C for long term. .o
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Immunohistochemistry Staining for HCV NS5A. Viral replication was assessed by NS5A e
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staining. Electroporated cells were seeded into 24-well plates containing cover slips and m
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immunostained for NS5A when they were subconfluent. Following fixation of cells in 4% r
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paraformaldehyde for 20 min, cells were washed 3 times with PBS, then permeabilized using 2
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0.1% Triton-X 100 in PBS for 7 min. Following 2 further washes with PBS, cells were incubated 8

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with an in-house derived polyclonal sheep anti-NS5A serum (provided by Mark Harris, g
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University of Leeds) diluted 1:5000 in 10% FCS PBS for 1 hour. After cells were washed 2 t
times with PBS, bound NS5A-specific antibody was detected by 1 h incubation with Alexa Fluor
488 donkey anti-sheep IgG (Invitrogen) diluted 1:1000 in PBS. Cells were further washed 2
times before NS5A-positive cells were detected using a fluorescence microscope. Three images
per coverslip were taken from two coverslips per sample and the percentage of HCV-positive
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