The human hepatoma cell line Huh7.5 was generously provided by Dr. Charles M. Rice[22] (Rockefeller University) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin, non-essential amino acids, and 10% fetal bovine serum (Invitrogen) at 37 °C in 5% CO₂. All the experiments described in this study were performed using these cells.

The monoclonal antibody to NS5A protein (Abcam), the goat anti-mouse IgG conjugated with horseradish peroxidase (Sigma), and goat anti-mouse IgG conjugated with Alexa Fluor 594 (Invitrogen) were all obtained commercially.

Plasmid constructs were based on the consensus sequence of HCV pJFH1, which was kindly provided by Dr. Wakita[10]. JFH1-∆V3-EGFP and JFH1-AM120 plasmids were kindly provided by Dr. C.H. Hagedorn and Shuang-Hu Liu[19]. The mutations located in HCV genomic RNA are shown in Figure 1. A series of primers for construction of adaptive variants of wild-type HCV JFH1 listed in Table 1 were designed using the pJFH1 sequence and mutations. The pJFH1 plasmid was used as a template for subsequent PCR with Phushion High-Fidelity PCR Master Mix with GC buffer (New England Biolabs) according to the manufacturer’s instructions. The preliminary PCR products (mE2-1, mE2-2, mp7-1, mp7-2, mNS4B-1, mNS4B-2, mNS5A-1, mNS5A-2, mNS5A-3, and mNS5A-4) were analyzed by 1% agarose gel electrophoresis, and used for overlap PCR following the combination showed in Tables 2 and 3 to obtain adaptive mutation fragments. The above fragments (mE2, mp7, mNS4B, mNS5A, mE2/NS5A, mp7/NS5A, mNS4B/NS5A, and mE2/p7/NS5A) were sub-cloned into pJFH1 using the appropriate unique restriction enzyme sites such as Bsiw I, Kpn I, Nsi I, Rsr II, or BsrG I, to produce JFH1-mE2, JFH1-mp7, JFH1-mNS4B, JFH1-mNS5A, JFH1-mE2/ NS5A, JFH1-mp7/NS5A, JFH1-mNS4B/NS5A, JFH1-mE2/p7/ NS5A, and also mJFH1, which contained all the six mutations. All new clones were sequenced using an ABI 3700-XL (Shanghai Sangon Biotech).

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Figure 1 Schematic representation of adaptive mutations used in this study (A) and the electrophoresis results of each mutant virus RNA (B). A: Both nucleotide substitutions (2310, 2681, 6132, 7160, 7359, and 7658) and amino acid substitutions (D657G, H781Y, N1931S, C2274R, I2340T, and V2440L) are shown; B: HCV RNA (500 ng) was analyzed using formaldehyde agarose gel electrophoresis. Lane 1: JFH1; Lane 2: JFH1-mE2; Lane 3: JFH1-mP7; Lane 4: JFH1-mNS4B; Lane 5: JFH1-mNS5A; Lane 6: JFH1-mE2/NS5A; Lane 7: JFH1-mp7/NS5A; Lane 8: JFH1-mNS4B/NS5A; Lane 9: mJFH1; Lane 10: JFH1-mE2/p7/NS5A; M: RNA marker. HCV: hepatitis C virus.

To generate the full-length genomic RNA, pJFH-1 and all plasmids were linearized with Xba I. The linearized plasmid DNA was purified and then used as a template for T7 in vitro transcription (MEGAscript; Ambion). The RNA genomes were detected by formaldehyde agarose gel electrophoresis as described previously[23], and transfected into cells by electroporation[13].

Cells seeded on glass coverslips were infected with HCV. After 48 h, the slips were washed with PBS. Then, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 1% BSA and 1% normal goat serum. The NS5A in cells was detected with a monoclonal antibody and a secondary goat anti-mouse Alexa Fluor 594 antibody (Invitrogen) and visualized by fluorescence microscopy.

The titer of infectious HCV was determined by immunofluorescence assay[7]. Virus titers from supernatants and cell lysates as well were determined using FFUs.

Cell lysates were prepared as described previously[24]. Briefly, cell pellets harvested after trypsinization were washed with PBS, re-suspended in completed culture medium, and lysed in four freeze/thaw cycles at -80 °C and 37 °C. The cell lysates were centrifuged at 4000 rpm for 5 min prior to inoculation into naïve Huh7.5 cells.

Virus titration analysis was conducted by serially diluting the cell supernatants or cell lysates 10-fold in DMEM. The supernatants were used to infect 1 × 10⁴ naïve Huh7.5 cells in 96-well plates. The cells were incubated with virus for 2 h at 37 °C, washed, and incubated with complete DMEM. The level of HCV infection was analyzed 3 and 9 d post-infection by immune- fluorescence staining for NS5A.

The Huh7.5 cells infected with HCV RNA were lysed in 50 mmol/L Tris-HCl (pH 7.5) containing 150 mmol/L sodium chloride, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, and proteinase inhibitors (Complete Mini, Roche). Samples were separated by 10% SDS-PAGE and then transferred onto nitrocellulose membranes. The HCV NS5A (p56/p58) was analyzed as described previously[13].

Total RNA was extracted with TRIzol (Invitrogen). HCV RNA was measured by qPCR analysis as described previously[25]. β-actin was used as the internal control. The relative quantity of HCV RNA in control and HCV samples was calculated by the comparative Ct (cycling threshold) method using LightCycler480.

Cells transfected with HCV RNA with adaptive mutations were seeded onto 24-well plates with cover slips. The cells were treated as previously described[13]. After 48 h, the cells were washed with PBS, fixed with 4% paraformaldehyde, and then permeabilized with 0.2% Triton X-100. Fixed cells were blocked with 1% bovine serum albumin and 1% normal goat serum in PBS. Next, HCV NS5A was analyzed in cells using a NS5A monoclonal antibody and a secondary goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, dilution of 1:1000). LipidTOX Deep Red (Invitrogen) was used to detect neutral lipids present in lipid droplets (LDs). The slides were counterstained using DAPI (Invitrogen), and examined using an Zeiss LSM 510 Meta confocal laser scanning microscope.

A previous study demonstrated that JFH-∆V3-EGFP variant produces a higher titer of reporter virus[19]. The six adaptive mutations in this variant were located in the E2 (D657G), p7 (H781Y), NS4B (N1931S), and NS5A (C2274R, I2340T, and V2440L) (Figure 1A), respectively. To determine the individual or synergistic combination of these mutations responsible for the increased viral production, recombinant JFH1 genomes containing only one of the selected mutations or four different combinations as shown in Figure 1A were constructed.

Next, ten in vitro-transcribed mutant JFH1 RNAs (Figure 1B) were electroporated into Huh7.5 cells to produce recombinants of adapted virus. The transfected cells were sub-cultured every three days. The infectivity titers of supernatants at day 3 (P1) and day 9 (P3) were measured (Figure 2). Viral titers are expressed as FFUs/mL and were assayed in duplicate, which was repeated three times. The data are presented as mean ± SD (n = 3). The HCV titers of wild type JFH1 (JFH1) were extremely low, with a typical titer of 10² FFUs/mL. The adaptive mutations in E2, p7, NS4B, and NS5A individually increased the production of infectious HCV titers 2- to 4-fold compared with the levels of JFH1. The NS5A mutations exhibited the greatest effect on the production of infectious HCV particles, with a titer of 1.30 × 10⁶ FFUs/mL at P3. The p7 mutation followed closely, generating a titer of 2.10 × 10⁵ FFUs/mL. Briefly, except for E2, the HCV titers of other variants at P3 were partially higher than those at P1.

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Figure 2 Generation of high titer cell culture-adaptive JFH1 virus. Hepatitis C virus RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus in cell culture. The transfected cells were passaged every three days. The infectivity titers of the culture supernatants at day 3 (P1) and day 9 (P3) were measured. Viral titers are expressed as focus-forming units per milliliter (FFUs/mL). The data are presented as mean ± SD (n = 3). HCV: Hepatitis C virus. ^aP < 0.05; ^bP < 0.01.

To further determine any synergistic effect of the six adaptive mutations on HCV production, we focused on the recombinant viruses with adaptive mutations in different combinations. As shown in Figure 2, JFH1-mE2/NS5A, JFH1-mp7/NS5A, JFH1-mNS4B/NS5A, and JFH1-mE2/p7/NS5A remarkably enhanced the production of infectious HCV, and the mJFH1 produced infectious HCV particles with a robust titer of 1.61 × 10⁶ FFUs/mL 9 d post-transfection.

These results suggest that all the six adaptive mutations increase the HCV particle production. NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) are the critical adaptive mutations.

HCV RNA genome replication and structural or nonstructural protein expression are early steps in the HCV life cycle. To further confirm our speculation that the robust HCV titers and enhanced virion release were both related to up-regulated RNA replication and protein expression, we determined the relative HCV RNA, NS5A immunofluorescence, and NS3 protein levels in the RNA-transfected Huh7.5 cells on day 3 (P1) and day 9 (P3).

As shown in Figure 3, the expression of NS5A (Figure 3A) and NS3 (Figure 3B) in mutants was up-regulated at different levels during serial passages. The trend was extraordinary obvious in NS5A or p7 mutants. Anti-NS3 Western blot analysis, which was the most widely used for quantitative experiment, yielded consistent results.

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Figure 3 Effects of the adaptive mutations on the hepatitis C virus RNA replication. A: Hepatitis C virus (HCV) RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus. The transfected cells were passaged every 3 d. Cells were fixed 48 h after passage and infected cells were identified by fluorescence immunostaining and microscopy. Nuclear DNA was stained with DAPI (blue); B: HCV RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus in cell culture. The transfected cells were passaged every 3 d. Cells were lysed at 72 h after passage. The HCV NS3 protein levels were analysis by Western blot. ^bP < 0.01; C: HCV RNA levels in cells 3 d after transfection. Intracellular HCV RNA levels were analyzed by quantitative RT-PCR. The mean ± SD for three independent experiments are presented (qPCR assays, n = 3). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

As shown in Figure 3C, the RNA levels of all the mutants were increased compared with JFH1, and mJFH1 was increased 18.7-fold. Interestingly, the results indicated that JFH1-mNS4B expression increased 6.1-fold, and the combination of mutants showed a 7.4-16.8-fold increase.

Taken together, we confirmed that the effect of NS5A (C2274R, I2340T and V2440L), p7 (H781Y), and NS4B (N1931S) on infectious HCV titers was robust, and started with HCV RNA replication and protein expression, followed by virion release.

Virion release is the last step of the HCV life cycle. To further explore the mechanism underlying the enhanced virus production, the role of adaptive mutations was examined. Ten HCV RNAs (JFH1, JFH1-mE2, JFH1-mP7, JFH1-mNS4B, JFH1-mNS5A, JFH1-mE2/NS5A, JFH1-mp7/NS5A, JFH1-mNS4B/NS5A, mJFH1, and JFH1-mE2/p7/NS5A) were electroporated into Huh7.5 cells. After 3 d, we collected the supernatants and cell lysates, and measured the HCV titers using NS5A immunofluorescence assays. Furthermore, to confirm the infectivity of virions, we carried out immunoblotting of supernatants and cell lysates with anti-NS3, which was extraordinarily consistent with the infectious HCV titers (Figure 4A). As shown in Figure 4B, we also calculated the proportion of extracellular (supernatant) or intracellular (cell lysate) HCV titers using total titers as 100%. Typically, the supernatant and intracellular HCV titers of JFH1 were 76.31% and 23.69%, respectively, and those of mJFH1 were 94.00% and 6.00%, respectively. Taken together, these findings provide evidence suggesting that the ten variant viruses enhanced the virion release, and the high viral production was linked to the effective virion release. NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) showed the highest levels compared with the others.

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Figure 4 Effect of the adaptive mutations on the virion release. A: Hepatitis C virus (HCV) RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus. At 72 h after transfection, the infectivity titers of the culture supernatants and cell lysates were measured. Viral titers are expressed as FFUs/mL. The data are presented as mean ± SD (n = 3); B: HCV RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus. At 72 h after transfection, the infectivity titers of the culture media and cell lysates were measured. The extracellular and intracellular viral titers were measured. The relative ratios of infectious virions are shown. The results were from three independent experiments; C: The naive Huh7.5 cells were infected with the culture media and cell lysates. At 72 h after infection, cells were lysed with RIPA buffer, and analyzed by Western blot.

LDs have been reported to play an important role in the HCV virion assembly process[26]. To determine if the six adaptive mutations increased the assembly of HCV at this step, LDs and NS5A were stained in JFH1 and mJFH1 transfected cells and the co-localization of LDs with NS5A was measured. As shown in Figure 5, the LDs were totally covered with NS5A in all cases. However, no significant difference was observed between JFH1 and mJFH1 groups using Image J software and Pearson’s correlation coefficient analysis. These results indicated that the six adaptive mutations were not required for LD localization of the NS5A proteins.

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Figure 5 Colocalization analysis of lipid droplets and hepatitis C virus NS5A. JFH1 and mJFH1 RNA was electroporated into Huh7.5 cells to produce the recombinants of adapted virus. At 48 h after transfection, the cells were fixed. Lipid droplets were stained with LipidTOXRed (Red). The HCV NS5A was stained with anti-NS5A antibody (Green). The nucleus was stained with DAPI (Blue). Each triplicate sample of 25 cells was analyzed using Image J software. The degree of co-localization was quantified and compared using Pearson’s correlation coefficients.

Previous studies showed that a ratio between the p56 and p58 phosphoforms of NS5A is required for optimal HCV RNA replication[13,27-29]. JFH1-AM120 is a robust adaptive mutant selected by Liu et al[13]. which displays significant switch of p56/p58. In this study, JFH1, JFH1-AM120, and mJFH1 RNA were transfected into Huh7.5 cells, and the total protein was used for Western blot analysis after 3 d (Figure 6). However, we observed no difference in p56 and p58 between the two groups. These results demonstrated that the phosphorylation level of NS5A was not affected by adaptive mutations.

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Figure 6 Phosphorylation of NS5A during JFH1 and mJFH1 replication. Huh7.5 cells were transfected with JFH1 or mJFH1 RNA. After three days of culture, cells were lysed for western blot using anti-NS5A and anti-β-actin antibodies. The quantity of p56 and p58 was determined using Image J software and the ratios of p56/p58 are shown. Data are presented as mean ± SD (n = 3). JFH1-AM120 was used as the positive control.

Hepatitis C virus (HCV) causes acute and chronic hepatitis, and leads to permanent liver damage and hepatocellular carcinoma. The infectious HCV JFH1 cell culture system represents a major advance in anti-HCV drug discovery research and facilitates the study of HCV life cycle. However, HCV JFH1 (genotype 2a) merely generates relatively low viral titers. JFH1-∆V3-EGFP, which includes six mutations located in the E2, p7, NS4B, and NS5A regions, could produce higher titers of HCV-EGFP reporter virus. However, there were no data about which mutations or combinations thereof are responsible for enhanced viral production and the underlying mechanisms.

This JFH1 model generated infectious viral particles in cell culture and facilitated the study of the HCV life cycle, but the low infectious virion titer limits its application range. Some previous studies have confirmed that adaptive mutations could enhance the virion titer, but the mechanism has not yet been fully elucidated. In this study, we focused on the positive effect of six adaptive mutations located in the E2, p7, NS4B, and NS5A regions, and found that the mechanism was different among them during the procession. These results gave us some new insights into the infectious HCV cell culture system and adaptive mutations.

The main objective of this study was to establish an infectious HCV cell culture system with a robust titer, and to discuss the underlying mechanisms of the adaptive mutations found in previous studies. The results of this study have supplied the researchers with a useful tool. We hope it will be used for the study of viral structure, virus-host interaction, anti-HCV drug discovery, and vaccine development.

We investigated JFH1-mE2, JFH1-mp7, JFH1-mNS4B, JFH1-mNS5A, JFH1-mE2/NS5A, JFH1-mp7/NS5A, JFH1-mNS4B/NS5A, JFH1-mE2/p7/ NS5A, and mJFH1, carrying all the six mutations. We analyzed the infectious HCV titer, HCV RNA and NS3 protein levels, viral release capacity, assembly and hyper-phosphorylation of NS5A to determine the role of these mutations in the HCV life cycle. These methods were the routine ways adopted widely in virological and molecular biological research.

The main findings in this study were as follows: (1) we generated infectious HCV particles with a robust titer of 1.61 × 10⁶ FFUs/mL; (2) The six adaptive mutations increased the HCV particle production at varying levels. The NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) are critical adaptive mutations. The effect of NS5A (C2274R, I2340T, and V2440L), p7 (H781Y), and NS4B (N1931S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release; and (3) the six adaptive mutations were all not required for the lipid droplet localization of NS5A proteins or the phosphorylation of NS5A. To our knowledge, this is a new robust titer related to adaptive mutations from JFH1. The problems that remain to be solved in the future include: (1) how could the adaptive mutations be translated to clinical conditions? (2) are these mutation patterns observed in vivo? and (3) would these results be relevant to the resistance to direct-acting antivirals (DAAs)?

First, this study generated infectious HCV particles with a robust titer of 1.61 × 10⁶ FFUs/mL. Second, all of the six adaptive mutations increased the HCV particle production at varying levels. Third, the NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) were critical adaptive mutations, but they were not required for the LD localization of NS5A proteins or the phosphorylation of NS5A. Based on the new findings of this study, we proposed that more important adaptive mutations would be addressed in the future, and unknown mechanism of the HCV life cycle would be explained.

This study re-confirmed that the JFH1 was still a promising system to study the HCV life cycle. To use adaptive mutations was an effective way to establish a new system with higher infectious HCV virion titer. In addition, we also re-confirmed that the molecular mechanism of interaction between viral proteins and/or host-cell proteins is more complex and important.