aac accepts, published online ahead of print on 18...

1

Robust and Persistent Replication of Genotype 6a Hepatitis C Virus Replicon in 1

Cell Culture 2

Short Title – Development of Genotype 6a HCV Replicons 3

Mei Yu, Betty Peng, Katie Chan, Ruoyu Gong, Huiling Yang, William Delaney IV, 4

Guofeng Cheng 5

Biology Department, Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404 6

Grant Support - none 7

Abbreviations - List alphabetically abbreviations not mentioned in the Style Guide, 8

which follows the Instructions to Authors. 9

Correspondence - Guofeng Cheng, Gilead Sciences, 333 Lakeside Dr. Foster City, CA 10

94404. [email protected] Tel: 650 522-4236 Fax: 650 522-5890 11

12

Disclosures- All authors on this manuscript are stockholders and employees of Gilead 13

Sciences. This manuscript contains information describing the in vitro activity of Gilead 14

compounds currently in development as treatments for HCV. 15

Transcript Profiling-none 16

Writing Assistance- none 17

Author Contributions - MY was involved in study design, data acquisition, and 18

manuscript preparation; BP was involved in study design and data acquisition; KC was 19

involved in analysis and interpretation of data, and manuscript preparation; RG and HY 20

were involved in acquisition and analysis of data; WD and GC were involved in study 21

concept, analysis and interpretation of data, study supervision and manuscript 22

preparation. 23

AAC Accepts, published online ahead of print on 18 February 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.01780-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.

on July 27, 2018 by guesthttp://aac.asm

.org/D

ownloaded from

http://aac.asm.org/

2

Abstract 24

Genotype (GT) 6 hepatitis C virus (HCV) is prevalent in Southeast Asia and Southern 25

China, where it can constitute up to 50% of HCV infections. Despite this, no direct-acting 26

antivirals are approved to treat GT6 HCV and no cell culture systems have been 27

described. In this study we aimed to develop a GT6 HCV subgenomic replicon to 28

facilitate the identification and development of new HCV therapies with pan-genotype 29

activity. A subgenomic replicon cDNA encoding a GT6a consensus sequence plus an 30

NS5A amino acid substitution S232I was synthesized. Electroporation of RNA encoding 31

the GT6a replicon into Huh7-derived cells consistently yielded 20-100 stable replicon 32

colonies. Genotypic analyses of individual replicon colonies revealed new adaptive 33

mutations across multiple viral nonstructural proteins. E30V and K272R in NS3 and 34

K34R in NS4A were observed most frequently and were confirmed to enhance GT6a 35

replication in the presence of the NS5A amino acid substitution S232I. These new 36

adaptive mutations allowed establishment of robust luciferase-encoding GT6a replicons 37

for reproducible quantification of HCV replication; luciferase-encoding replicons enabled 38

efficient determination of antiviral activity for HCV inhibitors in a 384-well assay 39

format. While nucleos/tide NS5B inhibitors and cyclophilin A inhibitors had similar 40

antiviral activity against both GT6a and GT1b, some NS5B non-nucleoside inhibitors, 41

NS3 protease inhibitors and NS5A inhibitors had less antiviral activity against GT6a. In 42

conjunction with other genotype replicons, this robust GT6a replicon system will aid in 43

the development of pan-genotypic HCV regimens. 44

Keywords: genotype 6a HCV replicon; K272R in NS3; K34R in NS4A; pan-genotype; 45

direct-acting antivirals; 46

47


.org/D

ownloaded from

http://aac.asm.org/

3

INTRODUCTION 48

Chronic hepatitis C virus (HCV) infection affects an estimated 170 million people 49

worldwide and represents a significant global health burden (1, 2). Until recently, the 50

standard of care was 24- to 48-week courses of pegylated interferon-α (PegIFN) plus 51

ribavirin (RBV) (3). Due to the partial efficacy and poor tolerability of this regimen, the 52

discovery and development of new antiviral agents has been intensely pursued. These 53

efforts have culminated in the recent FDA approval of two NS3 protease inhibitors 54

(boceprevir and telaprevir) for use in combination with PegIFN and RBV for the 55

treatment of chronic genotype (GT) 1 HCV infection (4). 56

HCV is a positive-strand RNA virus that exhibits extraordinary genetic diversity. Six 57

major genotypes (i.e. genotypes 1-6) along with multiple subtypes (e.g. genotypes 1a, 1b) 58

have been reported (5). Genotype 1, 2 and 3 are common throughout the world (6-8). 59

However, GT6 is prevalent in Southeast Asia and Southern China and can constitute up 60

to 50% of HCV infections in many of these areas (9, 10). Despite its limited geographical 61

presence, GT6 represents a significant portion of the global unmet medical need 62

associated with chronic HCV infection, due to the high HCV disease burden in Southeast 63

Asia and Southern China (more than 32 million people infected). Furthermore, in contrast 64

to North America and Europe, new incidence of HCV infection also remains high in 65

these regions due to higher risk of exposure to contaminated blood products and 66

intravenous drug use (9, 10). 67

Currently, the standard treatment for HCV GT6 patients remains PegIFN and RBV for 24 68

to 48 weeks (10). Although GT6 infection is more responsive to PegIFN/RBV than GT1 69


.org/D

ownloaded from

http://aac.asm.org/

4

(sustained virologic response 86% and 52%, respectively) (11), this treatment is still 70

partially efficacious and contra-indicated in many patients. No direct-acting antivirals 71

(DAAs) have been approved to treat GT6 HCV infection (4). Many HCV DAAs are in 72

advanced clinical development, but few are being developed to treat GT6 infections. 73

Thus, there is an urgent need to develop novel therapeutic agents for the treatment of 74

chronic GT6 HCV infection. This need also aligns with the tremendous interest in 75

developing “pan-genotypic” drugs that are active against all HCV genotypes to simplify 76

the treatment of HCV (12, 13). 77

GT6 is the most genetically diverse HCV genotype, with at least 23 currently known 78

subtypes and new subtypes expected to be identified continuously (14). It is well 79

documented that individual HCV genotypes respond differently to direct-acting antivirals 80

due to high HCV genetic diversity between and within genotypes (3, 15). For example, 81

essentially all HCV NS3 protease inhibitors, although potent against GT1, have 82

significantly reduced antiviral activity against GT3; this is largely due to GT3 83

polymorphs at known drug resistance sites within NS3 protease including residue 168 84

(16). For NS5A inhibitors, earlier compounds often inhibit GT2a JFH-1 virus efficiently 85

but have much weaker antiviral activities (> 200-fold) against more common GT2 strains 86

encoding the M31 polymorph in NS5A (17). The considerable genetic diversity of GT6 87

combined with a limited virological characterization compared to common GT1 strains, 88

create significant challenges to DAA development against this genotype. Meeting this 89

challenge will require the establishment of efficient GT6 tools for the identification and 90

development of new therapies. 91


.org/D

ownloaded from

http://aac.asm.org/

5

Hepatitis C virus (HCV) replicons are self-replicating viral RNAs that have served as 92

workhorses for molecular virology studies and drug discovery (18). These replicons have 93

been crucial in the identification of novel inhibitor classes, the optimization of clinical 94

candidates, and the characterization of clinical resistance. Despite initial successes in 95

generating replicons derived from genotypes 1a, 1b, or 2a (19-21), it has proven difficult 96

to generate efficiently replicating RNAs from other genotypes (22, 23). Not until very 97

recently, GT3 and GT4 replicons were successfully established, representing a significant 98

expansion of virological tools for HCV research and drug development (24-26). 99

Here we report the establishment of the first GT6a replicons that efficiently replicate in 100

hepatoma cell lines. We demonstrate that robust replication requires novel adaptive 101

mutations either in HCV NS3 or NS4A combined with the NS5A amino acid substitution 102

S232I. By incorporating adaptive mutations into luciferase encoding constructs, efficient 103

replication of GT6a HCV RNA was established in both stably replicating and transiently-104

transfected cultures. The replicons support efficient activity profiling of antiviral 105

compounds and will provide as valuable tools for molecular virology and drug 106

development applications.107


.org/D

ownloaded from

http://aac.asm.org/

6

MATERIALS AND METHODS 108

Cell culture. Huh-7 Lunet cells were obtained from ReBLikon GmbH (Mainz, 109

Germany) (27). 51C cells were derived by curing a Huh-7 Lunet-based GT1a replicon 110

clone and were described previously (28). 1C cells were derived by curing a GS-5885-111

resistant GT1a replicon clone derived from 51C cells, and showed much higher 112

permissiveness to GT1a replicon replication (24). All cell lines were propagated in 113

Dulbecco’s modified Eagle’s medium (DMEM) as described previously (29). Replicon 114

cell lines were selected and maintained in complete DMEM containing 0.25 to 0.5 mg/ml 115

G418 (Geneticin; Invitrogen). 116

Construction of plasmids encoding GT6a HCV subgenomic replicons. A plasmid 117

(pGT6aNeoSG) encoding a subgenomic GT6a replicon based on the consensus sequence 118

(Supplementry Figure S1) of sixteen GT6a genomes available in the European HCV 119

database (Supplementary Table S1), was prepared by DNA synthesis and cloning 120

(GeneScript, Piscataway, NJ). The synthesized replicon incorporated following elements 121

from 5′ to 3′ (Fig. 1): (1) the GT6a consensus 5′UTR (342 nt), plus the first 48 122

nucleotides of core, (2) a linker with the nucleotide sequence 5′-GGCGCGCCCA-3′, 123

which introduces the AscI restriction site (underlined), (3) the neomycin 124

phosphotransferase II (neo) gene, (4) a linker with nucleotide sequence 5′-125

GGCCGGCCA-3′, which introduces the FseI restriction site (underlined), (5) the 126

encephalomyocarditis virus (EMCV) IRES, (6) a linker with nucleotide sequence 5′-127

ACGCGTATG-3′, which introduces the MluI restriction site (underlined) and an ATG 128

start codon for HCV polyprotein expression, (7) the NS3 – NS5B polyprotein region of 129

GT6a consensus sequence including an NS5A amino acid substitution S232I (equivalent 130


.org/D

ownloaded from

http://aac.asm.org/

7

to S2204I in GT1 polyprotein), and (8) the 3′-UTR of GT2a JFH-1 (239 nt; no GT6a 3’-131

UTR available). The synthetic DNA fragment encoding the GT6a replicon was inserted 132

into pUC57 between EcoRI and XbaI restriction sites. 133

A second plasmid (pGT6aRlucNeoSG) encoding a subgenomic replicon that incorporated 134

the humanized Renilla luciferase (hRluc) reporter gene was generated as follows: the 135

pGT6aNeoSG plasmid (described above) was cut using AscI and MluI restriction 136

enzymes (to remove the neo gene) and gel purified using a commercial kit (Qiagen, 137

Valencia, CA). A gene fragment encoding the hRluc gene fused with the neo gene along 138

with the EMCV region from phRlucNeoSG2a plasmid (described below) were PCR 139

amplified using Accuprime super mix I (Invitrogen) with the following primers: 140

2aRlucNeoAscIFor: 5′-AAC ACC ATC GGC GCG CCC ATG GCT TCC AAG GTG 141

TAC GAC -3′ (AscI site is introduced by the primer and is underlined), 142

2aEMCVIRESMluIRev: 5′ -TCGGGG CCA TAC GCG TAT CGT GTT TTT CAA 143

AGG -3′ (MluI site underlined). The subsequent PCR fragment was cut with AscI and 144

MluI and gel purified using a commercial kit (Qiagen). The vector and insert pieces were 145

ligated using the LigaFast Rapid DNA Ligation System per the manufacturer’s protocol 146

(Promega, Madison, WI). The resulting vector, pGT6aRlucNeoSG, was sequenced to 147

confirm the correct orientation and sequence of the hRluc-Neo. The phRlucNeoSG2a 148

plasmid was constructed by replacing the Luc-Neo fragment in the plasmid 149

pLucNeoSG2a with the hRluc-Neo gene amplified from the plasmid hRluc-Neo Flexi(R) 150

(Promega) as previously described (28, 29). 151

A third plasmid (pGT6aPiRlucSG), encoding a bicistronic replicon with the hRluc 152

reporter gene downstream of the poliovirus IRES (Pi) and the GT6a consensus HCV 153


.org/D

ownloaded from

http://aac.asm.org/

8

nonstructural genes (NS3-NS5B) downstream of the EMCV IRES was used for transient 154

transfection studies. The plasmid was generated as follows: The pGT6aRlucNeoSG 155

plasmid (described above) was cut using AscI and MluI restriction enzymes (to remove 156

the Rluc-Neo gene) and gel purified using a commercial kit (Qiagen). A gene fragment 157

encoding the PI, hRluc gene and EMCV region was PCR amplified from a GT1b Pi-Rluc 158

plasmid using Accuprime super mix I (Invitrogen) with the following primers: 159

6aPiRlucAscIFor: 5′-AAC ACC ATC GGC GCG CCA AAC CAA GTT CAA TAG-3′ 160

(AscI site is introduced by the primer and is underlined), 1bEMCVIRESMluIRev: 5′-161

TCG GGG CCA TAC GCG TAT CGT GTT TTT CAA AGG -3′ (MluI site underlined). 162

The subsequent PCR fragment was cloned and ligated as described above to generate the 163

resulting vector, pGT6aPiRlucSG. 164

Construction of mutant replicons. Adaptive mutations were introduced into the 165

pGT6aNeoSG, pGT6aRlucNeoSG or pGT6aPiRlucSG replicons by site-directed 166

mutagenesis using a QuikChange Lightening kit (Stratagene, La Jolla, CA). A 167

replication-defective NS5B polymerase GND mutant was constructed by introducing a 168

point substitution in the nucleotide sequence that encodes the amino acid 318 in NS5B 169

from GAT to AAT, therefore altering the GDD motif of the NS5B polymerase active site 170

to GND and abolishing the polymerase activity (GenScript) (20). All substitutions were 171

confirmed by DNA sequencing by Elim Biopharm (Hayward, CA). 172

RNA transcription. Plasmids encoding GT6a subgenomic HCV replicons were 173

linearized with XbaI and purified using a PCR purification kit (Qiagen). RNA was 174

synthesized and purified with T7 MEGAScript (Ambion, Austin, TX) and RNeasy kits 175

(Qiagen), respectively, according to the manufacturer’s instructions. RNA concentrations 176


.org/D

ownloaded from

http://aac.asm.org/

9

were measured using optical density at 260 nm and confirmed by 0.8% agarose gel 177

electrophoresis (Invitrogen). 178

RNA transfection and isolation of stable replicon cell lines. Ten micrograms of in 179

vitro-transcribed RNA were transfected into Huh-7 Lunet or 1C cells by electroporation 180

as previously described (28). Briefly, cells were collected by trypsinization and 181

centrifugation, then washed twice with ice-cold phosphate buffered saline (PBS) and 182

resuspended in Opti-MEM medium (Invitrogen) at a concentration of 107 cells/ml. 183

Replicon RNA was added to 400 μl of cell suspension in a Gene Pulser (BioRad, 184

Hercules, CA) cuvette (0.4-cm gap). Cells were electroporated at 270 V and 960 μF, 185

incubated at room temperature for 10 minutes, resuspended in 30 ml complete DMEM 186

and then plated into two 100-mm-diameter dishes. Forty-eight hours after plating, 187

medium was replaced with complete DMEM supplemented with 0.25 mg/ml G418, 188

which was refreshed twice per week. After three weeks, cell clones were isolated, 189

expanded with 0.5 mg/ml G418, and cryopreserved at early passages. 190

Replicon colony formation assays. To determine the efficiency of replicon colony 191

formation, cells were electroporated with the indicated amounts of replicon RNA or 192

extracted cellular RNA, and plated at multiple densities ranging from 2 × 105 to 2 × 106 193

cells/100-mm dish. Forty-eight hours after plating, media were replaced with complete 194

DMEM supplemented with 0.5 mg/ml G418, which was refreshed twice per week. Three 195

weeks later, colony plates were used for cell expansion or G418-resistant foci were fixed 196

with 4% formaldehyde and stained with 0.05% crystal violet. 197


.org/D

ownloaded from

http://aac.asm.org/

10

Extraction, amplification, and genotypic analysis of HCV RNA. HCV RNA isolation, 198

RT-PCR, and population sequencing were performed as described previously (29). 199

Briefly, HCV replicon cellular RNA were extracted and purified using an RNeasy kit 200

(Qiagen) according to the manufacturer’s protocol. RT-PCR was performed using the 201

SuperScript III first-strand synthesis system (Invitrogen), and PCR products were 202

subsequently sequenced by Elim Biopharm (Hayward, CA). 203

Replicon Cell NS3-4A Protease Assay. Replicon cells were seeded in 96-well plates at a 204

concentration of 1 x 104 cells per well. The cells were incubated for 24 hours, after which 205

culture media were removed. Replicon cells were then lysed with 90 µl of 1x Promega 206

luciferase lysis buffer supplemented with 150 mM NaCl at room temperature for 20 min 207

on a plate shaker. 10 µl of 1 µM europium-labeled NS3-4A substrate diluted in the above 208

lysis buffer was added to each well. Protease activity data were collected and analyzed as 209

previously described (29). 210

211

Detection of NS5A protein by indirect immunofluorescence. Replicon cells were 212

plated in 96-well plates at a density of 1 × 104 cells per well. After incubation for 24 213

hours, cells were then stained for NS5A protein as described previously (29). Briefly, 214

cells were fixed in 4% paraformaldehyde for 20 minutes. Cells were then washed three 215

times with PBS, blocked with 3% bovine serum albumin, 0.5% Triton X-100, and 10% 216

FBS. NS5A protein staining was performed using a 1:10,000 dilution of mouse 217

monoclonal antibody 9E10 (Apath, Brooklyn, NY). After washing in PBS three times, a 218

secondary anti-mouse antibody conjugated to Alexa Fluor 555 was used to detect anti-219

NS5A antibody labeled cells (Invitrogen). Nuclei were stained with 1 µg/ml Hoechst 220


.org/D

ownloaded from

http://aac.asm.org/

11

33342 (Invitrogen). Cells were washed with PBS and imaged with a Zeiss fluorescence 221

microscope (Zeiss, Thornwood, NY). 222

Replicon antiviral assays. Replicon RNA were electroporated into 1C cells as described 223

above. After transfection, cells were quickly transferred into 100 mL of pre-warmed 224

culture medium, and 90 µl was seeded in 384-well plates at a density of 2,000 cells/well. 225

Cells were treated with three-fold serial drug dilutions at 10 different concentrations. Cell 226

plates were incubated at 37°C for 3 days, after which culture medium was removed and 227

cells were assayed for luciferase activity as markers for replicon levels. Luciferase 228

expression was quantified using a commercial luciferase assay (Promega). Luciferase 229

levels were converted into percentages relative to the levels in the untreated controls 230

(defined as 100%), and data were fitted to the logistic dose-response equation y = 231

a/[1+(x/b)c] using XLFit4 software (IDBS, Emeryville, CA). 232

Antiviral compounds. Telaprevir, boceprevir, and 2-C-methyl adenosine (2-CMeA) 233

were purchased from Acme Bioscience (Belmont, CA). Cyclosporine A (CsA) was 234

purchased from Sigma-Aldrich (St. Louis, MO). The Wyeth HCV NS5B site IV inhibitor 235

HCV-796 was synthesized by Curragh Chemistries (Cleveland, OH). Ledipasvir (GS-236

5885) (30), tegobuvir (GS-9190) (31), GS-9451 (32), GS-9669 (33), and sofosbuvir (GS-237

7977) (34), Pfizer NS5B thumb site II inhibitor filibuvir, Merck NS5B thumb site I 238

inhibitor MK-3281 and protease inhibitor MK-5172, and the Bristol-Myers Squibb NS5A 239

inhibitor daclatasvir (BMS-790052) were synthesized by Gilead Sciences. 240

241


.org/D

ownloaded from

http://aac.asm.org/

12

RESULTS: 242

Construction of a GT6a subgenomic replicon. The consensus sequence from sixteen 243

GT6a genomes available in the European HCV database was used to construct a GT6a 244

subgenomic replicon (GT6a-Neo) as previously described by Lohman et al (22) (Figure 245

1A). Due to the lack of complete GT6a 3’-UTR sequence available in the database, the 246

3’-UTR sequence from the highly replicative GT2a HCV isolate JFH-1 was used to 247

construct the GT6a replicon. In addition, the NS5A amino acid substitution S232I, which 248

was previously shown to enhance the replication of HCV replicons for multiple 249

genotypes, was included to determine if it could enhance the basal replication of the 250

GT6a replicon (23, 24, 26). 251

252

Selection of stable GT6a subgenomic replicon colonies. In vitro transcribed GT6a-Neo 253

replicon RNAs with or without the NS5A amino acid substitution S232I were 254

electroporated into Huh7-derived Lunet or 1C cells and subjected to G418 selection until 255

stable colonies emerged. In the absence of the S232I, no G418 resistant colonies emerged 256

in either cell background (Figure 2A). In contrast, in the presence of the S232I a modest 257

number of G418-resistant colonies was selected in both Lunet cells (average of 35 258

colonies per transfection) and 1C cells (average of 75 colonies per transfection) (Figure 259

2A). These results indicate that the NS5A amino acid substitution S232I enhances the 260

replication of the parental GT6a replicon and was vital to the selection of stable replicon 261

colonies. Colony formation efficiency was consistently 2-fold greater in 1C cells 262

compared to Lunet cells, suggesting that 1C cells provided a slightly more permissive 263

environment for GT6a replication. 264


.org/D

ownloaded from

http://aac.asm.org/

13

To confirm that the selected colonies harbored replicating GT6a replicons, colonies were 265

pooled and assessed for their intracellular NS3-4A expression by measuring NS3/4A 266

protease activity (Fig. 2B) and NS5A expression by immunofluorescence staining (Fig. 267

2C). Both the pooled GT6a-neo 1C and GT6a-neo Lunet colonies had readily detectable 268

NS3-4A protease activity, producing signal to background ratios >100-fold and signals 269

within 2-fold of those produced by a highly adapted GT1b replicon (28). Similarly, 270

NS5A expression was also readily detectable when the pooled cells were stained with an 271

anti-NS5A antibody (Fig. 2C). Together these results suggest that self-replicating GT6a 272

subgenomic replicons were successfully established in these stable colonies. 273

274

Identification of adaptive mutations for GT6a subgenomic replicons. To determine 275

whether adaptive mutations were acquired by the replicons in the selected colonies, 11 276

Lunet and 12 1C stable colonies were isolated and expanded for genotypic analysis. A 277

diverse set of mutations were identified in the viral nonstructural genes (full list of 278

emerging mutations provided in supplementary Table S2). Seventeen of 23 clones had 279

single mutations in the NS3 gene including one with an additional mutation in NS5A. 280

Five clones had a mutation in NS4A gene and one had a single mutation in NS5B. 281

Furthermore, all replicon clones retained the NS5A amino acid substitution S232I, 282

suggesting this NS5A S232I may be essential for the GT6a replicon replication. 283

284

Interestingly, single mutations of E30V or K272R in NS3 or K34R in NS4A appeared 285

independently at least twice. To determine if these three mutations enhanced replicon 286

replication, they were introduced individually into a GT6a renilla luciferase neo construct 287


.org/D

ownloaded from

http://aac.asm.org/

14

(GT6a-RlucNeo; Fig. 1B) harboring the S232I amino acid substitution. In vitro 288

transcribed RNA from each mutant was electroporated into permissive Huh-7 Lunet cells 289

and subjected to G418 selection until colonies were visible (Fig. 3). Incorporation of 290

renilla luciferase to the GT6a-Neo replicon resulted in significant reduction of stable 291

colony formation (35 for the GT6a-Neo replicon vs 1 colony for the GT6a-Rlucneo 292

replicon). Importantly, all three of the putative adaptive mutations significantly enhanced 293

replication of GT6a replicons as evidenced by the formation of large number of stable 294

colonies relative to the parental 6a-RlucNeo replicon (Fig. 3). These data indicate that the 295

mutations E30V and K272R in NS3 and K34R in NS4A function as adaptive mutations 296

for GT6a replicon replication. 297

298

Establishment of luciferase-encoding GT6a transient transfection replication 299

systems. Transient transfection replication systems facilitate rapid and direct analysis of 300

replication efficiencies and kinetics of HCV RNAs, independent of clonal cell 301

differences. A GT6a transient replication construct was generated by replacing the 302

RlucNeo gene in the parental GT6a-RlucNeo replicon with a Rluc reporter gene that is 303

much shorter and under translational control of a polivirus IRES (Pi-IRES), instead of the 304

HCV IRES (GT6a-PiRluc; Fig. 1C) (35). The three adaptive mutations identified in NS3 305

(E30V and K272R) and NS4A (K34R) were introduced individually into the GT6a-306

PiRluc replicon harboring the S232I amino acid substitution in NS5A. The impact of 307

each mutation on replication efficiency and kinetics was determined as shown in Figure 308

4. In vitro transcribed GT6a-PiRluc parental replicon RNA and replicons RNAs with 309

indicated mutations, along with the highly adapted GT1b PiRluc replicon, were 310


.org/D

ownloaded from

http://aac.asm.org/

15

electroporated into both Huh-7 Lunet and 1C cells. HCV replication was quantified 311

through measurement of luciferase activity in cell lysates over 7 days. All mutations in 312

either NS3 or NS4A significantly enhanced GT6a replication as evidenced by their higher 313

luciferase activity (> 30-fold) compared to the parental GT6a-PiRluc construct and 314

replication-defective NS5B polymerase GND mutant. Particularly in 1C cells, although 315

the GT6a-PiRluc replicons with adaptive mutations had slower replication kinetics 316

compared to the GT1b-PiRluc replicon, their replication capacity eventually all reached 317

to a level comparable to that of the GT1b-PiRluc by day 5 after transfection. These 318

results indicate that all three adaptive mutations in combination with S232I in NS5A 319

could support transient transfection replication studies of GT6a replicons in both Huh-7 320

Lunet and 1C cells. 321

322

Interestingly, the replication level of all GT6a-PiRluc replicons was approximately 10-323

fold lower in Huh-7 Lunet cells than in 1C cells (Fig. 4). This is in contrast to GT1b-324

PiRluc replicon that replicates at a similar level between these two Huh-7 derived cell 325

lines, suggesting that 1C cells are likely more permissive to GT6a replicon replication 326

than Lunet cells. To determine whether the adaptive functions of the selected mutations 327

were dependent on the NS5A amino acid substitution S232I, we reverted the S232I back 328

to wild-type (S232) in GT6a-PiRluc replicons encoding adaptive mutation NS3 K272R or 329

NS4A K34R. Figure 5 shows that in the absence of NS5A S232I, while the wild-type 330

sequence GT6a replicon has a very weak replication activity (~5-fold above the GND 331

negative control), neither NS3 K272R nor NS4A K34R alone enhanced the basal level 332

replication of GT6 replicon. On the contrary, the S232I alone could boost replicon 333


.org/D

ownloaded from

http://aac.asm.org/

16

replication ~ 10-fold. Together these data suggest the NS5A S232I is critical and that the 334

mutations K272R in NS3 and K34R in NS4A are synergistic with S232I for GT6a 335

replication. 336

Antiviral activity of known HCV inhibitors against GT6a. To evaluate GT6a 337

replicons for antiviral inhibitor screening, we assessed the response of two GT6a-PiRluc 338

replicons in parallel with the well-established GT1b-PiRluc replicon against five different 339

classes of HCV inhibitors (Table 1). 1C cells were transiently transfected with GT6a-340

PiRluc-NS3-K272R or GT1b-PiRluc HCV RNA and seeded in 384-well plates for 341

antiviral assays in high-throughput assay format. Compounds were added to the cells and 342

incubated for 72 hours, after which luciferase activity was measured to determine 343

replication levels. EC50 values were successfully generated for all inhibitors and are 344

shown in Table 1. Similar results were also generated with replicon GT6a-PiRluc-NS4A-345

K34R (EC50 values varying < 2-fold from those generated with GT6a-PiRluc-NS3-346

K272R, data not shown). Cyclosporin A (CsA), which targets host cyclophilin A, and the 347

NS5B nucleoside inhibitors sofosbuvir and 2’C-methyl Adenosine (2-CMeA) had similar 348

activity against GT6a and GT1b. The non-nucleoside NS5B (NNIs) inhibitors targeting 349

thumb site I (MK-3281) and palm site IV (HCV 796) of NS5B also had similar potency 350

against GT6a and GT1b. In contrast, the thumb site II inhibitors filibuvir and GS-9669 351

and palm site III/IV inhibitor tegobuvir, were approximately 30-, 40- and 200- fold less 352

active against GT6a, respectively compared to GT1b. The NS5A inhibitor daclatasvir had 353

slightly lower antiviral activity (~7-fold) against GT6a than GT1b; ledipasvir was active 354

against GT6a but approximately 300-fold less active relative to GT1b. The lower 355

antiviral activity of ledipasvir against GT6a is not specific in 1C cells because similar 356


.org/D

ownloaded from

http://aac.asm.org/

17

EC50 values were obtained in Lunet cells and ledipasvir has equivalent activity against 357

GT1b in 1C and Lunet cells (data not shown). The NS3 protease inhibitors currently 358

approved for treatment of GT1 HCV, boceprevir and telaprevir, each had similar 359

activities against GT6a and GT1b. MK-5172, a potentially pan-genotypic protease 360

inhibitor maintained potency against GT6a, within 4-fold of GT1b. Other protease 361

inhibitors including BILN-2061 and GS-9451 were less active against GT6a compared to 362

GT1b (17- and 8- fold respectively). Overall these results confirm that the GT6a replicon 363

system is a valuable tool for evaluating a wide range of HCV inhibitors against GT6a. 364

365


.org/D

ownloaded from

http://aac.asm.org/

18

DISCUSSION 366

GT6 HCV is most common among patients in Southeast Asia and the surrounding 367

regions, where there is a high prevalence for HCV infection. In this study, a robust GT6a 368

replicon was established to aid in the discovery and development of new therapies for 369

GT6 HCV infection. A subgenomic replicon cDNA encoding a GT6a consensus 370

sequence plus an NS5A S232I was synthesized. In Huh7-derived cells, this replicon 371

construct could consistently yield a modest number of stable GT6a colonies, and allow 372

identification of multiple novel adaptive mutations for GT6 replicon replication, 373

including E30V and K272R in NS3 and K34R in NS4A. These new adaptive 374

substitutions enabled establishing robust luciferase-encoding GT6a replicons for 375

reproducible quantification of HCV replication, and efficient antiviral activity profiling 376

of different classes of HCV inhibitors in a high throughput assay format. 377

Multiple reasons have been attributed to the difficulties of establishing HCV replicons, 378

including significant genetic divergence between genotypes, poor basal replication levels 379

for other genotypes, or the lack of suitable host cell lines. For example, Huh7.5, a cell 380

line derived from Huh-7 cells, became significantly more permissive to HCV replication 381

due to an inactivating mutation in the retinoic acid inducible gene-I (RIG-I) (36). 382

Compared to our recent GT4a replicon development, Huh7-derived Lunet cells were 383

more permissive to GT6a replicon with an average 35 colonies per transfection vs an 384

average of 0.5 colonies for GT4a (Fig. 2A) (24). Nevertheless, Lunet cells were still less 385

permissive than 1C cells to the GT6a HCV replication in both colony formation (Fig. 2A) 386

and transient transfection replicon replication assays (Fig. 4). The 1C cell line was a key 387

to our success in GT4a replicon development (24). Its high permissiveness to GT6a 388


.org/D

ownloaded from

http://aac.asm.org/

19

replicon replication, as well as GT3a replicon (37), suggests that 1C cells very likely have 389

some unique cellular background to support HCV replicon replication and have the 390

potential to further contribute to the establishment of additional HCV replicons of other 391

genotypes and subtypes. 392

Inclusion of the S232I in NS5A (2) was essential to the success of establishing the GT6a 393

replicons. Without S232I, no GT6a replicon colonies emerged in either 1C or Lunet cells 394

(Fig. 2A). Furthermore, the identified GT6a adaptive substitutions require S232I to 395

confer efficient HCV RNA replication (Fig. 5). In the absence of S232, neither K272R in 396

NS3 nor K34R in NS4A was sufficient to meaningfully enhance the basal replication of 397

GT6a replicon. In contrast, S232I alone was able to boost the replicon replication 398

significantly. These results are very similar to what we have observed in the 399

establishment of genotype 3a and 4a HCV replicons (24). Together, these data indicate 400

that the NS5A S232I could confer replication competence across HCV genotypes and 401

should be strongly considered to aid in the establishment of replicons for other HCV 402

genotypes/subtypes that have yet demonstrated replication in vitro. 403

Of the adaptive substitutions identified, K272R in NS3 helicase was the most frequently 404

observed, appearing independently in six colonies out of 23 colonies analyzed. Residue 405

K272 is located in domain 1 of the NS3 helicase protein and is highly conserved (99.9%) 406

across all HCV genotypes listed in the EU HCV database. Crystallography studies of the 407

HCV NS3 RNA helicase domain complexed with a single-stranded DNA oligonucleotide 408

revealed that K272 lies outside the central DNA binding cavity and is involved in 409

phosphate interactions with DNA helping to stabilize its conformation (38). Therefore, 410


.org/D

ownloaded from

http://aac.asm.org/

20

the emergence of adaptive substitution K272R in NS3 helicase suggests that regulation of 411

helicase activity could be involved in cell culture adaption. 412

413

The adaptive substitution K34R in NS4A was also observed frequently, appearing in 414

three colonies out of 23 colonies analyzed. Residue K34 is 79% conserved with the 415

adapted residue R34 observed in 20% viral sequences across all HCV genotypes listed in 416

the EU database. K34 is located immediately downstream of the NS4A region that forms 417

a non-covalent complex with NS3 to stimulate protease activity (39). It has been 418

hypothesized that residue 34 substitutions may modulate NS3/4A complex formation; 419

however no direct evidence exists to support this hypothesis. Interestingly, the same 420

substitution was identified in both genotype 1a and 1b replicons (3, 21). Similarly, in our 421

GT4a development, an adaptive substitution at residue 34 of NS4A was also identified 422

(Q34K or Q34R), and was highly efficient at enhancing replication (24). These results 423

thus suggest that a potential protein-protein interaction involving the residue 34 in NS4A 424

could have profound effect on HCV RNA replication in cell culture across genotypes. 425

Using the robust 6a-Pi-Rluc transient transfection replicon systems encoding either 426

K272R in NS3 or K34R in NS4A, we evaluated five classes of HCV inhibitors for 427

antiviral activities against GT6a in parallel with GT1b in a 384-well assay format (Table 428

1). As expected, inhibitors targeting the NS5B active site or host factors had similar 429

potencies against both GT1b and GT6a, consistent with previous pan-genotypic studies 430

(3, 13). Non-nucleoside NS5B polymerase, NS3 protease and NS5A inhibitors exhibited 431

differential activity between genotypes depending on the compound and target binding 432

site. Often the compounds that did not maintain potency in GT6a compared to GT1b 433


.org/D

ownloaded from

http://aac.asm.org/

21

could be explained by genetic variations within the inhibitor target site between the 434

genotypes. For example, NS5B thumb site II inhibitor filibuvir was 32-fold less active in 435

GT6a compared to GT1b. Interestingly, NS5B amino acid substitutions L419I and I482L 436

known to be resistant to filibuvir in genotype 1b are commonly present in GT6a NS5B 437

sequences, and also in our GT6 replicon sequence (40). Regarding protease inhibitors, 438

GS-9451 and BILN-2061 were less active against GT6a relative to GT1b (8- and 17- fold 439

respectively). The loss of their antiviral activity against GT6a is likely, at least in part, 440

due to the polymorph Q80K. The Q80K amino acid substitution showed reduced 441

susceptibility to GS-9451 and BILN-2061 in GT1 (6- and 3- fold respectively; data not 442

shown) and is commonly present in GT6a NS3 sequences. Put together, these antiviral 443

activity results prove the utility and efficiency of this GT6a replicon system in assessing 444

activity for inhibitors across multiple classes. 445

Currently, a surrogate approach using chimeric replicons is used to study different GT6a 446

genes (e.g. NS3, NS5A and NS5B) in cell culture. These tools are limited as they often 447

have low replication capacities and rely on the interplay of heterologous HCV viral 448

sequences and proteins. The availability of this robust GT6a replicon system (in which all 449

HCV non-structural genes are GT6 sequences) will address the limitation of existing 450

surrogate approaches. This GT6a replicon will not only become a valuable tool for the 451

discovery and evaluation of HCV inhibitors targeting GT6a, but also can be used in 452

molecular virology studies and to evaluate resistant variants identified in both preclinical 453

and clinical studies. Lastly, in conjunction with existing replicon systems, the GT6a 454

replicon will contribute to the development of pan-genotypic HCV regimens for the 455

treatment of HCV worldwide. 456


.org/D

ownloaded from

http://aac.asm.org/

22

References 457

1. Negro F, Alberti A. 2011. The global health burden of hepatitis C virus 458

infection. Liver Int 31 Suppl 2:1-3. 459

2. Lavanchy D. 2011. Evolving epidemiology of hepatitis C virus. Clin 460

Microbiol Infect 17:107-115. 461

3. Ghany MG, Strader DB, Thomas DL, Seeff LB. 2009. Diagnosis, 462

management, and treatment of hepatitis C: an update. Hepatology 49:1335-463

1374. 464

4. Yee HS, Chang MF, Pocha C, Lim J, Ross D, Morgan TR, Monto A. 2012. 465

Update on the Management and Treatment of Hepatitis C Virus Infection: 466

Recommendations from the Department of Veterans Affairs Hepatitis C 467

Resource Center Program and the National Hepatitis C Program Office. Am 468

J Gastroenterol. 469

5. Kuiken C, Simmonds P. 2009. Nomenclature and numbering of the hepatitis 470

C virus. Methods Mol Biol 510:33-53. 471

6. Cornberg M, Razavi HA, Alberti A, Bernasconi E, Buti M, Cooper C, 472

Dalgard O, Dillion JF, Flisiak R, Forns X, Frankova S, Goldis A, Goulis I, 473

Halota W, Hunyady B, Lagging M, Largen A, Makara M, Manolakopoulos 474

S, Marcellin P, Marinho RT, Pol S, Poynard T, Puoti M, Sagalova O, Sibbel 475

S, Simon K, Wallace C, Young K, Yurdaydin C, Zuckerman E, Negro F, 476

Zeuzem S. 2011. A systematic review of hepatitis C virus epidemiology in 477

Europe, Canada and Israel. Liver Int 31 Suppl 2:30-60. 478


.org/D

ownloaded from

http://aac.asm.org/

23

7. Germer JJ, Mandrekar JN, Bendel JL, Mitchell PS, Yao JD. 2011. Hepatitis 479

C Virus Genotypes in Clinical Specimens Tested at a National Reference 480

Testing Laboratory in the United States. J Clin Microbiol. 481

8. Sievert W, Altraif I, Razavi HA, Abdo A, Ahmed EA, Alomair A, 482

Amarapurkar D, Chen CH, Dou X, El Khayat H, Elshazly M, Esmat G, 483

Guan R, Han KH, Koike K, Largen A, McCaughan G, Mogawer S, Monis A, 484

Nawaz A, Piratvisuth T, Sanai FM, Sharara AI, Sibbel S, Sood A, Suh DJ, 485

Wallace C, Young K, Negro F. 2011. A systematic review of hepatitis C virus 486

epidemiology in Asia, Australia and Egypt. Liver Int 31 Suppl 2:61-80. 487

9. Nguyen NH, Vutien P, Trinh HN, Garcia RT, Nguyen LH, Nguyen HA, 488

Nguyen KK, Nguyen MH. 2010. Risk factors, genotype 6 prevalence, and 489

clinical characteristics of chronic hepatitis C in Southeast Asian Americans. 490

Hepatol Int 4:523-529. 491

10. Chao DT, Abe K, Nguyen MH. 2011. Systematic review: epidemiology of 492

hepatitis C genotype 6 and its management. Aliment Pharmacol Ther 34:286-493

296. 494

11. Fung J, Lai CL, Hung I, Young J, Cheng C, Wong D, Yuen MF. 2008. 495

Chronic hepatitis C virus genotype 6 infection: response to pegylated 496

interferon and ribavirin. J Infect Dis 198:808-812. 497

12. Gane E, Stedman C, Hyland R, Sorensen R, Symonds W, Hindes R, Berrey 498

M. 2012. Once Daily GS-7977 Plus Ribavirin in HCV Genotypes 1-3: The 499

ELECTRON Trial. 47th Annual Meeting of the European Association for the 500

Study of the Liver. 501


.org/D

ownloaded from

http://aac.asm.org/

24

13. Hassanein T, Nelson DR, Lawitz E, Dieterich D, Jacobson I, Baker C. 2011. 502

PSI-7977 with PEG/RBV Elicits Rapid Declines in HCV RNA in Patients 503

with HCV GT-4 and GT-6. HEPDart. 504

14. Dunford L, Carr MJ, Dean J, Waters A, Nguyen LT, Ta Thi TH, Thi LA, Do 505

HD, Thi TT, Nguyen HT, Diem Do TT, Luu QP, Connell J, Coughlan S, Hall 506

WW, Nguyen Thi LA. 2012. Hepatitis C virus in Vietnam: high prevalence of 507

infection in dialysis and multi-transfused patients involving diverse and novel 508

virus variants. PloS one 7:e41266. 509

15. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, 510

Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK. 2001. 511

Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus 512

ribavirin for initial treatment of chronic hepatitis C: a randomised trial. 513

Lancet 358:958-965. 514

16. Liverton N. 2010. MK-5172, the 1st HCV Protease Inibitor with Potent 515

Acyivity Against Resistance Mutations In Vitro 45th Annual Meeting of the 516

European Association for the Study of the Liver. 517

17. Scheel TK, Gottwein JM, Mikkelsen LS, Jensen TB, Bukh J. 2011. 518

Recombinant HCV variants with NS5A from genotypes 1-7 have different 519

sensitivities to an NS5A inhibitor but not interferon-alpha. Gastroenterology 520

140:1032-1042. 521

18. Bartenschlager R. 2005. The hepatitis C virus replicon system: from basic 522

research to clinical application. Journal of hepatology 43:210-216. 523


.org/D

ownloaded from

http://aac.asm.org/

25

19. Blight KJ, McKeating JA, Marcotrigiano J, Rice CM. 2003. Efficient 524

replication of hepatitis C virus genotype 1a RNAs in cell culture. J Virol 525

77:3181-3190. 526

20. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K, Mizokami M, 527

Wakita T. 2003. Efficient replication of the genotype 2a hepatitis C virus 528

subgenomic replicon. Gastroenterology 125:1808-1817. 529

21. Lohmann V, Hoffmann S, Herian U, Penin F, Bartenschlager R. 2003. Viral 530

and cellular determinants of hepatitis C virus RNA replication in cell culture. 531

J Virol 77:3007-3019. 532

22. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. 533

1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell 534

line. Science 285:110-113. 535

23. Blight KJ, Kolykhalov AA, Rice CM. 2000. Efficient initiation of HCV RNA 536

replication in cell culture. Science 290:1972-1974. 537

24. Peng B, Yu M, Xu S, Lee YJ, Tian Y, Yang H, Chan K, Mo H, McHutchison 538

J, Delaney Wt, Cheng G. 2013. Development of robust hepatitis C virus 539

genotype 4 subgenomic replicons. Gastroenterology 144:59-61 e56. 540

25. Saeed M, Gondeau C, Hmwe S, Yokokawa H, Date T, Suzuki T, Kato T, 541

Maurel P, Wakita T. 2013. Replication of hepatitis C virus genotype 3a in 542

cultured cells. Gastroenterology 144:56-58 e57. 543

26. Saeed M, Scheel TK, Gottwein JM, Marukian S, Dustin LB, Bukh J, Rice 544

CM. 2012. Efficient replication of genotype 3a and 4a hepatitis C virus 545


.org/D

ownloaded from

http://aac.asm.org/

26

replicons in human hepatoma cells. Antimicrob Agents Chemother 56:5365-546

5373. 547

27. Friebe P, Boudet J, Simorre JP, Bartenschlager R. 2005. Kissing-loop 548

interaction in the 3' end of the hepatitis C virus genome essential for RNA 549

replication. J Virol 79:380-392. 550

28. Robinson M, Yang H, Sun SC, Peng B, Tian Y, Pagratis N, Greenstein AE, 551

Delaney WEt. 2010. Novel hepatitis C virus reporter replicon cell lines enable 552

efficient antiviral screening against genotype 1a. Antimicrob Agents 553

Chemother 54:3099-3106. 554

29. Cheng G, Chan K, Yang H, Corsa A, Pokrovskii M, Paulson M, Bahador G, 555

Zhong W, Delaney Wt. 2011. Selection of clinically relevant protease 556

inhibitor-resistant viruses using the genotype 2a hepatitis C virus infection 557

system. Antimicrob Agents Chemother 55:2197-2205. 558

30. Link JO, Taylor JG, Xu L, Mitchell M, Guo H, Liu H, Kato D, Kirschberg T, 559

Sun J, Squires N, Parrish J, Keller T, Yang ZY, Yang C, Matles M, Wang Y, 560

Wang K, Cheng G, Tian Y, Mogalian E, Mondou E, Cornpropst M, Perry J, 561

Desai MC. 2014. Discovery of Ledipasvir (GS-5885): A Potent, Once-Daily 562

Oral NS5A Inhibitor for the Treatment of Hepatitis C Virus Infection. 563

Journal of medicinal chemistry Epub Date 2013/12/11. 564

31. Shih IH, Vliegen I, Peng B, Yang H, Hebner C, Paeshuyse J, Purstinger G, 565

Fenaux M, Tian Y, Mabery E, Qi X, Bahador G, Paulson M, Lehman LS, 566

Bondy S, Tse W, Reiser H, Lee WA, Schmitz U, Neyts J, Zhong W. 2011. 567

Mechanistic characterization of GS-9190 (Tegobuvir), a novel nonnucleoside 568


.org/D

ownloaded from

http://aac.asm.org/

27

inhibitor of hepatitis C virus NS5B polymerase. Antimicrob Agents 569

Chemother 55:4196-4203. 570

32. Sheng XC, Appleby T, Butler T, Cai R, Chen X, Cho A, Clarke MO, Cottell 571

J, Delaney WEt, Doerffler E, Link J, Ji M, Pakdaman R, Pyun HJ, Wu Q, 572

Xu J, Kim CU. 2012. Discovery of GS-9451: an acid inhibitor of the hepatitis 573

C virus NS3/4A protease. Bioorg Med Chem Lett 22:2629-2634. 574

33. Fenaux M, Eng S, Leavitt SA, Lee YJ, Mabery EM, Tian Y, Byun D, Canales 575

E, Clarke MO, Doerffler E, Lazerwith SE, Lew W, Liu Q, Mertzman M, 576

Morganelli P, Xu L, Ye H, Zhang J, Matles M, Murray BP, Mwangi J, 577

Hashash A, Krawczyk SH, Bidgood AM, Appleby TC, Watkins WJ. 2013. 578

Preclinical characterization of GS-9669, a thumb site II inhibitor of the 579

hepatitis C virus NS5B polymerase. Antimicrob Agents Chemother 57:804-580

810. 581

34. Sofia MJ, Bao D, Chang W, Du J, Nagarathnam D, Rachakonda S, Reddy 582

PG, Ross BS, Wang P, Zhang HR, Bansal S, Espiritu C, Keilman M, Lam 583

AM, Steuer HM, Niu C, Otto MJ, Furman PA. 2010. Discovery of a beta-d-584

2'-deoxy-2'-alpha-fluoro-2'-beta-C-methyluridine nucleotide prodrug (PSI-585

7977) for the treatment of hepatitis C virus. Journal of medicinal chemistry 586

53:7202-7218. 587

35. Friebe P, Lohmann V, Krieger N, Bartenschlager R. 2001. Sequences in the 588

5' nontranslated region of hepatitis C virus required for RNA replication. J 589

Virol 75:12047-12057. 590


.org/D

ownloaded from

http://aac.asm.org/

28

36. Sumpter R, Jr., Loo YM, Foy E, Li K, Yoneyama M, Fujita T, Lemon SM, 591

Gale M, Jr. 2005. Regulating intracellular antiviral defense and 592

permissiveness to hepatitis C virus RNA replication through a cellular RNA 593

helicase, RIG-I. J Virol 79:2689-2699. 594

37. Yu M, Corsa AC, Xu S, Peng B, Gong R, Lee YJ, Chan K, Mo H, Delaney 595

Wt, Cheng G. 2013. In vitro efficacy of approved and experimental antivirals 596

against novel genotype 3 hepatitis C virus subgenomic replicons. Antiviral 597

research 100:439-445. 598

38. Kim JL, Morgenstern KA, Griffith JP, Dwyer MD, Thomson JA, Murcko 599

MA, Lin C, Caron PR. 1998. Hepatitis C virus NS3 RNA helicase domain 600

with a bound oligonucleotide: the crystal structure provides insights into the 601

mode of unwinding. Structure 6:89-100. 602

39. Yao N, Reichert P, Taremi SS, Prosise WW, Weber PC. 1999. Molecular 603

views of viral polyprotein processing revealed by the crystal structure of the 604

hepatitis C virus bifunctional protease-helicase. Structure 7:1353-1363. 605

40. Herlihy KJ, Graham JP, Kumpf R, Patick AK, Duggal R, Shi ST. 2008. 606

Development of intergenotypic chimeric replicons to determine the broad-607

spectrum antiviral activities of hepatitis C virus polymerase inhibitors. 608

Antimicrob Agents Chemother 52:3523-3531. 609

610 611

612


.org/D

ownloaded from

http://aac.asm.org/

29

Table 1. Antiviral activities of HCV inhibitors against GT6a replicons 613

Drug Classes Compounds GT6a EC50 (nM) GT1b EC50 (nM) GT6a/1b EC50 ratio

NS3 Protease

Telaprevir 208 ± 41 428 ± 22 0.5

Boceprevir 185 ± 42 181 ± 42 1.0

GS-9451 67 ± 15 8.3 ± 1.6 8.1

MK-5172 3.0 ± 0.4 0.74 ± 0.07 4.1

BILN-2061 17 ± 1.6 0.99 ± 0.1 17

NS5A Ledipasvir 1.1 ± 0.08 0.004 ± 0.003 275

Daclatasvir 0.11 ± 0.05 0.016 ± 0.010 6.9

NS5B Nuc Sofosbuvir 22 ± 6.5 98 ± 38 0.2

2-CMeA 22 ± 8 86 ± 0.1 0.3

NS5B Non-Nuc

GS-9669 736 ± 18 3.5 ± 0.3 210

MK-3281 1311 ± 635 534 ± 47 2.5

Tegobuvir 110 ± 21 2.6 ± 0.2 42

Filibuvir 3536 ± 93 112 ± 13 32

HCV-796 21 ± 0.5 20 ± 5.5 1.1

Host Target Cyclosporin A 65 ± 4.4 81 ± 1.4 0.8

614 Replicons GT6a-PiRluc-NS3-K272R (with S232I in NS5A) and GT1b-PiRluc were 615

transiently transfected in 1C cells and antiviral assays were performed in a high 616

throughput 384-well format. All EC50 values represent an average of at least two 617

independent experiments with standard deviations.618


.org/D

ownloaded from

http://aac.asm.org/

30

Figure 1. Schematic diagram of GT6a replicon constructs 619

GT6a subgenomic replicons used in these studies. Elements engineered into the 620

replicons include the neomycin phosphotransferase II gene (neo) (A), a Renilla luciferase 621

(Rluc)- neomycin phosphotransferase II gene fusion reporter (RlucNeo) (B) or a 622

poliovirus IRES (PI-IRES)-driven Rluc reporter (C). All replicons encoded the GT6a 5’-623

UTR, an EMCV IRES controlling translation of NS3 – NS5B polyprotein region of GT6a 624

consensus sequence including an NS5A amino acid substitution (S232I); and the 3’-UTR 625

of GT2a JFH-1 virus. Black bars indicates GT6a core N-terminal sequence. Gray bars 626

indicates HCV polyprotein sequence. “ ”indicates the S232I. Neo and RlucNeo 627

translation are controlled by an HCV IRES (A and B) whereas Rluc translation is 628

controlled by a Pi-IRES (C). 629


.org/D

ownloaded from

http://aac.asm.org/

31

Figure 2. Establishment of stable GT6a replicons 630

(A) Selection of stable GT6a replicon colonies. In vitro transcribed RNA encoding GT6a-631

Neo replicons with and without the NS5A S232I were transfected into Lunet or 1C cells 632

and subjected to G418 selection (0.5 mg/ml). G418-resistant colonies emerged after 633

approximately four weeks of treatment and were counted. The values represent the mean 634

of three independent assays with standard errors. (B) Intracellular NS3-4A protease 635

activity in pooled GT6a replicon cells. Cells were plated at a concentration of 5x104 cells 636

per well in 96-well plates, and NS3-4A protease activity was determine 24 hours post cell 637

seeding. (C) Expression of NS5A protein in pooled GT6a replicon cells. Pooled GT6a-638

Neo 1C or Lunet cells were stained with anti-NS5A antibody (red) and Hoechst 33342 639

(blue, indicating nuclei). Parental 1C cells were stained as a negative control. 640


.org/D

ownloaded from

http://aac.asm.org/

32

Figure 3. NS3 and NS4A substitutions enhance replication of GT6a replicons. 641

In vitro transcribed GT6a-RlucNeo replicon RNAs, either from the parental replicon or 642

the replicons engineered to encode the indicated NS3 substitutions (E30V or K272R) or 643

NS4A substitution (K34R), were transfected into Lunet cells and subjected to selection 644

by G418 (0.5mg /ml). G418-resistant colonies harboring GT6a replicons emerged after 645

approximately three weeks and were visualized by staining with crystal violet. All 646

replicons encode NS5A amino acid substitution S232I. 647


.org/D

ownloaded from

http://aac.asm.org/

33

Figure 4. NS3 and NS4A substitutions confer high GT6a HCV replication 648

NS3 (E30V and K272R) or NS4A (K34R) substitutions were introduced into the GT6a-649

PiRluc replicon harboring S232I in NS5A. In vitro transcribed replicons RNAs were 650

transfected into either Lunet or 1C cells and plated in 96-well plates at a density of 5x103 651

cells/well. Renilla luciferase activity (RLUs) was measured as a marker for HCV 652

replication at the indicated time points. A replication-defective NS5B polymerase GND 653

mutant was included as a negative control. The background of the assay is ~ 100 RLUs. 654

All values represent the mean of two independent assays with standard errors. 655


.org/D

ownloaded from

http://aac.asm.org/

34

Figure 5. The NS5A amino acid substitution S232I is required for efficient GT6a 656

HCV replication. The NS3 K272R and NS4A K34R substitutions were introduced 657

individually into the GT6a-PiRluc replicon with or without S232I in NS5A. In vitro 658

transcribed replicon RNAs were transfected into 1C cells and plated in 96-well plates at a 659

density of 5x103 cells/well. Renilla luciferase activity (RLUs) was measured as a marker 660

for HCV replication at the indicated time points. A replication-defective NS5B 661

polymerase GND mutant was included as a negative control. The background of the 662

assay is ~ 100 RLUs. All values represent the mean of two independent assays with 663

standard errors. 664


.org/D

ownloaded from

http://aac.asm.org/


.org/D

ownloaded from

http://aac.asm.org/

aac accepts, published online ahead of print on 18...

Documents