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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2012 Nov 14;109(48):19757–19762. doi: 10.1073/pnas.1218260109

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

Yi-Ping Li a,b, Santseharay Ramirez a,b, Sanne B Jensen a,b, Robert H Purcell c,1, Judith M Gottwein a,b, Jens Bukh a,b,c,1
PMCID: PMC3511766  PMID: 23151512

Abstract

Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture patient isolates representing HCV genotypes 1–7 and subtypes; only a recombinant 2a genome (strain JFH1) spontaneously replicated in vitro. Recently, we identified three mutations F1464L/A1672S/D2979G (LSG) in the nonstructural (NS) proteins, essential for development of full-length HCV 2a (J6) and 2b (J8) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5′ untranslated region (5′UTR)-NS5A and JFH1 NS5B-3′UTR; recovered viruses acquired two adaptive mutations located in NS3 and NS4B. Introduction of these changes into a replication-deficient TN full-length genome, harboring LSG, permitted efficient HCV production. Additional identified NS4B and NS5B mutations fully adapted the TN full-length virus. Thus, a TN genome with 8 changes (designated TN cell-culture derived, TNcc) replicated efficiently and released infectious particles of ∼5 log10 focus-forming units per mL; passaged TNcc did not require additional changes. IFN-α and directly acting antivirals targeting the HCV protease, NS5A, and NS5B, each inhibited full-length TN infection dose-dependently. Given the unique importance of genotype 1 for pathogenesis, this infectious 1a culture system represents an important advance in HCV research. The approach used and the mutations identified might permit culture development for other HCV isolates, thus facilitating vaccine development and personalized treatment.

Hepatitis C virus (HCV) chronically infects an estimated 130–170 million people worldwide. The infection increases the risk of developing liver cirrhosis and liver cancer and results in more than 350,000 deaths annually. No HCV vaccine is available. Current standard treatment is based on IFN-α/ribavirin, which, however, has low efficacy against the most prevalent HCV variants (1). Incorporation of directly acting antivirals (DAAs) in treatment regimens improves sustained viral response rate, but a favorable outcome is challenged by fast emergence of drug resistance and differential responses of the different HCV genotypes (2). Thus, HCV infection continues to be a huge health and economic burden to the world population, and improved in vitro experimental systems would be important to permit additional studies of new antivirals and associated resistance patterns.

HCV is a small enveloped virus belonging to the genus Hepacivirus in the family Flaviviridae. The HCV genome is a positive-sense single-strand RNA (∼9.6 kb), consisting of a single ORF flanked by 5′ and 3′ untranslated regions (UTRs). The ORF encodes virus structural proteins (core, E1, and E2), p7, and six nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (1). HCV isolates are classified into seven major variants (genotypes 1–7) and numerous subtypes (a, b, etc.) differing by ∼30% and ∼20%, respectively, in nucleotide and amino acid sequences (3). Genotype 1 is the most prevalent in the world. In the United States, Japan, China, and southern Europe over 70% of HCV patients are infected with genotype 1 (4–7). In northern Europe, genotype 1 accounts for ∼50% of HCV infections (8). Genotype 1 infection is more resistant to IFN-based treatment than infection with other genotypes (1).

In vitro HCV culture systems are needed to study treatment regimens and associated viral resistance. However, drug and vaccine development have been greatly hampered by inability to culture patient isolates in vitro. Although a number of HCV full-length clones were shown to be infectious in chimpanzees (ref. 9 and cited references therein), only JFH1 (genotype 2a) spontaneously replicated in human hepatoma cells (Huh7 and its derivatives) and released infectious virus particles (10, 11). Efficient growth of JFH1 required culture adaptive mutations (12). Recently, we reported efficient J6cc (2a) and J8cc (2b) full-length culture systems (13); subsequently Date et al. reported on a full-length system for 2a strain JFH2 (14). A single full-length genotype 1a genome, H77-S, carrying mutations identified in the subgenomic replicon of the same strain, has been reported to release relatively small amounts of virus particles (15). The Con1 (1b) full-length culture system was reported, but a very low level of replication has limited its utility (16). Thus, efficient HCV full-length culture systems remained limited to genotype 2 isolates. The clinical importance of the marked genetic differences between HCV genotype isolates poses a critical need for development of robust full-length culture systems for HCV genotype 1 isolates.

The unique replication capacity of JFH1 has permitted the development of JFH1-based HCV recombinants (17); we and others have reported different inter- and intragenotypic recombinants including core-NS2 (17–24), 5′UTR-NS2 (25), NS3 protease/NS4A (26, 27), NS5A (28), and core-NS3 protease plus NS4A-NS5A (29) of various genotypes. These JFH1-based culture systems have been used for genotype-specific studies of HCV genes (19, 20, 22, 25–28, 30), for testing of HCV DAAs (26–28) and neutralizing antibodies (19, 20, 22, 30), and for studying aspects of virus–host interaction (25, 31, 32). Through studies of J6 (2a) recombinants with the entire or partial NS5B and 3′UTR from JFH1, we recently identified adaptive mutations F1464L in NS3 and A1672S in NS4A, designated LS (13). Combination of LS with selected mutations in NS5B and the 3′UTR allowed replication of the full-length J6 genome, which led to the identification of an additional unique mutation D2979G in NS5B, designated G (13). The LSG substitutions permitted the development of robust HCV full-length culture systems for J6 and for the prototype genotype 2b J8 strain (13). In this study, we used LSG as a critical component and a unique approach to develop a highly efficient full-length genotype 1a (strain TN) infectious culture system, named TN cell-culture derived (TNcc). The TNcc replicated efficiently in culture, released viral particles of ∼105 focus-forming units (FFU)/mL and did not require additional mutations after viral passage. We demonstrated that full-length TN responded dose dependently to IFN-α, as well as HCV DAAs already used in the clinic and being tested in clinical trials.

Results

HCV Genotype 2-Derived LSG Mutations Permitted Adaptation of Genotype 1a (Strain TN) Recombinant with JFH1 NS5B-3′UTR.

We recently developed full-length HCV genotype 2a (J6cc) and 2b (J8cc) infectious culture systems using mutations F1464L (NS3 helicase), A1672S (NS4A) and D2979G (NS5B), designated LSG (13). All positions of nucleotides and amino acids (aa) throughout this manuscript are according to the genome of strain HC-TN (GenBank accession no. EF621489). In the present study, we attempted to use the LSG mutations for development of a full-length HCV genotype 1 cell culture system.

We selected a genotype 1a strain, HC-TN, which was originally isolated from a patient with fulminant hepatitis (33), and for which we previously demonstrated that its consensus molecular clone was infectious in chimpanzees (34) but not in Huh7.5 cells (23, 34). Using JFH1-based recombinants, we demonstrated that TN core-NS2 (23) and NS5A (28) were functional in Huh7.5 cells. We recently also proved functionality of 5′UTR-NS3 protease and NS4A-NS5A regions by development of a culture adapted TN semi-full-length recombinant only depending on JFH1 helicase, NS5B, and 3′UTR. Thus, and also given our recent finding on viability of J6-JFH1 chimeras, in this study we generated a TN recombinant with only NS5B and 3′UTR from JFH1, designated TN(JFH1_5BX) (X indicating inclusion of the entire 3′UTR with 3′X region), and tested its viability by RNA transfection of Huh7.5 cells. No HCV positive (HCV+) cells were detected for up to 22 d by immunostaining for HCV core or NS5A.

Because the LSG mutations enhanced HCV RNA replication, virus particle assembly, and release of the full-length J6 virus (13), we next introduced them into TN(JFH1_5BX) (Fig. 1A). In two independent transfections of TN(JFH1_5BX)_LSG, HCV+ cells were detected at day 4 and the virus spread to most of the cultured cells in 24 and 26 d (Fig. 1A). In infected cultures, peak supernatant HCV infectivity titers were 104.2–104.4 FFU/mL, comparable to the titers of control cultures infected with J65′UTR-NS2/JFH1 (104.3–104.6 FFU/mL) (Fig. 1A) (13, 25). Viruses recovered from supernatants of transfection cultures could be passaged to naïve Huh7.5 cells, and the first passage viruses produced titers of 104.4 FFU/mL (Table 1). ORF sequence analysis of first passage viruses revealed two coding changes in both viruses, A1226G in the NS3 helicase (NS3 aa position 200) and Q1773H (NS4B aa 62) (Table 1 and Table S1), indicating their importance for the viability of TN(JFH1_5BX)_LSG.

Fig. 1.

Fig. 1.

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Development of HCV genotype 1a (strain TN) full-length infectious culture system. RNA transcripts of HCV genomes with indicated structure and mutations (genome illustrations) were transfected into Huh7.5 cells (graphs), HCV core, and/or NS5A antigens were detected by immunostaining, and percentage of positive cells was estimated (Left y axis; shown as line plots). HCV infectivity titers in supernatant at peak of infection (≥80% HCV+ culture cells) were determined by focus-forming-unit assay (mean of triplicate infections ± SEM, Right y axis; shown as bar graphs). Duplicate experiments are shown as “exp. 1” and “exp. 2.” J65′UTR-NS2/JFH1 (25) was used as a positive control. (A) Previously identified F1464L/A1672S/D2979G [illustrated by black dots; referred to as LSG (13)] initiated replication and adaptation of TN(JFH1_5BX) in Huh7.5 cells (graph). Mutations A1226G and Q1773H (yellow dots) were identified in first passage viruses (Table 1 and Table S1) and were able to adapt the TN(JFH1_5BX)_LSG for efficient growth. TN(JFH1_5BX) without mutations remained HCV antigen negative 22 d after transfection. Data were not necessarily from one experiment; representative positive control is shown. (B) Combination of LSGF [LSG (13) and Y2981F (13, 35); black dots] and TN-derived mutations A1226G or A1226G/Q1773H (yellow dots) permitted the TN full-length genome to replicate and produce infectious HCV in culture supernatant (graph, Left). Additional identified mutations N1927S/T and/or F2994S (yellow dots) further improved the viability of TN_LSGF/A1226G (Center) and TN_LSGF/A1226G/Q1773H (Center and Right). TN full-length viruses TN_LSGF/A1226G/Q1773H/N1927S/F2994S or TN_LSGF/A1226G/Q1773H/N1927T/F2994S (Right) produced peak supernatant infectivity titers of 4.6–4.9 log10 FFU/mL in duplicate experiments, similar to positive control J65′UTR-NS2/JFH1 (4.7–4.9 log10 FFU/mL).

Table 1.

Sequence analysis of the complete ORF of TN(JFH1_5BX) chimeric viruses and of TN full-length viruses

graphic file with name pnas.1218260109unfig01.jpg
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One milliliter of culture supernatant from TN(JFH1_5BX) and TN full-length transfections were passaged to naïve Huh7.5 cells (∼4 × 105 cells), and culture supernatant collected at peak of infection (≥80% cells infected) was subjected to ORF sequence analysis. Primers used for RT-PCR were previously described (18, 25, 28, 47, 52). Nucleotide and amino acid positions of the specific recombinant with mutations are listed; the corresponding position of H77 reference sequence (AF009606) is given. Shadings indicate the engineered mutations: J6-drived mutations LSGF [LSG, F1464L/A1672S/D2979G (13); F, Y2981F (13, 35)] are in dark shading; TN-derived mutations are in light shading. Two capital letters separated by a slash indicates a nucleotide quasispecies (50/50) in sequencing reads, whereas a capital letter separated from a lowercase letter indicates a dominant/minor ratio. Only coding changes found in at least two viruses are shown; remaining changes are shown in Table S1. Chimeric viruses TN(JFH1_5BX)_LSG/A1226G/Q1773H and TN(JFH1_5BX)_LSG/A1226G and TN full-length viruses TN_LSGF/A1226G/N1927S/F2994S and TNcc have neither coding nor noncoding changes (Table S1). Dots indicate identity with original sequence. Viral infectivity titers were determined for at least three consecutive time points at and after peak of infection, and representative peak infectivity titers (FFU/mL) are shown; RNA titers were between 7.1 and 7.9 log10 IU/mL (Table S1).

*A-to-G and A-to-C nucleotide changes correspond to amino acid changes N to S and N to T, respectively.

†TN(JFH1_5BX) recombinants originally have T at this position; indicated as (T).

‡Second, third, and fourth passage viruses showed supernatant HCV infectivity titers of 105.1, 105.5, and 105.4 FFU/mL, respectively; the amino acid substitution F2994S was complete in the fourth passage virus, and the original amino acid was not present.

§A virus stock pool made from first passage supernatants collected at days 9, 11, 13, and 15 was sequenced and used for antiviral treatment (Fig. 2).

Second passage virus had a peak titer of 4.6 log10 FFU/mL (Table S1).

Next, we introduced A1226G and Q1773H, singly or in combination, into TN(JFH1_5BX)_LSG (Fig. 1A). The TN(JFH1_5BX)_LSG/A1226G and TN(JFH1_5BX)_LSG/A1226G/Q1773H recombinants showed 20–30% HCV+ cells on day 1 and reached the peak of infection (≥80% HCV+ cells) on day 4 posttransfection, with supernatant peak infectivity titers of 104.1 and 104.5 FFU/mL, respectively. TN(JFH1_5BX)_LSG/Q1773H showed delayed spread kinetics and a peak titer of only 103.9 FFU/mL. Following passage to naïve Huh7.5 cells, TN(JFH1_5BX)_LSG/A1226G/Q1773H (peak titer 104.5 FFU/mL) and TN(JFH1_5BX)_LSG/A1226G (103.9 FFU/mL) did not acquire additional coding changes. TN(JFH1_5BX)_LSG/Q1773H (105.0 FFU/mL) acquired two mutations in NS3 helicase, including A1226G, indicating the importance of co-occurrence of A1226G and Q1773H (Table 1 and Table S1). Taken together, we demonstrated that previously identified genotype 2-adapting LSG mutations could initiate replication of a genotype 1-specific 5′UTR-NS5A recombinant. Mutations A1226G and Q1773H identified in cultures infected with the TN intergenotypic 5′UTR-NS5A recombinant further adapted the virus to efficient growth in Huh7.5 cells.

Development of Highly Efficient Full-Length TN Infectious Culture Systems Based on LSG and A1226G/Q1773H Mutations.

Because A1226G or the combination A1226G/Q1773H could efficiently adapt TN(JFH1_5BX) (Fig. 1A and Table 1), we engineered them into a TN full-length genome with LSG (13) and Y2981F (NS5B aa 561) (35), designated TN_LSGF. LSGF substitutions were previously shown to permit adaptation of full-length genotype 2 strains, J6 and J8 (13). However, after RNA transfection of TN_LSGF in Huh7.5 cells, we did not observe any HCV+ cells during 4 wk of follow-up. In contrast, transfection of TN_LSGF/A1226G and TN_LSGF/A1226G/Q1773H showed 5 and 20% HCV+ cells on day 1, reached peak infection within 13 and 8 d, and produced peak supernatant titers of 103.8 and 103.6 FFU/mL, respectively (Fig. 1B, Left). Transfection-recovered TN_LSGF/A1226G and TN_LSGF/A1226G/Q1773H could be passaged to naïve Huh7.5 cells, and first passage viruses reached peak titers of 104.5 and 104.9 FFU/mL, respectively (Table 1 and Table S1). The second, third, and fourth passage TN_LSGF/A1226G/Q1773H viruses reached peak titers of 105.1, 105.5, and 105.4 FFU/mL, respectively. Thus, we had adapted the TN full-length genome to efficiently replicate and produce infectious virus particles in Huh7.5 cells, using previously identified genotype 2 LSGF mutations (13, 35) combined with TN mutations A1226G/Q1773H identified in the present study.

ORF sequence analysis of first passage viruses revealed that both TN_LSGF/A1226G and TN_LSGF/A1226G/Q1773H had common changes as quasispecies at two positions: N1927N/S/T (NS4B aa 216) and F2994F/S (NS5B aa 574) (Table 1 and Table S1). F2994F/S became a complete change, F2994S, in the fourth passage TN_LSGF/A1226G/Q1773H virus (105.4 FFU/mL). Thus, we introduced F2994S singly into TN_LSGF/A1226G/Q1773H. Because the N1927N/S/T mutations were previously shown to improve the viability of J6_LSG (13), and coexisted here with F2994F/S in first and second passage TN_LSGF/A1226G (Table 1 and Table S1), we introduced combinations N1927S/F2994S or N1927T/F2994S into TN_LSGF/A1226G. In duplicate transfection experiments, TN_LSGF/A1226G/Q1773H/F2994S showed 10 and 20% HCV+ cells on day 1 and peak titers of 103.8 and 103.9 FFU/mL, respectively (Fig. 1B, Center). The TN_LSGF/A1226G/N1927S/F2994S and TN_LSGF/A1226G/N1927T/F2994S recombinants showed 50–60% HCV+ cells at day 1 in duplicate transfections, and reached peak of infection within 3 d (Fig. 1B, Center); infected cultures produced peak titers of 104.3–104.4 FFU/mL, similar to the titers of first passage TN_LSGF/A1226G (Table 1 and Table S1). Because combinations N1927S/F2994S or N1927T/F2994S greatly increased virus production of TN_LSGF/A1226G, we tested their effect in TN_LSGF/A1226G/Q1773H, viability of which was only marginally increased by single mutation F2994S. In duplicate transfection experiments, the TN_LSGF/A1226G/Q1773H/N1927S/F2994S and TN_LSGF/A1226G/Q1773H/N1927T/F2994S recombinants both showed 60–70% HCV+ cells on day 1 and reached peak infection within 3 d (Fig. 1B, Right). The infections produced peak titers of 104.6–104.9 FFU/mL, similar to the titers of first passage TN_LSGF/A1226G/Q1773H (Table 1 and Table S1) and comparable to the included transfection control J65′UTR-NS2/JFH1 (104.7–104.9 FFU/mL) (Fig. 1B, Right).

Transfection-derived adapted TN viruses were passaged to naïve Huh7.5 cells, with peak infectivity titers of 104.6–104.7 FFU/mL (Table 1). We sequenced viruses from one replicate passage culture for each recombinant. TN_LSGF/A1226G/N1927T/F2994S had no coding changes, but a single noncoding change (C4748C/T). In TN_LSGF/A1226G/N1927S/F2994S and LSGF/A1226G/Q1773H/N1927T/F2994S, we did not observe coding or noncoding changes (Table 1 and Table S1). TN_LSGF/A1226G/Q1773H/N1927T/F2994S showed specific infectivity of ∼1/400 [FFU/international unit (IU)] (Table S1), and we designated this virus TNcc. Thus, we established a highly efficient HCV genotype 1 full-length culture system with infectivity titers comparable to those from the most efficient JFH1-based systems, such as Jc1 and SA13/JFH1 (20, 21), using a unique approach that permits identification of efficient adaptive mutations.

Full-Length TN Virus Was Inhibited by HCV Protease-, NS5A-, and NS5B-Polymerase Inhibitors and by IFN-α2b in a Dose-Dependent Manner.

The TN full-length culture system allows testing of DAAs targeting any genomic element or protein expressed by the viral genome and of IFN-α in the context of the complete viral life cycle in vitro. To validate the TN full-length culture system, we here demonstrated that the TN full-length virus was dose-dependently inhibited by linear [telaprevir (VX-950) (36) and boceprevir (SCH503034) (37)] and macrocyclic [simeprevir (TMC435) (38)] NS3 protease inhibitors, by NS5A inhibitor daclatasvir (BMS-790052) (39), by nucleotide NS5B inhibitor PSI-7977 (40), and by IFN-α2b (Fig. 2A). The first passage TN_LSGF/A1226G/Q1773H virus stock was used for these experiments (Table 1). Median effective concentration (EC50) of NS5A inhibitor daclatasvir for TN full-length virus (0.032 nM) (Fig. 2B) was similar to that of J6/JFH1-based recombinant with TN NS5A (0.042 nM) (28), thus validating TN full-length and TN-specific NS5A recombinant culture systems in the studies of antiviral treatment. Compared with J6/JFH1, TN full-length virus was more sensitive to telaprevir, boceprevir, simeprevir, and daclatasvir, with about two- to fivefold lower EC50, whereas no apparent difference was observed for PSI-7977 and IFN-α2b (Fig. 2B).

Fig. 2.

Fig. 2.

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Dose-dependent efficacy of NS3/NS4A protease-, NS5A-, and NS5B-polymerase inhibitors and IFN-α2b against TN full-length virus. Naïve Huh7.5 cells were infected with first passage virus stock of TN full-length recombinant TN_LSGF/A1226G/Q1773H; the ORF sequence of this virus stock is shown in Table 1 and Table S1. A J6/JFH1 (17) virus stock was treated in parallel. Antivirals were administered 24 h postinfection at the indicated doses; IFN-α2b was also administered 48 h postinfection. The number of HCV+ cells per well was determined 72 h postinfection. (A) Dose–response curves for treatments with lead compound protease inhibitors telaprevir (VX-950), boceprevir (SCH503034), and simeprevir (TMC435), NS5A inhibitor daclatasvir (BMS-790052), and NS5B inhibitor PSI-7977, as well as IFN-α2b. Values are means of triplicate determinations with SEM. (B) EC50 of the used inhibitors against the indicated viruses. Values for EC50 and 95% confidence interval are shown.

Discussion

In this study, we developed a highly efficient full-length HCV genotype 1 cell culture system, TNcc. The TNcc replicated efficiently following transfection and produced HCV infectivity titers of ∼5 log10 FFU/mL. This titer level is orders of magnitude higher than published systems for other genotype 1 strains, and comparable to the most efficient JFH1-based chimeric culture systems. First passage TNcc did not require additional changes. Thus, the development of TNcc is a significant advance in HCV research and provides a valuable tool for HCV studies, especially on HCV genotype 1, the most prevalent genotype in the world.

Recently, we developed full-length infectious culture systems of HCV genotype 2a (J6cc) and 2b (J8cc) using mutations F1464L/A1672S/D2979G (LSG) identified through studies of J6 recombinants (13). These results suggested that a limited number of mutations could confer viability to a replication-deficient HCV genome in cultured cells, and that mutations identified for genotype 2 may enable adaptation of other HCV genotype isolates. Here, by a unique approach using the genotype 2-derived mutations and an in vivo functional HCV genome, HC-TN (34), we developed a robust full-length HCV genotype 1 culture system. We demonstrated that genotype 2-adapting mutations LSG (13) could initiate adaptation of a nonviable HCV genotype 1 (strain TN)-specific 5′UTR-NS5A recombinant in Huh7.5 cells, which led to identification of additional mutations A1226G (NS3 helicase) and Q1773H (NS4B). These TN mutations, in combination with genotype 2-adapting mutations, permitted efficient virus production and further adaptation of the TN full-length virus.

After discovery of HCV in 1989 (41), only a single HCV isolate (JFH1, genotype 2a) was found to be able to replicate autonomously in a hepatoma-derived cell line, Huh7, and release infectious virus (11). This breakthrough was not accomplished until 2005. Other HCV culture systems have been reported (42–44); however, none was found to release infectious virus at a level high enough to study the HCV life cycle. The H77-S (1a) system with H77 replicon mutations was the only genotype 1a isolate reported to be able to, albeit inefficiently, infect Huh7.5 cells and release infectious virus particles (15). Apparently, infectivity could be improved by introducing additional mutations, but the details have not been published, and the titers indicated remain relatively low (45). A genotype 1b isolate, Con1, was reported to produce virus particles in vitro, but quantitative virological assays could not be performed due to low level of replication (16). Thus, the TNcc system permits detailed virological studies and applications previously difficult or not possible for genotype 1 viruses.

There is apparently a low probability of obtaining from a patient an HCV genome with replication capacity in vitro. Host restriction is believed to account for a distinct and narrow tissue and host species tropism of HCV. Therefore, it is important to develop approaches that will enable researchers to overcome the host barrier for the growth of the pathogen in vitro and in vivo. Development of full-length HCV infectious genotype 1a, 2a, and 2b culture systems by use of a limited number of mutations has exemplified that introducing specific mutations into a consensus full-length genome could overcome such host barriers. This knowledge could be of overall interest for understanding virus–host interactions and for the development of culture systems or animal models for other viruses or pathogens that have similar host restrictions.

Identification of adaptive mutations permitting replication of full-length HCV genomes in vitro has been difficult. Mutations identified in the Con1 (1b) replicon enhanced RNA replication but impaired in vitro virus assembly (16) and in vivo infectivity (46). Although other mutations identified from strain-specific subgenomic replicons permitted the replication of H77-S (1a) (15) and JFH2 (2a) (14), their effect on different isolates of HCV genotypes has not been reported. Because host restriction could affect any step of the HCV life cycle, mutations selected from productive infectious culture systems must harbor the ability to bypass each of those steps. Possibly such mutations could overcome some key universal host restrictions, thus having a pangenotypic effect. The LSG mutations were identified from infectious culture systems and have been proven to adapt different genotype 2 strains, J6 (2a) and J8 (2b) (13) and in this study, genotype 1a strain TN (Fig. 1B). Thus, the LSG mutations apparently have adaptation effects across HCV genotypes. Therefore, compared with replicon-derived mutations, infection-selected mutations may be advantageous in aiding replication of full-length HCV genomes.

Acquiring replication capacity is a critical step for evolutionary selection of beneficial mutations that can overcome the blocks of the viral life cycle. The JFH1 NS5B polymerase could initiate the replication of recombinants of various HCV genotypes (13, 17–29, 47), which led to the identification of numerous adaptive mutations, including those adapting full-length HCV genomes (Fig. 1A) (13). Therefore, the use of the unique replication capacity of the JFH1 NS5B RNA polymerase to initiate the replication of a strain-specific 5′UTR-NS5A recombinant may be an efficient approach to identify mutations permitting replication of full-length HCV genomes, as we here demonstrated for TNcc (Fig. 1). In future studies it would be of great interest to examine this approach for culture development of other HCV genotype isolates.

We here identified TN-adapting mutations A1226G in the NS3 helicase, Q1773H in NS4B, and F2994S in NS5B (Fig. 1B and Table 1). A1226 (NS3 aa 200) is highly conserved among HCV genotype 1 and 4 isolates, whereas glycine was found at this position for genotype 2, 3, 5, 6, and 7 isolates (Los Alamos HCV Sequence Database). In a recent study, an A1226G substitution was shown to enhance replication of an ED43 (4a) subgenomic replicon (48). Q1773 (NS4B aa 62) localizes to the N-terminal amphipathic α-helix AH2 domain of NS4B; this position is conserved for all HCV genotypes. The α-helix AH2 contributes to NS4B association with membranes (49) and is a major determinant for NS4B oligomerization, which is required for the formation of a functional replication complex (50). Interestingly, the changes N1927S/T (NS4B aa 216), which we previously found to improve the J6 full-length system (13), were also identified in several TN full-length viruses and enhanced TN viral infectivity (Fig. 1B and Table 1). Thus, N1927S/T has cross-genotype adaptive activity. N1927 is located in the NS4B C-terminal end and may also regulate the HCV infection cycle in JFH1 and JFH1-based recombinant Jc1 (51). F2994 is located in the C-terminal transmembrane segment of NS5B, a region important for HCV production (13) and is conserved among HCV genotype 1a isolates, whereas tryptophan and leucine are dominant in genotype 1b and other genotypes. It should be noted that mutations identified in this study are different from those previously found in TN-infected chimpanzees (34). Thus, these mutations may be specific for cell culture. Previously identified LSG are highly conserved among all HCV genotypes (Los Alamos HCV Sequence Database), and enhanced HCV RNA replication and virus assembly (13). In future studies it would be of interest to investigate the specific mechanism of the identified TN mutations for adaptation of TN isolate and the utility of these mutations, or various combinations, for development of other full-length HCV culture systems. It is also a possibility that the TN-derived mutations are specific for adaptation of genotype 1 isolates, thus in future studies it would be of interest to test the effect of these mutations in genotype 1a isolate H77 or 1b isolate Con1; as mentioned above, both genomes were previously shown to be viable in culture, with low level titers, after introduction of other mutations (15, 16).

With TNcc, all HCV inhibitors can now be tested at an isolate-specific level. To validate the TN full-length culture system, we here demonstrated that TN full-length viruses responded to NS3/4A protease-, NS5A-, and NS5B inhibitors, respectively, and to IFN-α2b in a dose-dependent manner (Fig. 2). TNcc (1a) and J6/JFH1 (2a) responded similarly to IFN-α2b treatment. Thus, the observed difference in the effect of IFN in HCV genotype 1 and 2 patients was not found in our in vitro assay. Interestingly, compared with J6/JFH1, TN full-length virus was more sensitive to the protease inhibitors and the NS5A inhibitor, but with similar sensitivity to the NS5B inhibitor. Higher efficacy against genotype 1 may result from the fact that the development of these HCV-specific drugs was based on genotype 1 sequences. No NS5B inhibitor was previously tested for full-length HCV genotype 1 virus in culture. Here, we demonstrated that a lead NS5B inhibitor PSI-7977 could efficiently inhibit HCV of genotypes 1 and 2, thus confirming its cross-genotype antiviral effect (Fig. 2). None of the mutations in TN full-length virus were located in the NS3 protease sequence (Table 1), thus lower EC50 for TN full-length most likely reflect the actual drug sensitivity of the TN isolate. However, it cannot be excluded that adaptive mutations outside protease regions could have an impact on sensitivity to the tested drugs. Thus, a better understanding of the specific interactions between the different HCV proteins in drug resistance is needed and may be achieved by the development of additional full-length HCV culture systems for different genotypes and subtypes.

In conclusion, we have developed a highly efficient full-length HCV genotype 1 culture system, TNcc. This system may prove to be a major asset to the hepatitis C field by directly contributing to drug development and preclinical screening of drugs that will optimize treatment regimens in HCV genotype 1-infected patients. The TNcc, in combination with other genotype culture systems, may facilitate development of drugs with universal effect for different HCV genotypes. The approach and identified mutations used for developing the TNcc culture system may also facilitate the culture development of HCV full-length systems for most HCV patient isolates, with application for HCV vaccine and drug development, and for better individualized treatments.

Materials and Methods

Plasmids.

Partial TN NS5A and JFH1 NS5B-3′UTR (BbvCI-XbaI digested) of J6/JFH1(TN-NS5A) (28) was cloned into full-length HC-TN clone (GenBank accession no. EF621489) (34) to make TN(JFH1_5BX). Mutations in TN(JFH1_5BX) or in full-length TN genomes were generated by PCR, site-directed mutagenesis by QuikChange (Agilent Technologies) or chemically synthesized (GenScript). T7 promoter was inserted immediately upstream of the TN 5′UTR to initiate in vitro transcription (34). All final plasmid preparations were sequenced covering T7 promoter and the entire HCV genome.

Transfection and Infection of Huh7.5 Cells.

The human hepatoma cell line Huh7.5 was cultured as described (17, 18). Twenty-four hours before transfection or infection, cells were seeded in six-well plates (∼4.0 × 105 cells per well) and had reached 80–90% confluence at the time of transfection. Transfection and infection procedures were previously described (13). The transfected or infected cultures were left for ∼16 h and subcultured every 2–3 d; the supernatant was collected, filtered (0.45 µm), and stored at −80 °C.

Analysis of HCV in Cultured Cells.

Monoclonal anticore antibodies B2 (Anogen) or C7-50 (Enzo Life Sciences) and anti-NS5A antibody 9E10 (17) were used for immunostaining for HCV, as previously described (13, 18, 28, 47). The percentage of HCV antigen positive cells was estimated under fluorescence microscopy and used as an approximate indication of the status of HCV infection in the culture. HCV infectivity titers were determined by focus-forming-unit assay as previously described (19, 28, 47). Anti-NS5A 9E10 (17) was used in 1/1,000 dilutions for TN(JFH1_5BX) recombinant viruses. A combination of C7-50 (1/500) and 9E10 (1/1,000) was primarily used for full-length viruses; the combination staining overall increased the intensity of immunostaining for HCV infected cells without affecting the infectivity titer, as determined for control J65′UTR-NS2/JFH1 (25). The number of focus-forming units was automatically counted with an ImmunoSpot Series 5 UV Analyzer with customized software (CTL Europe) (9, 28). Supernatant HCV RNA titers were determined using real time RT-PCR TaqMan assay as previously described (18). ORF sequence analysis of the TN recombinant viruses with JFH1 NS5B-3′UTR sequences and TN full-length virus was previously described (18, 25, 28, 47, 52).

HCV Antiviral Treatment.

HCV DAAs were purchased from Acme Bioscience and dissolved in dimethyl sulfoxide. IFN-α2b was purchased from Schering-Plough. The EC50 value of telaprevir (VX-950), boceprevir (SCH503034), simeprevir (TMC435), daclatasvir (BMS-790052), and IFN-α2b against positive control J6/JFH1 were comparable to our previous determinations (13, 26, 28), verifying the reproducibility of our treatment assays. Briefly, Huh7.5 cells grown in poly-d-lysine–coated 96-well plates (Nunc) were infected with HCV and treated with DAAs 24 h postinfection. Treatment with IFN-α2b was performed at 24 and 48 h postinfection (28). Single HCV core and NS5A+ cells (13, 28) were determined 72 h postinfection by immunostaining with combination of C7-50 (Enzo Life Sciences) and 9E10 antibodies. Dose–response curves were fitted and EC50 values with 95% confidence interval were calculated as described (26). A cytotoxicity assay (Promega) (26) was performed for PSI-7977, as we had not used this drug previously. The noncytotoxic dose range for telaprevir, boceprevir, simeprevir, daclatasvir, and IFN-α2b was determined previously (26, 28). No cytotoxic effects were observed at the doses used in this study.

Supplementary Material

Supporting Information
supp_109_48_19757__index.html (5.7KB, html)

Acknowledgments

We thank L. S. Mikkelsen for technical assistance, A. L. Sørensen and L. Ghanem for general laboratory support, J. O. Nielsen and O. Andersen for providing valuable support (all from Copenhagen University Hospital, Hvidovre), and C. M. Rice (Rockefeller University, New York) and T. Wakita (National Institute of Infectious Diseases, Tokyo) for providing reagents. This study was supported by research grants from the Lundbeck Foundation (J.G.M. and J.B.), the Danish Cancer Society (J.M.G. and J.B.), the Novo Nordisk Foundation (Y.-P.L., J.M.G., and J.B.), the A. P. Møller og Hustru Chastine Mc-Kinney Møllers Fondation (J.B.), the Danish Council for Independent Research–Medical Sciences (Y.-P.L., S.R., and J.B.), and in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (R.H.P.). S.R. is the recipient of an individual postdoctoral stipend from the Danish Council for Independent Research–Medical Sciences.

Footnotes

The authors declare no conflict of interest.

Data deposition: The nucleotide sequence of pTNcc has been deposited in the GenBank database (accession no. JX993348).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218260109/-/DCSupplemental.

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