Intracellular Hepatitis C Virus RNA Research Articles

Analysis of Interferon Signaling by Infectious Hepatitis C Virus Clones with Substitutions of Core Amino Acids 70 and 91

Substitution of amino acids 70 and 91 in the hepatitis C virus (HCV) core region is a significant predictor of poor responses to peginterferon-plus-ribavirin therapy, while their molecular mechanisms remain unclear. Here we investigated these differences in the response to alpha interferon (IFN) by using HCV cell culture with R70Q, R70H, and L91M substitutions. IFN treatment of cells transfected or infected with the wild type or the mutant HCV clones showed that the R70Q, R70H, and L91M core mutants were significantly more resistant than the wild type. Among HCV-transfected cells, intracellular HCV RNA levels were significantly higher for the core mutants than for the wild type, while HCV RNA in culture supernatant was significantly lower for these mutants than for the wild type. IFN-induced phosphorylation of STAT1 and STAT2 and expression of the interferon-inducible genes were significantly lower for the core mutants than for the wild type, suggesting cellular unresponsiveness to IFN. The expression level of an interferon signal attenuator, SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type. Interleukin 6 (IL-6), which upregulates SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type, suggesting interferon resistance, possibly through IL-6-induced, SOCS3-mediated suppression of interferon signaling. Expression levels of endoplasmic reticulum (ER) stress proteins were significantly higher in cells transfected with a core mutant than in those transfected with the wild type. In conclusion, HCV R70 and L91 core mutants were resistant to interferon in vitro, and the resistance may be induced by IL-6-induced upregulation of SOCS3. Those mechanisms may explain clinical interferon resistance of HCV core mutants.

Inhibitors of Endoplasmic Reticulum α-Glucosidases Potently Suppress Hepatitis C Virus Virion Assembly and Release

α-Glucosidases I and II are endoplasmic reticulum-resident enzymes that are essential for N-linked glycan processing and subsequent proper folding of glycoproteins. In this report, we first demonstrate that downregulation of the expression of α-glucosidase I, II, or both in Huh7.5 cells by small hairpin RNA technology inhibited the production of hepatitis C virus (HCV). In agreement with the essential role of α-glucosidases in HCV envelope glycoprotein processing and folding, treatment of HCV-infected cells with a panel of imino sugar derivatives, which are competitive inhibitors of α-glucosidases, did not affect intracellular HCV RNA replication and nonstructural protein expression but resulted in the inhibition of glycan processing and subsequent degradation of HCV E2 glycoprotein. As a consequence, HCV virion assembly and secretion were inhibited. In searching for imino sugars with better antiviral activity, we found that a novel imino sugar, PBDNJ0804, had a superior ability to inhibit HCV virion assembly and secretion. In summary, we demonstrated that glucosidases are important host factor-based antiviral targets for HCV infection. The low likelihood of drug-resistant virus emergence and potent antiviral efficacy of the novel glucosidase inhibitor hold promise for its development as a therapeutic agent for the treatment of chronic hepatitis C.

ME3738 enhances the effect of interferon and inhibits hepatitis C virus replication both in vitro and in vivo

ME3738 (22β-methoxyolean-12-ene-3β, 24-diol), a derivative of soyasapogenol B, attenuates liver disease in several animal models of acute and chronic liver injury. ME3738 is thought to inhibit replication of hepatitis C virus (HCV) by enhancing interferon (IFN)-β production, as determined using the HCV full-length binary expression system. We examined the effect of ME3738 combined with IFN-α on HCV replication using the genotype 1b subgenomic replicon system and an in vivo mouse HCV model. HCV replicon cells (ORN/3-5B/KE cells and Con1 cells) were incubated with ME3738 and/or IFN-α, and then intracellular IFN-stimulated genes (ISGs) and HCV RNA replication were analyzed by reverse-transcription-real time polymerase chain reaction and luciferase reporter assay. HCV-infected human hepatocyte chimeric mice were also treated with ME3738 and/or IFN-α for 4 weeks. Mouse serum HCV RNA titer, HCV core antigen, and ISGs expression in the liver were measured. ME3738 induced gene expression of oligoadenylate synthetase 1 and inhibited HCV replication in both HCV replicon cells. The drug enhanced the effect of IFN to significantly increase ISG expression levels, inhibit HCV replication in replicon cells, and reduce mouse serum HCV RNA and core antigen levels in mouse livers. The combination treatment was not hepatotoxic as evident histologically and did not reduce human serum albumin in mice. ME3738 inhibited HCV replication, enhancing the effect of IFN-α to increase ISG expression both in vitro and in vivo, suggesting that the combination of ME3738 and IFN might be useful therapeutically for patients with chronic hepatitis C.

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

Host cell factors are critical to all stages of the hepatitis C virus (HCV) life cycle. While many cellular proteins that regulate HCV genome synthesis have been identified, the mechanisms engaged in this process are incompletely understood. To identify novel cellular proteins involved in HCV RNA replication, we screened a library of small interfering RNAs (siRNAs) targeting 299 cellular factors, which principally function in RNA interactions. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tricistronic subgenomic replicons (SGRs). We found that the U2OS cell line gave lower levels of intracellular HCV RNA replication compared with Huh-7 cells and was more readily transfected by siRNAs. Consequently, increased gene silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells provided a suitable and more sensitive alternative to Huh-7 cells for siRNA studies on HCV RNA replication. From the screen, several cellular proteins that enhanced and suppressed HCV RNA replication were identified. One of the genes found to downregulate viral RNA synthesis, ISG15, is expressed in response to alpha interferon and may therefore partly contribute to the clearance of virus from infected individuals. A second gene that inhibited HCV RNA levels was the 5'-3' exoRNase XRN1, which suggested a role for cellular RNA degradation pathways in modulating the abundance of viral genomes. Therefore, this study provides an important framework for future detailed analyses of these and other cellular proteins.

Ethanol Enhances Hepatitis C Virus Replication through Lipid Metabolism and Elevated NADH/NAD+

Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD(+) ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD(+). Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting beta-oxidation with beta-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. Therefore, lipid metabolism and alteration of cellular NADH/NAD(+) ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress.

Hepatitis B virus and hepatitis C virus interaction in Huh-7 cells

Co-infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) increases the risk of development and the severity of chronic liver disease. Although dominant and suppressive effects of each virus over the other have been reported in vivo, in vitro studies of HBV/HCV co-infection have been limited to analysis of the effects of over-expression of HCV proteins on HBV replication. We have re-examined HBV/HCV interactions in Huh-7 cells following co-infection with cell culture-propagated HCV (HCVcc; genotype 2a) and a recombinant adenovirus vector capable of delivering a replication-competent HBV genome (AdHBV; genotype A). While intracellular HCV RNA levels were significantly increased when cells were pre-infected with AdHBV, HCV replication and virion secretion were not altered by simultaneous infection with AdHBV or AdHBV superinfection of HCV-infected cells. Likewise intracellular and secreted HBV DNA levels and HBV promoter activities were either unchanged or modestly increased by HCVcc infection. Despite this, HCV E2 and HBsAg proteins colocalized extensively in co-infected cells suggesting shared stages in viral egress. These studies indicate that there is little direct interaction of HBV and HCV in co-infected hepatocytes and imply that indirect effects of host-viral interactions dictate viral dominance in HBV/HCV co-infected individuals.

The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.

Modeling Subgenomic Hepatitis C Virus RNA Kinetics during Treatment with Alpha Interferon

Although replicons have been used to demonstrate hepatitis C virus (HCV) inhibition by alpha interferon (IFN-alpha), the detailed inhibition kinetics required to mathematically model HCV RNA decline have been lacking. Therefore, we measured genotype 1b subgenomic replicon (sg1b) RNA levels under various IFN-alpha concentrations to assess the inhibition kinetics of intracellular HCV RNA. During nine days of IFN-alpha treatment, sg1b RNA decreased in a biphasic, dose-dependent manner. Using frequent measurements to dissect these phases during IFN-alpha treatments of 100 and 250 U/ml revealed that the first-phase sg1b RNA decline began approximately 12 h posttreatment, continued for 2 to 4 days, and then exhibited a distinct flat or slower second phase. Based on these data, we developed a mathematical model of IFN-alpha-induced intracellular sg1b RNA decline, and we show that the mechanism(s) mediating IFN-alpha inhibition of HCV acts primarily by reducing sg1b RNA amplification, with an additional effect on HCV RNA stability/degradation detectable at a dose of 250 U/ml IFN-alpha. While the extremely slow or flat second phase of viral RNA inhibition observed in vitro, in which there is little or no cell death, supports the in vivo modeling prediction that the more profound second-phase decline observed in IFN-alpha-treated patients reflects immune-mediated death/loss of productively infected cells, the second-phase decline in viral RNA with a dose of 250 U/ml IFN-alpha suggests that a further inhibition of intracellular HCV RNA levels may contribute as well. As such, dissection of HCV IFN-alpha inhibition kinetics in vitro has brought us closer to understanding the mechanism(s) by which IFN-alpha may be inhibiting HCV in vivo.

Intragenotypic JFH1 based recombinant hepatitis C virus produces high levels of infectious particles but causes increased cell death

The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone. Subsequent infection of naive Huh-7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility of the chimeric junction. Moreover, NTRNS2 a chimeric virus equivalent to the previously reported FL-J6/JFH chimera, showed a 10-fold enhancement of virus titers compared to CNS2. NTRNS2 differs from CNS2 by three nucleotide differences residing in the 5′ NTR and core coding sequence and all three nucleotide changes were necessary for increased virion production. Importantly, cells producing Cp7 virus showed increased apoptosis compared with JFH1, an effect correlating with virion production. These studies begin to unravel requirements for robust virus replication and the relationship between increased virion production and host cell viability.

Equal Amounts of Intracellular and Virion‐Enclosed Hepatitis C Virus RNA Are Associated with Peripheral‐Blood Mononuclear Cells In Vivo

Hepatitis C virus (HCV) replicating in peripheral-blood mononuclear cells (PBMCs) may represent an extrahepatic viral reservoir. Quantitation of HCV RNA with regard to its subcellular distribution and longitudinal course is needed for better understanding of the largely unexplored in vivo dynamics and potential pathogenetic significance of HCV in PBMCs. Plasma and PBMCs from 30 patients coinfected with HCV and human immunodeficiency virus were evaluated in cross-sectional and longitudinal analyses, for up to 40 months. Differential extraction of virion-enclosed HCV RNA associated with cells was performed in parallel with extraction of total cellular HCV RNA. HCV RNA of either orientation was quantified by real-time polymerase chain reaction. HCV RNA was detected only in PBMCs from patients with viremia and at relatively stable quantities over time. Intracellular HCV RNA corresponding to ~60% of total cellular HCV RNA was strongly correlated with virion-enclosed HCV RNA but was only weakly associated with viral loads in plasma. In contrast, the ratio of HCV RNA load in PBMCs versus that in plasma was patient specific and stable over time. The substantial and patient-specific amounts of intracellular HCV RNA found by the present study support a concept of low-level replication in PBMCs. There was no evidence for persistent HCV infection in PBMCs after clearance of viremia in plasma.

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro.

To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively. The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system

We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV.

Hepatitis C virus replication in stably transfected HepG2 cells promotes hepatocellular growth and tumorigenesis.

HepG2 cells stably transfected with a full-length, infectious hepatitis C virus (HCV) cDNA demonstrated consistent replication of HCV for more than 3 years. Intracellular minus strand HCV RNA was present. Minus strand synthesis was NS5B dependent, and was sensitive to interferon alpha (IFN alpha) treatment. NS5B and HCV core protein were detectable. HCV stimulated HepG2 cell growth and survival in culture, in soft agar, and accelerated tumor growth in SCID mice. These mice became HCV RNA positive in blood, where the virus was also sensitive to IFN alpha. The RNA banded at the density of HCV, and was resistant to RNase prior to extraction. Hence, HCV stably replicates in HepG2 cells, stimulates hepatocellular growth and tumorigenesis, and is susceptible to IFN alpha both in vitro and in vivo.

Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein.

We constructed dicistronic, subgenomic hepatitis C virus (HCV) replicons in which the sequence encoding the human immunodeficiency virus (HIV) tat protein was placed in the upstream cistron, between the HCV 5'NTR and a picornaviral 2A proteinase sequence fused to the selectable marker Neo. Stably transformed Huh7 cells expressing secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV LTR promoter actively secreted SEAP following transfection with these replicon RNAs. Extracellular SEAP activity correlated closely with intracellular HCV RNA levels, as determined by Northern blotting and real-time RT-PCR analysis. These RNAs replicated efficiently despite the absence of core-protein-coding sequence downstream of the HCV IRES. The replication efficiency of replicons derived from the HCV-N strain of HCV was significantly greater than those derived from Con1 in transiently transfected cells. Using this reporter system, we have demonstrated significant differences in the response to interferon alpha-2b in cell lines containing replicons derived from these two strains of HCV.

In vitro infection of adult normal human hepatocytes in primary culture by hepatitis C virus.

In vitro infection of adult normal human hepatocytes in primary culture has been performed for investigating the replication cycle of hepatitis C virus (HCV) in differentiated cells. Hepatocytes were prepared from liver tissue resected from donors who tested negative for HCV, and inoculation was performed 3 days after plating with 33 HCV serum samples of different virus load and genotype. The presence of intracellular HCV RNA, detected by a strand-specific rTth RT-PCR assay, was used as evidence of infection. A kinetics analysis of HCV replication revealed that intracellular negative-strand RNA appeared at day 1 post-infection with a maximum level at days 3 and 5, followed by a decrease until day 14. At day 5, we estimated that the copy level of viral RNA was amplified at least 15-fold in infected cells. The level of intracellular HCV RNA in response to different serum samples was reproducible from one hepatocyte culture to another, suggesting that there is no inter-individual variability in the susceptibility of hepatocytes to HCV infection. These findings indicate that adult human hepatocytes in primary culture retain their susceptibility to in vitro HCV infection and support HCV RNA replication. This model should represent a valuable tool for the study of initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.

Human hepatocyte clonal cell lines that support persistent replication of hepatitis C virus

We previously found that a human T-cell leukemia virus type I infected T-cell line, MT-2, was susceptible to hepatitis C virus (HCV) infection, and that cloned MT-2C cells could support HCV replication more persistently than the parental MT-2 cells. Recently we found that a non-neoplastic hepatocyte line, PH5CH, showed good susceptibility to HCV infection. In this study, we cloned PH5CH cells to obtain cells that supported more persistent HCV replication, and consequently three clones (PH5CH1, PH5CH7 and PH5CH8) in which intracellular HCV RNA could be detected at least 25 days postinoculation (p.i.) were obtained. Semi-quantitative analysis of HCV RNA indicated that HCV replicated in these cloned PH5CH cells was released into the culture medium. Semi-quantitative analysis of internalized HCV RNA after treatment of cloned PH5CH cells and parental PH5CH cells with proteinase K immediately after virus inoculation revealed that PH5CH1, PH5CH7 and PH5CH8 cells contained 10-fold higher levels of HCV RNA than low susceptible cloned PH5CH or parental PH5CH cells. Furthermore, we demonstrated that HCV replication was maintained for 70–100 days in these three clonal lines when the temperature of cell culture after virus inoculation was reduced from 37 to 32°C. Moreover, we demonstrated that interferon α had antiviral effect on HCV-infected PH5CH8 cells. The three PH5CH clones obtained in this study will provide a useful tool for the study of HCV replication and proliferation, and for development of an assay system for antiviral agents.

Analysis of the cell tropism of HCV by using in vitro HCV-infected human lymphocytes and hepatocytes

Background/Methods: We recently established two hepatitis C virus (HCV) replication systems, using MT-2, a human T-cell leukemia virus type I-infected human T-cell line,a nd PH5CH, a non-neoplastic human hepatocyte line immortalized with simian virus 40 large T antigen. These HCV replication systems were used to assess the infective potencies of seven sera containing more than 10 6 HCV genomes per ml obtained from HCV-positive blood donors. Results: The results showed that these sera had different infectivities for MT-2 and PH5CH cells. One of the seven sera, 1B-1, was more infective for MT-2 cells than PH5CH cells, whereas all the sera except serum 1B-1 were more infective for PH5CH cells than for MT-2 cells. Intracellular HCV RNA could be detected at least 30 days after inoculation with three of the sera. These findings suggested that the infective potency of each serum depends on the type of target cells. To further investigate HCV replication in these cells, we examined the hypervariable region 1 (HVR1) populations of HCV recovered from both MT-2 and PH5CH cells at 8 days postinoculation. The results revealed that the shift to limited HVR1 populations from the quasi-species of HVR1 populations in both cells usually occurred within 8 days after virus inoculation. Furthermore, in two of four sera, the predominant HVR1 populations in MT-2 and PH5CH cells appeared to be different. Conclusion: These results suggest that HCV exhibits cell tropism.

Characterization of hepatitis C virus replication in cloned cells obtained from a human T-cell leukemia virus type 1-infected cell line, MT-2.

We recently found that a human T-cell leukemia virus type 1-infected cell line, MT-2, could support the replication of hepatitis C virus (HCV) (N. Kato, T. Nakazawa, T. Mizutani, and K. Shimotohno, Biochem. Biophys. Res. Commun. 206:863-869, 1995). In order to develop a culture system in which HCV replicates more efficiently, we examined the efficiency of HCV replication in cloned MT-2 cell lines by the limiting dilution method. Consequently, we obtained five clones in which intracellular positive-stranded HCV RNA could be detected until at least 21 days postinoculation (p.i.), as opposed to 15 days p.i. in uncloned MT-2 cells. MT-2C, one of the five clones which supported HCV replication up to 30 days p.i., was used for further characterization of HCV replication. Semiquantitative analysis of HCV by PCR revealed that RNA synthesis in infected cells increased after inoculation, reached a maximum level at 4 days p.i., and maintained this level until at least 11 days p.i. The 5' untranslated region of negative-stranded HCV RNA was also detected in the infected cells by two different methods with strand specificity. These results suggest that HCV replicated and multiplied in the MT-2C cells. HCV-infected MT-2C cells that were treated with antibiotics, such as G418 and hygromycin B, sustained HCV RNA for a longer period than did untreated cells. We demonstrated inhibitory effects on HCV replication by an antisense oligonucleotide complementary to the HCV core encoding region and by interferon-alpha. Furthermore, cell-free viral transmission was demonstrated by this culture system. These results suggest that our cell culture system will be useful for studying the mechanism of HCV replication, for screening antiviral agents, and for developing HCV vaccines.

Susceptibility of Human T-Lymphotropic Virus Type I-Infected Cell Line MT-2 to Hepatitis C Virus Infection

To obtain a hepatitis C virus (HCV) proliferation system, we examined the susceptibility of various cultured cell lines to HCV infection. We found that a human T-lymphotropic virus type I infected cell Line MT-2 was fairly sensitive to HCV infection. Using the polymerase chain reaction, intracellular positive-stranded HCV RNA was detected until at least 15 days postinoculation (p.i.). Intracellular negative-stranded HCV RNA was also detected at 10 days p.i., although not at 7 days p.i., suggesting that HCV is replicating in MT-2 cells 10 days p.i. Sequence analysis of hypervariable region 1 (HVR1) revealed that HVR1 sequences from cells 10 days p.i. had become homogeneous, although HVR1 sequences from the inoculum showed the typical quasi-species. We also found a lack of anti-HVR1 antibody against the HVR1 species which became homogeneous at 10 days p.i., although we easily detected antibody against the other HVR1 species obtained from the inoculum. These findings suggest that MT-2 cells are susceptible to HCV infection and are capable of supporting HCV replication.