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The Journal of General Virology logoLink to The Journal of General Virology
. 2014 Feb;95(Pt 2):423–433. doi: 10.1099/vir.0.055772-0

Direct visualization of hepatitis C virus-infected Huh7.5 cells with a high titre of infectious chimeric JFH1-EGFP reporter virus in three-dimensional Matrigel cell cultures

Shuanghu Liu 1,†, Ren Chen 1,†, Curt H Hagedorn 1,2,✉
PMCID: PMC3917068  PMID: 24243732

Abstract

Identification of the hepatitis C virus (HCV) JFH1 isolate enabled the development of infectious HCV cell culture systems. However, the relatively low virus titres and instability of some chimeric JFH1 reporter viruses restricts some uses of this system. We describe a higher-titre JFH1-EGFP reporter virus where the NS5A V3 region was replaced with the EGFP gene and adapted by serial passage in Huh7.5 cells. Six adaptive mutants were identified: one each in E2, P7 and NS4B, plus three in the NS5A region. These adaptive mutants increased the reporter virus titres to 1×106 immunofluorescent focus-forming units ml−1, which is the highest titre of JFH1-EGFP reporter virus reported to our knowledge. This chimeric virus did not lose EGFP expression following 40 days of passage and it can be used to test the activity of HCV antivirals by measuring EGFP fluorescence in 96-well plates. Moreover, this reporter virus allows living infected Huh7.5 cells in Matrigel three-dimensional (3D) cultures to be visualized and produces infectious viral particles in these 3D cultures. The chimeric NS5A-EGFP infectious JFH1 reporter virus described should enable new studies of the HCV life cycle in 3D cell cultures and will be useful in identifying antivirals that interfere with HCV release or entry.

Introduction

Hepatitis C virus (HCV), a member of the virus family Flaviviridae family, infects approximately 3 % of the human population worldwide and remains a major public health problem. HCV infection frequently leads to chronic hepatitis, liver cirrhosis and, eventually, hepatocellular carcinoma (Alter & Seeff, 2000; Bialek & Terrault, 2006). A preventive vaccine has not been developed and although HCV antivirals are improving, there remains a need for additional antivirals (Bowen & Walker, 2005; Fried et al., 2002; Houghton & Abrignani, 2005).

HCV is an enveloped RNA virus and has a single-stranded 9.6 kb RNA genome of positive polarity containing a 5′ internal ribosome entry site (IRES) element (Penin et al., 2004; Reed & Rice, 2000). IRES-driven HCV RNA translationally produces a polyprotein of approximately 3000 amino acids. The polyprotein precursor is co- and post-translationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins (Yi et al., 2007). The structural proteins include the core protein, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The nonstructural proteins, NS2 through NS5B, include the NS2-3 auto protease, the NS3 serine protease, an RNA helicase located in the C-terminal region of NS3, the NS4A polypeptide, the NS4B and NS5A proteins and the NS5B RNA-dependent RNA polymerase (Jones et al., 2007; Moradpour et al., 2007).

The infectious HCV JFH1 cell culture system represented a major advance (Kato et al., 2001; Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005). This cell culture model allows all stages of the HCV life cycle to be studied. However, viral protein immunostaining used in detecting and measuring virus replication can make assays using this system time consuming. A wide variety of chimeric infectious HCV luciferase reporter systems have been developed to solve this problem (Koutsoudakis et al., 2006; Liu et al., 2011, 2012; Phan et al., 2009; Tscherne et al., 2006). HCV-EGFP reporter systems that can be used in three-dimensional (3D) culture systems to study HCV infection have been challenging. Although chimeric EGFP JFH1 viruses with EGFP inserted into the NS5A C-terminal region have produced infectious chimeric HCV systems (Kim et al., 2007, 2011; Liu et al., 2012), they have a relatively low titre that is more comparable to JFH-1 WT and make high-throughput 96-well assays impractical. Development of a stable higher titre chimeric infectious EGFP HCV reporter system would enable a number of new studies, such as identifying inhibitors of viral entry, more approachable than using other EGFP JFH-1 chimeras that produce low titres of infectious virus or have other limits for such studies.

The NS5A protein of HCV is multifunctional and contains an amphipathic α-helix and zinc-binding domain at the amino terminus, both of which are required for viral replication (Appel et al., 2005; Brass et al., 2002; Evans et al., 2004; Tellinghuisen et al., 2004, 2005). NS5A of both HCV-1a and 1b has a highly variable region of 24 amino acids, designated the V3 region (Inchauspe et al., 1991). Our previous study demonstrated that the V3 region of JFH1 HCV 2a is located in aa 2356–2383 of NS5A and that replacing V3 with the Renilla luciferase gene did not disrupt HCV replication and the production of infectious virus (Liu et al., 2011).

Recent studies have suggested the importance of 3D hepatoma cell cultures in studying HCV (Molina-Jimenez et al., 2012; Sainz et al., 2009). Matrigel is a gelatinous protein mixture secreted by Engelbreth–Holm–Swarm mouse sarcoma cells that resembles the complex extracellular environment found in many tissues and produces a thick matrix for 3D cell culture (Kleinman & Martin, 2005). Huh7.5 cells can grow in Matrigel and these cell line-based, Matrigel-embedded cultures can be used to study HCV infection and virion production in a context of polarized hepatocyte-like cells. The development of a stable, relatively high-titre cell culture of infectious chimeric EGFP HCV JFH1 reporter virus would expand the possible uses of 3D HCV cell culture systems. We reported that replacing the V3 region of NS5A of HCV JFH1 with the EGFP gene followed by serial passage in cell culture cells produced such an infectious chimeric virus that allowed infected cells to be visualized and studied in 96-well plates and in 3D Matrigel cultures.

Results

Replacement of the JFH1 V3 region with EGFP does not disrupt viral replication and the production of infectious HCV particles

Our previous study demonstrated that the V3 region of JFH1 could be replaced with the Renilla luciferase gene (930 bp) (Liu et al., 2011). In this study the EGFP gene (720 bp) was used to replace the NS5A V3 region of JFH1, designated JFH1-ΔV3-EGFP, with the aim of developing an infectious reporter virus that would enable intact infected cells to be directly visualized (Fig. 1a). RNA was transcribed from JFH1-WT and JFH1-ΔV3-EGFP plasmids and was used to transfect Huh7.5 cells to determine whether cells transfected with JFH1-ΔV3-EGFP RNA expressed HCV proteins and released infectious viral particles. Three days after transfection, cell lysates were prepared and the levels of NS5A protein were evaluated by Western blot analysis. Three days after RNA transfection of cells, wild-type NS5A and the NS5A-ΔV3-EGFP fusion protein were easily detected by Western blotting of cell lysates (Fig. 1b). Cells transfected with RNA were grown to approximately 90 % confluence, subcultured at 3-day intervals for 18 days and titres of infectious virus in culture supernatants measured by inoculation of naïve Huh7.5 cells. Maximum virus titres of 4.6×104 focus-forming units (ffu) ml−1 for wild-type JFH1 and 1.0×104 ffu ml−1 for JFH1-ΔV3-EGFP were observed at 15 days post-transfection (Fig. 1c), demonstrating that the titres of infectious viral particles produced by JFH1-ΔV3-EGFP-transfected cells were not dramatically different. These results demonstrate that the replacement of the V3 region of NS5A in JFH1 with the EGFP gene does not abrogate viral replication and only moderately decreases the production of infectious viral particles.

Fig. 1.

Fig. 1.

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Analysis of HCV replication and production of infectious virus following JFH1-ΔV3-EGFP RNA transfection. (a) Schematics of JFH1-wt and JFH1-ΔV3-EGFP where the V3 region was replaced with the EGFP gene. The location of the V3 in the NS5A region of JFH1 was previously described (Liu et al., 2011). (b) Analysis of NS5A protein expression following RNA transfection. The NS5A, NS5A-EGFP fusion and β-actin proteins were identified by Western blotting. Experiments were done three times and a representative example is shown. (c) Titre of infectious HCV produced following RNA transfection. Huh7.5 cells were infected with supernatants collected 15 days after transfection with JFH-wt and JFH1-ΔV3-EGFP RNA. Viral titres are expressed as supernatant ffu ml−1 as determined by the average number of NS5A-positive foci detected by immunofluorescence staining for NS5A. Assays were done in triplicate in 96-well plates, performed twice and the data represent the mean±sd (n = 6).

Generation of cell culture-adapted high-titre JFH1-ΔV3-EGFP reporter virus during serial passage

The JFH1-ΔV3-EGFP virus titre of 1×104 limited the use of this reporter virus for many experiments, including 96-well plate cell culture studies. To address this problem we attempted to produce an adapted higher-titre infectious JFH1-ΔV3-EGFP reporter virus by serial passage. In vitro transcribed JFH1(WT)-ΔV3-EGFP RNA was electroporated into Huh7.5 cells which were subcultured (passaged) every 3 days. Five passages were defined as one cycle. The culture supernatant from the fifth passage of each cycle was used to infect fresh Huh7.5 cells. A total of four cycles, 20 passages (60 days), was performed. The HCV titre was found to be increased by day 30 and reached 1.0×106 ffu ml−1, suggesting that JFH1-ΔV3-EGFP acquired adaptive mutations increased the production of infectious virus. Cells continued to be infected and passaged as described, and the virus titre was observed to plateau at approximately 1.0×106 ffu ml−1 following another 30 days of passage. At that time the passaging of cells was stopped and virus stocks prepared from these cells were used for subsequent experiments. The adapted virus was designated Ad-JFH1-ΔV3-EGFP (Adapted JFH1-ΔV3-EGFP). To identify the mutations responsible for the enhanced production of infectious Ad-JFH1-ΔV3-EGFP, HCV RNA isolated from infected cells was reverse transcribed and PCR amplified in four overlapping fragments as described previously (Liu et al., 2012). The PCR fragments were subcloned and sequenced as described in Methods. A total of six non-synonymous mutations was found in Ad-JFH1-ΔV3-EGFP. One mutation each in the E2, P7 and NS4B genes was identified and the remaining three mutations were found in the NS5A gene (Fig. 2a). No synonymous mutations in Ad-JFH1-ΔV3-EGFP were identified.

Fig. 2.

Fig. 2.

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Growth comparison of the HCV JFH1-ΔV3-EGFP and the cell culture-adapted virus (Ad-JFH1-ΔV3-EGFP). (a) The position of adaptive mutations in HCV Ad-JFH1-ΔV3-EGFP. Amino acids are numbered based on the HCV polyprotein. (b) Titres of infectious Ad-JFH1-ΔV3-EGFP compared to the JFH1-ΔV3-EGFP virus. Huh7.5 cells were transfected with 2 µg of wild-type or Ad-JFH1-ΔV3-EGFP RNAs. The supernatants were collected at 24, 48, 72 and 96 h post-transfection. Assays were done in triplicate in 96-well plates, performed twice and the data presented as the mean±sd (n = 6). (c) Growth curves of JFH1-ΔV3-EGFP and Ad-JFH1-ΔV3-EGFP in Huh7.5 cells. Huh7.5 cells in 24-well plates were infected with JFH1-ΔV3-EGFP or Ad-JFH1-ΔV3-EGFP at a m.o.i. of 0.3. Infectious HCV titres at different time points (24, 48, 72 and 96 h) following infection are shown. Assays were done in triplicate in 96-well plates, performed twice and presented as the mean±sd (n = 6).

In order to confirm the effect of adaptive mutations on the production of infectious virus, Huh7.5 cells were transfected with Ad-JFH1-ΔV3-EGFP RNA and virus titres were measured. Ad-JFH1-ΔV3-EGFP produced infectious viral titres that were two orders of magnitude higher than JFH1-ΔV3-EGFP virus at 48, 72 and 96 h (Fig. 2b). The enhanced production of infectious Ad-JFH1-ΔV3-EGFP, relative to JFH1-ΔV3-EGFP, was confirmed by measuring infectious titres at different time points after infecting cells with the same m.o.i. (Fig. 2c).

Adaptive mutations were not necessary for the intracellular co-localization of NS5A on lipid droplets

Lipid droplets have been reported to play a critical role in the intracellular assembly of HCV (Miyanari et al., 2007). In addition, the recruitment of NS5A to lipid droplets is a prerequisite for HCV assembly in host cells (Aizaki et al., 2008; Miyanari et al., 2007). To determine whether the adaptive mutations in NS5A increased the production of HCV at this step, lipid droplets and NS5A were stained in JFH1-ΔV3-EGFP- and Ad-JFH1-ΔV3-EGFP-transfected cells and the co-localization of NS5A with lipid droplets was analysed. Analysis showed that the lipid droplets were covered with NS5A in cells infected with both viruses (Fig. 3a). The degree of co-localization of NS5A and lipid droplets was quantified using Image J software and Pearson’s correlation coefficient analysis. No significant difference was observed between JFH1-ΔV3-EGFP- and Ad-JFH1-ΔV3-EGFP-infected cells (Fig. 3b), providing evidence that the adaptive mutations did not have an effect on this step of virus assembly.

Fig. 3.

Fig. 3.

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Co-localization of the NS5A-EGFP fusion protein with lipid droplets (LDs). (a) Huh7.5 cells were transfected with JFH1-ΔV3-EGFP and Ad-JFH1-ΔV3-EGFP RNA, fixed after 48 h of incubation and LDs were stained with LipidTOX Red (red) and the NS5A–EGFP fusion protein (green) visualized by fluorescence confocal microscopy. A 5 µm scale bar is shown. (b) The degree of co-localization of LDs and NS5A-EGFP was quantified using Pearson’s correlation coefficient. Fifty cells in each sample were analysed using ImageJ software. P-values were calculated using Student’s t-test.

Stability of the HCV Ad-JFH1-ΔV3-EGFP reporter virus after multiple passages

To determine whether cells infected with the progeny virus of Ad-JFH1-ΔV3-EGFP could undergo serial passage in naïve Huh7.5 cells without the loss of EGFP, we infected naïve cells at a very low m.o.i. of 0.005 and followed the production of the NS5A-EGFP fusion protein and infectious virus titres. The low m.o.i. inoculum infected a relatively small percentage of cells until day 6. Cells were passaged every 2 days and monitored for expression of the NS5A-EGFP fusion protein and infectious virus titres. Lysates of the infected cells were analysed by Western blotting and a fusion protein of the predicted molecular mass of 84.5 kDa was detected, showing no evidence of decreased expression after 40 days of passage (Fig. 4a).

Fig. 4.

Fig. 4.

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Kinetics of infectious virus production following infection of Huh7.5 cells with Ad-JFH1-ΔV3-EGFP. (a) Huh7.5 cells were infected with Ad-JFH1-ΔV3-EGFP virus at a low m.o.i. of 0.005. Cells were passaged every 2 days and, following four passages, cell supernatants were used to infect naïve Huh7.5 cells. Cell lysates were analysed by Western blotting for NS5A-EGFP at the indicated times. Experiments were performed twice and representative results are shown. (b) Virus titres in culture supernatants collected at the indicated times after infection are shown in NS5A-positive ffu. Assays were done in triplicate, performed twice and the data presented as the mean±sd (n = 6). (c) Co-localization of NS5A and EGFP after the fifth cycle (40 days) of passaging cells. Fixed whole cells were immunostained with anti-NS5A antibody. Experiments were performed twice and representative results are shown.

Cell supernatants were collected every 2 days, clarified and supernatants from each passage were added to naïve Huh7.5 cells to measure the titre of infectious virus. Three days after inoculation of cells, infectious virus particles accumulated exponentially in the supernatants, reaching a maximal titre of 1.0×106 ffu ml−1 on day 8 (Fig. 4b). To further access the genomic stability of serially passaged Ad-JFH1-ΔV3-EGFP, the co-localization of the NS5A protein and EGFP was examined by immunofluorescence assays 40 days after infection. Images showing cells that were only red (NS5A positive) would mean that EGFP was lost during serial passaging. Image analysis showed that all NS5A-positive cells (red) also expressed EGFP, resulting in a yellow colour after merging images (Fig. 4c). These results provide evidence that Ad-JFH1-ΔV3-EGFP can be passaged in Huh7.5 cells without a major loss of infectivity and that EGFP expression remains high over 40 days of passaging.

Using EGFP produced by Ad-JFH1-ΔV3-EGFP-infected cells to more rapidly measure activity of HCV antivirals

IFN-α has been well documented as inhibiting HCV replication in cell culture (Cai et al., 2005; Guo et al., 2001). Increasing concentrations of IFN-α were added to naïve Huh7.5 cells 2 h after infection with Ad-JFH1-ΔV3-EGFP virus. Two days post-inoculation, cells were fixed with 4 % paraformaldehyde and EGFP-positive cells were directly visualized by fluorescence microscopy and images captured. EGFP-positive cells decreased progressively with increasing concentrations of IFN-α (Fig. 5a). IFN-α markedly inhibited virus replication, as measured by EGFP-positive by Ad-JFH1-ΔV3-EGFP-infected cells (Fig. 5b). To verify that the change in EGFP fluorescence corresponded to a similar change in HCV replication, the NS5A-EGFP fusion protein and HCV RNA were quantified by Western blotting and qPCR, respectively. The results showed that both the NS5A-EGFP fusion protein (Fig. 5c) and HCV RNA levels (Fig. 5d) were inhibited in a dose–response manner and that this corresponded to changes in EGFP fluorescence measurements. Thus, the more rapid measurement of EGFP fluorescence in Ad-JFH1-ΔV3-EGFP-infected cells can replace the more time-intensive HCV protein Western blotting, and RNA qPCR assays and immunostaining of ffu. The utility of the Ad-JFH1-ΔV3-EGFP reporter virus was further tested using a 2′-C-methyladenosine nucleoside analogue HCV RNA-dependent RNA polymerase (NS5B) inhibitor that has been demonstrated to inhibit recombinant NS5B in vitro and HCV replication in hepatoma cells (Eldrup et al., 2004; Liu et al., 2011). This nucleoside analogue inhibited replication of Ad-JFH1-ΔV3-EGFP in a concentration-dependent manner (Fig. 5e). These results provide evidence that the HCV Ad-JFH1-ΔV3-EGFP reporter virus we describe should be useful in identifying and studying other HCV antivirals, particularly those that inhibit the release or transmission of infectious virus.

Fig. 5.

Fig. 5.

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Effect of interferon-alpha (IFN) on HCV Ad-JFH1-ΔV3-EGFP replication in cell culture. (a) Two hours after infection of Huh7.5 cells with the Ad-JFH1-ΔV3-EGFP reporter virus (m.o.i. of 0.3), increasing concentrations of IFN were added followed by 2 days of incubation. Cells were fixed with 4 % paraformaldehyde and directly visualized by fluorescence microscopy. Experiments were performed twice and representative results are shown. (b) Two hours after infection of Huh7.5 cells in 96-well plates with Ad-JFH1-ΔV3-EGFP (m.o.i. of 0.3), increasing concentrations of IFN were added followed by 2 days of incubation. Cells were lysed and fluorescence was measured. Experiments were performed three times and the data presented as the mean±sd (n = 9). (c) Two hours after infection of Huh7.5 cells in 12-well plates with Ad-JFH1-ΔV3-EGFP (m.o.i. of 0.3), increasing concentrations of IFN were added followed by 2 days of incubation. Cells were lysed in RIPA buffer and Western blots were done with anti-NS5A monoclonal antibodies. Experiments were performed twice and representative results are shown. (d) Two hours after infection of Huh7.5 cells in six-well plates with Ad-JFH1-ΔV3-EGFP (m.o.i. of 0.3), IFN was added and followed by 2 days of incubation. Total RNA was extracted from cells and qPCR analysis of HCV RNA was performed. Experiments were performed three times and the data presented as the mean±sd (n = 9). (e) Two hours after infection of Huh7.5 cells in 96-well plates with Ad-JFH1-ΔV3-EGFP (m.o.i. of 0.3), increasing concentrations of NS5B inhibitor, 2-C-methyladenosine nucleoside analogue NS5B polymerase, were added followed by 2 days of incubation. Cells were lysed and fluorescence was measured. Experiments were performed three times and the data presented as the mean±sd (n = 9).

Infection of Huh7.5 cells in 3D Matrigel cultures with the Ad-JFH1-ΔV3-EGFP reporter virus

3D cell culture systems are likely to have advantages in studying at least some aspects of HCV biology in a less artificial environment than two-dimensional (2D) cultures (Molina-Jimenez et al., 2012; Sainz et al., 2009). To determine whether Ad-JFH1-ΔV3-EGFP infection allowed direct visualization of infected cells in 3D cultures, we used a Matrigel-embedded 3D Huh7.5 cell culture system. Huh7.5 cells were seeded in 12-well plates, cultured for 18 h, infected with Ad-JFH-ΔV3-EGFP (m.o.i. of 0.1) and incubated for an additional 24 h. Cells were infected with Ad-JFH1-ΔV3-EGFP virus and after 48 h of culture EGFP positive cells were visualized by fluorescence microscopy. Huh7.5 cells in Matrigel cultures assembled into spheroids, whereas standard 2D-cultured Huh7.5 cells formed typical epithelial monolayers, and cells cultured under both conditions expressed EGFP (Fig. 6a). Confocal images of 3D cultures were processed using Volocity image software. EGFP-positive cells in Matrigel assembled into 3D spheroids (Fig. 6c). Supernatants were collected and used to test virus titres. The virus titres in 3D cultures were similar to 2D cultures.

Fig. 6.

Fig. 6.

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3D Matrigel cultures of Huh7.5 cells infected with HCV Ad-JFH1-ΔV3-EGFP. (a) Confocal phase-contrast images (20× magnification) of 2D and 3D Huh7.5 cell cultures 48 h after Ad-JFH1-ΔV3-EGFP infection. Ad-JFH1-ΔV3-EGFP infected Huh7.5 cells were seeded in 96-well plates without Matrigel for 2D cultures (control). Ad-JFH1-ΔV3-EGFP infected Huh7.5 cells were incubated for 24 h, trypsinized and mixed with Matrigel (1 : 1 v/v) for 3D cultures . After 48 h of incubation images were captured with fluorescence microscopy (scale bar, 100 µm; bright indicates bright field; EGFP indicates green fluorescence). (b) Assays of infectious virus produced in 2D and 3D cultures 48 h after infection. Huh7.5 cells were infected times after infection with Ad-JFH1-ΔV3-EGFP. Assays were done in triplicate in 96-well plates, performed twice and with supernatants collected at 48 h after infection with Ad-JFH1-ΔV3-EGFP from 2D and 3D cultures. Assays were done in triplicate in 96-well plates, experiments performed twice and the data presented as the mean±sd (n = 6). (c) Cells were grown in 3D cultures for 48 h and processed for confocal analysis. A representative example of two separate experiments is shown.

The kinetics of Ad-JFH1-ΔV3-EGFP virus replication in 3D Matrigel cultures were determined. 3D Matrigel Huh7.5 cell cultures were prepared and incubated as described. Live cell confocal images were taken at 24, 48 and 72 h of culture and supernatants were collected to measure viral titres. These images showed that EGFP-positive cells increased from 24 to 72 h (Fig. 7a). The production of infectious virus increased with time in 3D cultures (Fig. 7b).

Fig. 7.

Fig. 7.

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Kinetics of infectious virus produced by HCV Ad-JFH1-ΔV3-EGFP in 3D Matrigel Huh7.5 cell cultures. (a) Ad-JFH1-V3-EGFP-infected Huh7.5 cells in 3D Matrigel cultures were prepared in 96-well plates as in Fig. 6. Images of live cells in 3D cultures were taken at 24, 48 and 72 h and supernatants were collected to measure infectious viral titres. Images of 3D cultures of EGFP expression in infected cells reconstructed using NIS-Element AR software. (b) Titres of infectious virus produced over time in 3D cultures. Huh7.5 cells were infected with supernatants collected at the times indicated; data presented as the mean±sd (n = 6).

Discussion

Previous studies have shown that the highly variable 28 amino acid V3 region located within domain III of NS5A (aa380 to 407 of NS5A or aa2356 and 2383 of the polyprotein) in JFH1 strain of HCV genotype 2a is not essential for viral replication or the release of infectious virus particles (Liu et al., 2011). The V3 region was replaced with the Renilla luciferase gene, resulting in an infectious chimeric virus that has proven to be useful in screening and studying HCV antivirals (Liu et al., 2011). However, one limitation of the chimeric infectious Renilla luciferase reporter virus is that cells must be lysed to measure the reporter molecule and intact cells cannot be monitored for viral infection over time. In this study, we demonstrated that the V3 region of JFH1 can also be replaced with the EGFP gene to produce an infectious chimeric reporter virus that can be used to directly visualize, quantify and monitor HCV infection over time in 3D cultures of Huh7.5 cells. This new reporter virus retains expression of EGFP following multiple passages, produces relatively high titres of infectious chimeric report virus and can monitor the spread of HCV infection between living cells in 3D cultures in 96-well plates.

Problems with chimeric EGFP JFH1 reporter viruses have included the loss of the reporter gene with serial passage or the production of relatively low titres of infectious virus, limiting their experimental use. Although JFH1-ΔV3-EGFP initially had a relatively low titre of 1×104 ffu ml−1, serial passage allowed adaptive mutations to occur resulting in a 100-fold increase in titres of infectious Ad-JFH1-ΔV3-EGFP (1×106 ffu ml−1). Moreover, EGFP expression was retained at a high level following 20 passages (40 days) of infected cells. This higher-titre EGFP chimeric reporter virus should have uses in high-throughput HCV antiviral screening that does not require lysis of cells, and may be adapted to screening of antivirals that impair the release or uptake of HCV. To our knowledge, Ad-JFH1-ΔV3-EGFP is the highest-titre HCV-EGFP chimeric reporter virus described to date and should allow questions that were previously difficult to approach to be answered.

A total of six mutations was identified in Ad-JFH1-ΔV3-EGFP and at least some of these are likely to explain the increased titres of infectious chimeric virus produced in cell culture. One mutation each occurred in the E2, P7 and NS4B regions and three in the NS5A region. The adaptive mutations D657G, H781Y and I2340T have not been reported previously. The adaptive mutations V2440L, C2294R and N1931S have been described with other cell culture-adapted HCV variants (Kaul et al., 2007; Li et al., 2012; Liu, et al., 2012). Cell culture adaptation of viruses can enhance the production of infectious virus by a number of mechanisms. For example, adaptive mutations that shorten the replication cycle of HCV can increase the titres of infectious virus produced in culture (Kaul et al., 2007). Alternatively, adaptive mutations in HCV that enhance protein–protein interactions between structural and nonstructural proteins can promote the assembly of infectious virus particles (Jiang & Luo, 2012). In addition, adaptive mutations that increase the titre of infectious HCV can correlate with a decrease in NS5A p58 (hyperphosphorylated form of NS5A) relative to p56 that may alter virus assembly (Liu et al., 2012). HCV nonstructural proteins, including NS5A and replication complexes are recruited to membranes associated with lipid droplets by the core protein, and this is critical in the assembly of infectious virus (Aizaki et al., 2008; Miyanari et al., 2007). However, analysis of this critical step in HCV replication and viral assembly showed no difference between WT-JFH1-ΔV3-EGFP and Ad-JFH1-ΔV3-EGFP. The mechanism of how adaptive mutations in Ad-JFH1-ΔV3-EGFP increase the titre of infectious HCV is under further study.

Although monolayer (2D) cell culture systems have been the standard in studying HCV in cell culture, they represent a reductionist model where the loss of extracellular matrix (ECM) and other physical properties, such as polarization, differentiation and cell–cell communication contacts are absent. 3D cultures such as Matrigel cultures can overcome some of the limitations of standard 2D cell culture systems (Härmä et al., 2010). For example, Matrigel-cultured Huh7 cells assemble into 3D spheroids, whereas standard 2D-cultured cells form epithelial monolayers (Molina-Jimenez et al., 2012). In vivo, the HCV life cycle occurs in a much more complex environment than in standard 2D cultures. In general, the interaction of viruses with polarized epithelial cells in the host is one of the key steps in the viral life cycle. Matrigel-embedded Huh7 3D cultures have been used to perform initial studies on the contribution of cell polarity and tight junction proteins (CD81, claudin-1 and occludin) to HCV infection, and demonstrated that infectious HCV particles are produced in such systems (Molina-Jimenez et al., 2012). Other 3D Huh7 cell culture systems provide evidence that they more closely mimic the differentiated and polarized state of hepatocytes in vivo, suggesting that such 3D systems may be more appropriate for studying some aspects of HCV biology (Sainz et al., 2009). One limitation of these 3D culture systems has been a requirement for fixation of the cells in the matrix to identify HCV-infected cells using immunohistochemical methods and the inability to monitor HCV replication and the spread of infection between cells in real time. The infectious chimeric Ad-JFH1-ΔV3-EGFP reporter virus described in this report overcomes a number of these limitations, including monitoring the spread of HCV infection between cells in 3D cultures in real time. This system may have advantages in studying aspects of HCV biology, such as virus assembly and budding, and also provides a system for screening antivirals that inhibit the release or transmission of infectious HCV.

Methods

Cell culture.

Human hepatoma cells, Huh7.5, were generously provided by Dr Charles M. Rice (Blight et al., 2002) and maintained in DMEM (Invitrogen) supplemented with 100 U ml−1 of penicillin, 100 µg ml−1 of streptomycin, nonessential amino acids and 10 % FBS (Invitrogen) at 37 °C in 5 % CO2. All experiments described in this study were performed using these cells.

Antibodies.

The monoclonal antibody to the NS5A protein (9E10) was a gift from Dr Charles Rice (Rockefeller University), and goat anti-mouse conjugated with HRP (Sigma) and goat anti-mouse IgG conjugated with Alexa Fluor 594 (Invitrogen) were obtained commercially.

Plasmids.

Plasmid constructs were based on the consensus sequence of HCV.

pJFH1 was kindly provided by Dr Wakita (Kato et al., 2001). JFH1-ΔV3-MluI was constructed as described previously (Liu et al., 2011). The entire EGFP gene was amplified by PCR using primers (forward) 5′-TTATATACGCGTGTGAGCAAGGGCGAGGAG-3′ and (reverse) 5′-CTCTGAACGCGTCTTGTACAGCTCGTCCAT-3′ and subcloned into the MluI site of JFH1-ΔV3-MluI. This construct was named JFH1-ΔV3-EGFP (with the JFH1-WT ‘backbone’) and full-length clones of the correct sequence were used to produce RNA, infect cells and develop the cell culture-adapted chimeric EGFP virus (designated Ad-JFH1-ΔV3-EGFP) that produced a higher titre of infectious virus.

Construction of the plasmid encoding Ad-JFH1-ΔV3-EGFP.

The general approach and primers for preparing this plasmid were described previously in preparing a plasmid encoding an adapted JFH1-ΔV3-Rluc chimeric virus (Liu et al., 2012). Cells infected with JFH1-ΔV3-EGFP RNA were passaged as described in the results to produce the adapted (Ad) JFH1-ΔV3-Rluc virus. Briefly, total RNA was isolated from a 6 cm-diameter dish of confluent Huh7.5 cells infected with the Ad-JFH1-ΔV3-EGFP virus by Trizol (Invitrogen). Total RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen) and random primers. The resulting cDNA was used as a template for subsequent PCR amplification with Platinum Pfx DNA polymerase (Invitrogen) and the four pairs of primers previously described (Liu et al., 2012). The four fragments were sequentially subcloned into pJFH1 using the unique restriction enzyme sites described in Liu et al., (2012) and sequenced. A total of six non-synonymous mutations were found in Ad-JFH1-ΔV3-EGFP. One mutation each in the E2, P7 and NS4B genes was identified and the remaining three mutations were found in the NS5A gene (Fig. 2a). No synonymous mutations in Ad-JFH1-ΔV3-EGFP were identified.

Transfection of HCV RNA.

To generate the full-length genomic RNA, pJFH-1 and pJFH-ΔV3-EGFP were linearized at the 3′ end of the HCV cDNA with XbaI. The linearized plasmid DNA was purified and used as a template for T7 in vitro transcription (MEGAscript; Ambion). In vitro-transcribed RNAs above were transfected into cells by electroporation as previously described (Krieger et al., 2001; Liu et al., 2011).

Immunofluorescence assays (IFA).

Cells infected with HCV was washed with PBS, fixed with 4 % paraformaldehyde, and permeabilized with 0.2 % Triton X-100. Cells were blocked with 1 % BSA and 1 % normal goat serum. NS5A in cells was detected with a monoclonal antibody and a secondary goat anti-mouse Alexa Fluor 594 antibody (Invitrogen, 1 : 1000) and visualized by fluorescence microscopy.

Titration of infectious HCV.

The titre of infectious HCV was determined by IFA, where the number of NS5A-positive cell foci was determined as described previously (Zhong et al., 2005). Cell supernatants were serially diluted 10-fold in DMEM. The supernatants were used to infect 1×104 naïve Huh7.5 cells in 96-well plates. Cells were incubated with virus for 2 h at 37 °C, washed and incubated with complete DMEM. The level of HCV infection was determined 3 days post-infection by immunofluorescence staining for NS5A. Viral titres are expressed as supernatant ffu ml−1.

Western blot analysis.

The HCV-infected Huh7.5 cells were lysed in 50 mM Tris/HCl, pH 7.5, 150 mM sodium chloride, 1 % Nonidet P40, 0.5 % sodium deoxycholate and 0.1 % SDS with proteinase inhibitors (cOmplete Mini, Roche). NS5A was detected with a NS5A monoclonal antibody (9E10, Apath) as described previously (Liu et al., 2012).

Use of EGFP fluorescence to measure viral replication and infectivity.

In viral replication assays, the adapted JFH-ΔV3-EGFP virus (m.o.i. of 0.3) was used to infect cells in 96-well plates in triplicate. After interferon α (Fitzgerald Industries Int.) or NS5B inhibitor, 2′-C-methyladenosine nucleoside analogue HCV NS5B polymerase (Eldrup et al., 2004) treatment, cells were lysed with 100 µl lysis buffer (20 mM Tris/HCl, 2 mM EGTA, 2 mM EDTA, 30 mM NaF, 30 mM Na4O7P2, 2 mM Na2VO4, 1 % Triton X-100, 0.03 % SDS, pH 7.4) with the protease inhibitor cocktail (Roche) at 4 °C, and the EGFP fluorescence signal was measured using a Synergy HT microplate reader (Bio-Tek). An excitation of 485/20 nm and emission of 528/20 nm were used.

Quantification of HCV RNA by qPCR.

Total RNA was extracted with Trizol (Invitrogen) and HCV RNA was measured by qPCR (quantitative PCR) analysis as described previously (Papic et al., 2012). The relative quantity of HCV RNA in control and HCV samples was calculated with the comparative Ct (cycling threshold) method. A reference gene (β-actin) was used as the control.

Assays for inhibition of HCV replication using JFH-ΔV3-EGFP.

Huh7.5 cells were grown overnight in 12-well plates (for qPCR and Western blotting analysis), 24-well plates (for EGFP photographs), or 96-well plates (for EGFP quantification by fluorescence) under standard conditions. Cells were infected for 2 h with adapted JFH-ΔV3-EGFP at a m.o.i. of 0.3. Interferon-α (IFN-α) (Fitzgerald) was serially diluted in complete DMEM and added to incubations. Following 2 days of incubation at 37 °C, HCV replication was measured by visualization of EGFP-positive cells by fluorescence microscopy, EGFP reporter assays, qPCR and Western blotting of the NS5A-EGFP fusion protein.

Laser-scanning confocal microscopy.

Cells transfected with adapted JFH1-ΔV3-EGFP were seeded onto 24-well plates with coverslips and cultured as previously described. The cells were washed twice with PBS after 48 h of culture, fixed with 4 % paraformaldehyde and permeabilized with 0.2 % Triton X-100. Fixed cells were blocked with 1 % BSA and 1 % normal goat serum in PBS. HCV NS5A was detected in cells using a NS5A monoclonal antibody and a secondary goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, 1 : 1000 dilution). Neutral lipids present in lipid droplets were visualized by staining with LipidTOX Deep Red (Invitrogen). Coverslips were mounted onto slides with DAPI (Vectorshield mounting media, Vector Laboratories) and slides were examined with an Olympus FV1000 laser-scanning confocal microscope.

3D cell cultures.

Huh7.5 cells were seeded in 12-well plates, cultured for 18 h, infected with adapted JFH-ΔV3-EGFP (m.o.i. of 0.1) and incubated for 24 h as previously described. Cells were trypsinized and 2×104 of infected Huh7.5 cells in 50 µl of complete DMEM were mixed with 50 µl of Matrigel (BD Biosciences) (per well) and seeded into 96-well plates with black walls and clear bottoms (Costar). The cells were incubated at 37 °C for 30 min allowing the Matrigel to solidify, 100 µl of complete DMEM was added to cover the cells embedded in Matrigel and culture for 48 h performed as described. 2D cell culture controls were prepared with infected Huh7.5 cells (same m.o.i.) and cultured in DMEM without Matrigel in the same 96-well plates under the same conditions. EGFP-positive cells were visualized after 48 h of culture and photographed with a Nikon A1 fluorescence confocal microscope.

Live 3D imaging of Matrigel-embedded Huh7.5 cells infected with JFH-ΔV3-EGFP.

Huh7.5 cells were seeded in 12-well plates, cultured for 18 h, infected with adapted JFH-ΔV3-EGFP (m.o.i. of 0.1) and incubated for 24 h as previously described. Cells were trypsinized, and 2×104 of infected Huh7.5 cells in 50 µl of complete DMEM were mixed with 50 µl Matrigel and added to 96-well-plates with black walls and clear bottoms. Cells embedded in Matrigel were incubated at 37 °C for 30 min to allow the Matrigel to solidify, 100 µl of complete DMEM was added per well and cells were cultured at 37 °C as previously described. Live cell images were taken after 24, 48 and 72 h of culture and supernatants were collected for viral titre assays. Images of 3D cultures were taken using a confocal fluorescence microscope as described. 3D reconstructions corresponding to images were created with NIS-Element AR and Volocity 5.3 software.

Acknowledgements

We thank Dr T. Wakita for providing the plasmid containing the HCV JFH1 plasmid. We also thank Dr C. M. Rice for providing Huh7.5 cells and the anti-NS5A monoclonal antibody. We also thank S. Young for her illustrations. R. C. was supported in part by a fellowship from Guangzhou General Hospital, Guangzhou, China. This work was supported in part by NIH Grant CA63640 (CHH).

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