Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

Abstract

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro.

METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RT-PCR, in situ hybridization and immunohisto-chemistry respectively.

RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS₃, CP₁₀ antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed.

CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

Key Words: hepatitis C virus; cell culture; cell model; carcinoma, hepatocellular/pathology; tumor cells, cultured; hepatitis B virus; virus replication

INTRODUCTION

Hepatitis C virus (HCV) is the major cause of transfusion-associated non-A, non-B hepatitis[1]. Acute HCV infection leads, in more than 70% of the patients, to the development of chronic hepatitis[2], and then cirrhosis and hepatocellular carcinoma[3,4]. HCV is an enveloped positive-stranded RNA virus which contains a genome of about 9500 nts encoding a polyprotein by host and viral proteases results in the production of structural and nonstructual (NS) protein[5-8]. Despite increasing knowledge of genome structure and individual viral proteins, studies on virus replication and pathogenesis have been hampered by the lack of reliable and efficient cell culture systems. To identify critical epitopes that elicit a neutralizing antibody response and to understand the pathogenesis of persistent infection, efficient infection cell models for HCV are needed[8-10]. Many cell lines has been sought to support HCV infection and replication in vitro. Human T-lymphocyte cell lines[11-18], human fibroblast cells[19] (VH3), peripheral blood mononuclear cells[20,21] (PBMCs) and hepatocytes[22-28] have been shown to support HCV replication in vitro. These cell models have been proved to be useful for the study of HCV. To be mentioned, hepatocytes are the main target cells for HCV replication in vivo. Whether the replication of HCV in non-hepatocytes entirely mimics the behavior of human hepatocytes and whether the molecular characterization of growing HCV in these cell models has changed are still poorly understood[28]. In this study, we employed a human hepatoma cell line 7721 to test its susceptibility to HCV and established an infection cell model that can support HCV long-term replication in vitro. The presence of both plus- and minus- strand of HCV RNA and the expression of viral antigens in infected cells were demonstrated.

MATERIALS AND METHODS

Inoculum

The principal inoculum was a serum obtained from a 45 year old female chronic patient, who was positive for anti-HCV antibody (Abbott) and HCV-RNA (1b genotype). Sera from patients without any evidence of HCV infection or chronic liver diseases were also used as controls.

Cell line

The hepatoma cell line 7721 was obtained from Lab. of Gastroenterology in Third Military Medical University. Cells were maintained in PRMI1640 medium supplemented with non-essential amino acids and 100 mL•L¯¹ fetal bovine serum. The cells were fed fresh medium every 3 d to 5 d, and were subcultured at the ratio of 1:2 to 1:4.

Viral inoculation and sample collection

The HCV-positive serum (inoculum) was diluted at the ratio of 1:5. 7721 cells maintained in 25 cm2 polystyrene flasks and 6-well dishes were incubated with the inoculum at 37 °C for 8 h. Then all medium was removed and cells were washed 6 times with phosphate buffered saline (PBS). Cultures were refed with medium with 100 mL•L¯¹ fetal bovine serum and the media were changed every 4 d-5 d.

Cells and supernatants were harvested according to the method introduced by Iacovacci et al[27], with slight modification. Briefly, cells (approximately 2 × 10⁶ cells) and 1 mL aliquots of cell supernatants from infected and uninfected cell cultures were harvested at different periods after infection for up to 70 d, and slices grown with cells were also collected accordingly (for study of the localization of different HCV antigens and minus- strand of HCV RNA), replacing the medium removed at each collection. Supernatants were centrifuged at 10000 × g to remove cellular debris, and the ultracentrifuged pellets were stored at -20 °C until detection of HCV RNA. Cells were harvested for RNA extraction after 6 washes with phosphate buffered saline (PBS).

Detection of HCV RNA by RT-PCR

For detection of HCV RNA, total RNA was extracted from 1 mL of the ultracen trifuged supernatants and approximately 2 × 10⁶ cells by using a nucleotide and protein extraction kit (Tripure, Roche). Ten μL of the RNA solution was denatured at 72 °C for 4 min and incubated at 37 °C for 1 h with 200 U of murine moloney leukaemia virus reverse transcriptase (MMLV-RT, Promega) and 50 pmol of the outer antisense oligonucleotide primer (antisense, CACTCGCAAGCACCCTATCA-; nucleotides 302-285). Synthesis of cDNA was stopped by heating the samples at 95 °C for 10 min. Amplification of the cDNA was performed by using 15 μL cDNA solution and 50 pmol of one of the outer primers (sense, -GGCGACACTCCACCATAGAT-: nucleotides9-28). Thirty cycles of DNA amplification were carried out followed by and extension step for 10 min at 72 °C. Each cycle of PCR consisted of 94 °C for 60 s, 55 °C for 90 s and 72 °C for 120 s. The second PCR was carried out in the same way with 10 μL of the first PCR mixture and 50 pmol of each inner prime (sense, -CTGTGAGCAACTACTGTCT-; nucleotides 36-55 and antisense, -CGGTGTACTCACCGGTTCC-; nucleotides 161-143). The amplified DNA was visualized by 20 g•L¯¹ agarose gel electrphoresis and ethidium bromide staining.

Detection of HCV antigens by immunohistochemistry

A modification of immunohistochemical staining of viral antigens by streptavidin peroxidase (SP) method was performed. Monolayers of infected and uninfected cells grown on slides were fixed for 15 min in 40 g•L¯¹ formaldehyde pH(7.3) in PBS solution. After washing, endogenous peroxidase was blocked by a 10-minute incubation with 3 mL•L¯¹ H₂O₂ in methanol. Sections were washed in PBS, 15 min at 37 °C. Sections were washed in PBS (pH7.4); then 200 mL•L¯¹ normal goat serum was used for 15 minutes.

The sections were incubated with 1:200 dilution of the primary antibody (anti- NS3) or with 1:60 dilution of primary antibody (anti-CP₁₀) for 16 h at 4 °C in a moisture chamber. The immunostaining was also done without the primary antibodies. These reagents and all subsequent reagents were diluted in PBS with 1 g•L¯¹ bovine serum albumin (BSA) and used at a volume of 100 mL per slide. After a PBS rinse, biotinylated second antibody was applied for 30 min at room temperature. After another PBS rinse, streptavidin peroxidase was applied for 30 min. After a final PBS rinse, the cell sections were incubated with 0.6 g•L¯¹ diaminobenzidine (Sigma) and 0.1 mL•L¯¹ H₂O₂. The sections were then conterstained with hematoxylin, dehydrated with graded alcohols and xylene, and observed under coveslips.

In situ hybridization

The localization of intracellular minus-strand of HCV RNA by in situ hybridization was done according to the METHODS by Cribier et al[20] and Nouri-Aria et al[29] with modifications. The slides were rehydrated through an ethanol series (1000-500 mL•L¯¹) and rinsed in PBS for 5 min, and then the cells were digested with 1 mg•L¯¹ proteinase K (Roche) for 30 min at 37 °C. The slides were rinsed twice in PBS containing 2 g•L¯¹ glycine (10 min each), refixed in 40 g•L¯¹ PFA for 5 min at room temperature, then rinsed three times for 10 min in 2 × SSC buffer, and finally 10 mmol•L¯¹ DDT was then added to the slides. Dehydration in an ethanol series (500 - 1000 mL•L¯¹) was followed. The probe was denatured for 15 min at 85 °C and added to hyridization solution (500 g•L¯¹ formamide, 100 g•L¯¹ dextran sulphate, 2 × SSC, 2 × Denhardt’s solution, 1 g•L¯¹ Triton X-100, 0.01 mol•L ¯¹ DDT, 200 mg•L¯¹ herring sperm DNA). The final concentration of the probe was 1 mg•L¯¹. The slides were denatured for 2 min at 95 °C, and various amounts of probe, depending on the area, were added on the cell section. Samples were covered with siliconized coverslips, sealed with paraffin, and incubated in a humidified chamber at 37 °C for 10 h-20 h. The coverslips were removed and the slides were rinsed three times in 1 × SSC buffer and washed twice in 500 g•L¯¹ formamide, 1 × SSC for 15 min at 42 °C. After two washes at 42 °C in 1 × SSC and another wash in 0.1 × SSC (10 min each), the slides were dehydrated through an ethanol series and dried under vacuum. The sections were incubated with a streptavidin-biotin alkaline phosphatase complex and successively developed with nitroblue-tetrazolium and bromo-chloro-indolyl phosphate (NBT/BCIP), according to the manufacturer’s instructions. After being rinsed in water, the slides usually were stained briefly with eosin. Uninfected cells were hybridized with the HCV probes and served as a first negative control. The Dig-labeled genomic strand RNA transcript was 41mer and its sequences is 5’-CTGCTAGCCGACTAGTGTTGGGTCCGCGAAACC-GCTTGTGG-3’.

RESULTS

Detection of HCV RNA in the infected cells

The intracellular RNA was determined by RT-PCR in infected cells at various times during culture, ranging from d2 to d95, as shown in Table 1. Plus- strand of HCV RNA was intermittently detectable after infection, but not detectable on d 25, d 30, d 52 and d 75 after infection. Minus-strand RNA was detectable from d 3 after infection, and still detect able at d 95 after infection.

Table 1 Detection of HCV RNA in 7721 cells and supernatant.

Day p.i.	Cells		Supernatant
	Plus-strand RNA	Minus-strand RNA	Plus-strand RNA	Minus-strand RNA
2	+	-	+	-
3	++	++	+	-
10	+	+	+	ND
20	++	+	-	-
25	-	-	-	ND
30	-	-	-	-
40	+	-	-	-
52	-	-	+	-
60	+	-	-	-
75	-	+	-	-
82	++	++	++	-
90	++	-	+	-
95	+	+	-	-

^*+, Weakly positive; ++, Positive; -, Negative; ND, Not detection.

Detection of HCV RNA in the culture medium

The presence of HCV RNA in culture supernatant was also determined by RT-PCR. Plus-strand of HCV RNA in supernatant was less often than that in cells. The HCV negative-strand could not be detected in the supernatants and inoculum, as shown in Table 1. Immediately after incubation and six washes of the uninfected 7721 cells, the RT-PCR were negative on the buffer used in the final wash of the cells.

These results were confirmed by three other repeated experiments.

The expression of HCV antigens in infected cells

7721 cells were tested for the expression of virus-encoded proteins by SP method using mouse monoclonal antibodies directed against NS₃ and CP₁₀. Figure 1A, B show that infected 7721 cells were positive for different viral antigens. The positive cells were scattered and displayed diffuse cytoplasmic yellow staining similar to that reported in other cell culture systems and naturally infected liver cells[30-34]. Moreover, uninfected cells and cells incubated with normal serum were tested negative using these same antibodies (Figure 1C).

Figure 1 Immunohistochemical detection of HCV-encoded protein in infected 7721 cells. Positive reaction (yellow) with anti-NS₃ (A) and anti-CP₁₀ (B) in 7721 cells, were scattered in the lobules (SP method, original magnification, A × 200, B × 400). Lack of staining with the same antibody in uninfected 7721 cells processed at the same times and 7721 incubated with oormal serum (C).

In situ hybridization

Using the HCV positive-strand probe, positively labeled cells could be observed at various time after infection. The positive signals (blue) also mainly localized in cytoplasm, and were similar to that reported in other cell culture systems and naturally infected liver cells[35-40], as shown in Figure 2A, B. Uninfected 7721 as controls showed no labeling with the same probe (Figure 2C).

Figure 2 Detection of minus-strand of HCV RNA in cells using in situ hybridization. Positive reaction (blue) was mainly present with cytoplasm (A, original magnification, × 400). Higher magnification showed the positive cells looked morhologically normal (B, original magnification, × 1000). No signal in uninfected cells (C).

Infectivity of the released HCV in the culture supernatant

To determine the infectivity of HCV particles released into the culture supernatant, we harvested cell-free culture supernatant from primary infected-7721 culture on d6 after infection, as described by Mizutani et al[14-16]. Fresh (uninfected) 7721 cells were incubated with the culture supernatant containing HCV for 8 h at 37 °C and then the medium was washed. On d 4 and d 8 after initiating the culture, we harvested the medium and cells for the presence of HCV RNA and HCV antigens as above described. Figure 3A shows that minus-strand RNA could be detected in fresh 7721 cells on d4 and d8. HCV NS₃antigen could also be detected in cells on day 8 (Figure 3B).

Figure 3 Cell-free transmission of HCV in cell culture. HCV transmission to fresh/uninfected 7721 was carried out by using the supernatant of culture medium derived from HCV-infected cell culture. The presence of minus-strand of HCV RNA and NS 3 antigen in cells was detected by in situ hybridization (A, original magnification, × 400) and immunohistochem istry (B, original magnification, × 400), respectively.

Transmission of HCV in cell culture

In order to demonstrate whether this system is abortive or productive of infectious HCV, the transmission of the viral genome in HCV-infected 7721 cells to new cells was tested by coculture. For this purpose, we separated the PBMCs from healthy man as described[20]. PBMCs (2 × 10⁵) were mixed with an equal number of HCV-infected 7721 cells. The presence of HCV NS₃antigen and minus-strand RNA in PBMCs were tested on day 4 and 10 after coculture. The result showed that minus-strand RNA and NS₃ antigen of HCV could be detected by in situ hybridization and immunohistochemistry, indicating that the genome was successfully transmitted to PBMCs.

Cell growth and phenotyic change

7721 cells presented a spindle or round shape in culture and grew in a monolayer. Untreated cells and cells incubated with negative or positive serum, proliferated at the same rate with no visible phenotypic changes. Cell growth was not affected by incubation with the HCV-positive serum.

DISCUSSION

Although the knowledge of the molecular biology of HCV has progressed rapidly, our understanding of viral replication and pathogenicity is still hampered by the lack of reliable and efficient cell culture systems. Development of an efficient in vitro culture system for HCV will faeilitate the study of HCV. We have demonstrated the susceptibility of a hepatoma cell line 7721 to HCV and established a stable cell model that may support HCV long-term replication in vitro. Because HCV RNA in both cells and supernatant after infection may be the residue of the inoculated virus attached to the cell surface, it is necessary to identify that HCV sequences detected in the infected cultures are not from inoculum but newly produced.

We tested both plus-and minus-strands of HCV RNA, the expression of HCV antigens, the localization of minus-strand of HCV RNA, separately, and could show that HCV replication in the cultured cells was not the residue of inoculum. The data presented here indicate that the 7721 cells support HCV infection for at least 3 mo. The infected 7721 cultures released detectable levels of HCV into the medium as early as d 2 after infection. Reduction of adsorption time (from 8 h to 2 h) did not significantly reduce the efficiency of viral replication (not shown), suggesting that binding to cell receptors occurs in a relatively short time[14,28].

We found that both plus- and minus- strands were not often but intermittently detectable in cells and supernatant. Such intermittent detection of the HCV RNA was also reported in other culture systems[20,27,28] and the infection in vivo[41-45]. Interestingly, in our study, we showed that the fresh 7721 cells could be infected by the supernatant from cultured infected cells, and that the HCV-infected cells were able to transmit the virus to new cells (PBMCs) after coculture. These results suggested that HCV-infected 7721 cells could deliver infectious and mature virus as others cell models[11,28].

Our findings are consistent with a productive in vitro infection of 7721 cells by HCV and are in agreement with the results of Tagawa et al[26-28]. However, these authors did not study the expression of viral antigens in hepatoma cell lines after infection. Detection of the HCV protein in cultured 7721 cells by immunohistochemistry indicated that HCV RNA was translated and HCV proteins were produced in cultured cells. The frequency of antigen-positive was less than 30%. The expression of different viral antigens and the localization of minus-strand of HCV RNA were similar to the naturally infected liver cells[30-34]. The results of this study correlates well with experiments as shown in naturally infected hepatocyes, suggesting that the HCV infection and replication occurring in 7721 cultures closely mimic the infection occurring in vivo[46-50]. Blocking of viral attachment by a hyperimmune rabbit serum against hypervariable region 1 has been demonstrated on the basis of this model[51]. The mechanisms of HCV infection, replication, assembly, and neutralization can now be studied in detail in vitro. This system should also be useful for analysis of antigen epitopes and the evaluation of vaccine candidates, which is especially important because of the limited availability of chimpanzees for vaccine studies and the high degree of variability in the HCV glycoprotein domains.