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- MATERIALS AND METHODS
Although a cell culture system for HCV JFH-1 strain has been developed, no robust cell culture system for serum-derived HCV is available. In this study, we have established systems capable of monitoring infection with JFH-1 virus based on specific reporter gene expression through proteolysis of chimeric transcription factors by HCV NS3/4A protease. We utilized a transcriptional factor Gal4-TBP that synergistically enhances transcription of the GAL4UAS and HIV-1 LTR tandem promoter with the Tat protein. We constructed chimeric Tat and Gal4-TBP transcription factors containing the HCV NS3/4A cleavage sequence of a mitochondria-resident IPS-1, but not those of the HCV polyprotein, and manipulated them to localize in the ER. Upon infection with JFH-1 virus, the transcription factors were efficiently cleaved by HCV protease, migrated into the nucleus and activated the reporter gene under the tandem promoter. Upon infection with JFH-1 virus, the Huh7OK1/TG-Luc cell line carrying the transcription factors and a luciferase gene under the promoter expressed luciferase in a dose-dependent manner in close correlation with HCV RNA replication. Huh7OK1/TG-LNGFR cells carrying the transcription factors and a cDNA of human low affinity nerve growth factor receptor under the promoter were selectively concentrated by immunomagnetic cell sorting upon infection with JFH-1 virus. These results indicate that the chimeric constructs bearing the ER-resident IPS-1 sequence are specifically recognized and efficiently cleaved by HCV protease and are harnessed for detection of HCV replication and for recovery of HCV-infected cells. This strategy may be applicable for the establishment of cell culture systems for the isolation of serum-derived HCV.
HCV infects more than 170 million people worldwide, and is a major cause of chronic liver disease, including hepatic steatosis, cirrhosis, and hepatocellular carcinoma (1). Combination therapy with pegylated IFN and ribavirin has achieved a sustained virological response in 50% of individuals infected with HCV genotype 1 (2). The establishment of cell culture systems using an HCV genotype 2a JFH-1 strain isolated from a patient with fulminant hepatitis C (3–5) and genotype 1a H77 and H77-S strains (6, 7) marked an epoch in the history of HCV study. However, reliable and robust cell culture systems capable of propagating serum-derived HCV from hepatitis C patients have not been established, hampering the development of effective anti-viral measures for HCV.
HCV is an enveloped virus possessing a single-stranded positive-sense RNA genome, and belongs to the genus Hepacivirus in the family Flaviviridae. The genome encodes a large precursor polyprotein composed of about 3000 amino acids. The viral polyprotein is processed by cellular and viral proteases, resulting in structural proteins (core, E1, E2), a putative viropore protein (p7), and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). HCV proteins recruit various host cellular proteins for efficient viral propagation (8–10) and disturb host immune systems to establish persistent and chronic infection (11, 12). Viral serine protease NS3 participates in the processing of HCV nonstructural proteins through a non-covalent interaction with cofactor NS4A (13, 14) and plays a crucial role in HCV replication. In addition, HCV NS3/4A protease plays an important role in the inhibition of both host innate immune response (11, 12) and apoptosis induced by HCV infection (15) through cleavage of IPS-1 (also called MAVS, Cardif or VISA), a mitochondria-resident adaptor molecule of retinoic acid-inducible gene-I.
The infectious titer and amounts of viral RNA and core protein of HCV have so far been determined by time- and cost-consuming methods such as focus-forming assay, qRT-PCR, and enzyme-linked immunosorbent assay, respectively. Although replicon RNA or infectious viruses incorporating various reporter genes in their viral genomes have been developed for high-throughput screening of anti-HCV compounds (16–18), strategies for manipulating viral genomes are not useful for establishing a cell culture system for serum-derived HCV from patients. To identify and purify HCV-infected cells without any genetic manipulation of the HCV genome, several groups have reported HCV NS3/4A-mediated trans-activation of reporter gene systems based on chimeric transcription factors containing the cleavage sequence of the NS3/4A protease derived from the HCV polyprotein (19–21).
In this study, we established a novel indicator cell culture system for HCV based on activation of the chimeric transcription factors containing the HCV NS3/4A cleavage sequence of IPS-1, but not of the HCV polyprotein, as reported in previous studies (19–21). Our system is sensitive and quantitative enough to detect JFH-1 virus propagation with a log-scale dynamic range. The chimeric transcription factors were efficiently cleaved by HCV protease and harnessed for detection of HCV replication and for recovery of HCV-infected cells. The strategy employed herein for establishing an indicator cell culture system may provide clues toward the establishment of cell culture systems for serum-derived HCV from hepatitis C patients.
- Top of page
- MATERIALS AND METHODS
The establishment of indicator cell lines based on MAGI assays (30) has made a great contribution to our understanding of HIV-1 life cycles and the development of anti-HIV-1 reagents (41, 42). In this study, we have established indicator cell lines for HCV infection in the same manner using a MAGI assay. To establish indicator cell lines for HCV, HCV NS3/4A-mediated trans-activation of reporter gene systems based on chimeric transcription factors containing the cleavage sequence of the NS3/4A protease derived from HCV polyprotein have been reported (19–21). Breiman et al. (21) reported construction of a chimeric transcription factor consisting of a fusion protein of the Gal4 DB domain with the VP16 transactivator protein of herpes simplex virus (Gal4VP16), NS3/4A protease-cleavage sequences between HCV NS5A and NS5B proteins and a portion of the ER-resident protein PERK for anchoring at the ER membrane. Upon infection with HCV, the Gal4VP16 was cleaved off by the NS3/4A protease and activated the transcription of reporter genes. NS3/4A protease-cleavage sites from NS4A to NS5B junctions were inserted between green fluorescent protein and SEAP, and HCV infection was quantified by the SEAP activity secreted into culture supernatants (19, 20). However, in the previous studies these chimeric constructs containing NS3/4A protease-cleavage sites derived from HCV proteins exhibited only partial cleavage upon co-expression of NS3/4A protease, suggesting that processing of the HCV sequences in the fusion proteins is not efficiently recognized by HCV protease.
To overcome this obstacle, we utilized the C-terminal portion of the mitochondria-resident IPS-1 as a transmembrane anchor of the chimeric transcriptional factors. It has been reported that cleavages of IPS-1 are observed in cell cultures by expression of NS3/4A derived from several HCV strains of different genotypes (43) and also in the liver tissue of patients with chronic hepatitis C infection (43), suggesting that chimeric constructs fused with the C-terminal region of IPS-1 can be cleaved by NS3/4A protease in a broad range of HCV genotypes. Furthermore, to avoid induction of mitochondrial dysfunction and cell death, we modified the C-terminal residues of IPS-1of the chimeric transcription factors to achieve localization on the ER membrane. HCV NS3/4A is an ER-membrane associated protease, and subcellular localization and distance of the cleavage site of the substrates from the membrane could be crucial for efficient processing. Judging from the processing of the Tat/IPS-1ER and Gal4TBP/IPS-1ER proteins by co-expression of NS3/4A protease, the ER-anchored C-terminal domain of IPS-1 was efficiently cleaved by NS3/4A protease.
One of the difficulties in development of indicator cells is a low S/N ratio. When we examined the combination of Tat/IPS-1ER and an HIV-1 LTR promoter, a high background of luciferase expression was observed, probably due to spontaneous release of an undetectable population of the transcription factor from the ER and contingent activation of the target promoter (data not shown). A previous report has indicated that Gal4-TBP and Tat proteins accelerate transcription at different steps (33). Gal4-TBP and Tat accelerate recruitment of RNA polymerase II holoenzyme to initiate transcription, and of P-TEFb to enhance elongation of the polymerase reaction, respectively. As we expected, synergistic enhancement of luciferase expression with a high S/N ratio was achieved by the combination of the two chimeric transcription factors and the GAL4UAS and HIV-1 LTR tandem promoter. Furthermore, a close correlation between expression and viral RNA replication upon HCV infection was observed in Huh7OK1/TG-Luc cells. These results indicate that the Huh7OK1/TG-Luc cell line is useful for quantifying HCV replication with a high sensitivity and specificity and applicable for high-throughput screening of anti-HCV compounds. Among the 1300 compounds screened here, we identified fiduxosin hydrochloride and flunarizine dihydrochloride as anti-HCV agents. Fiduxosin is known as an alpha1-adrenoceptor antagonist (39). Previous screening of a whole-genome siRNA library has shown that knockdown of alpha-1 adrenoceptor A, B, or D expression significantly reduces the replication of the genotype 1b subgenomic replicon (44), suggesting that alpha-1 adrenoceptors participate in the replication of HCV. Flunarizine is an antagonist of T-type calcium ion channels (40) and has been shown to induce expression of HO-1 (which is an antioxidant defense and key cytoprotective enzyme) through PI3K/Nrf2 signaling in an auditory cell line (45). It has been reported that overexpression of HO-1 suppresses HCV replication in genotype 1b replicon cells (46). Although further studies are required, it is reasonable to speculate that HO-1induced by treatment with flunarizine participates in the inhibition of HCV replication.
Another advantage of the indicator system is its flexibility in the choice of reporter genes. For easy separation and concentration of HCV-infected cells by immunomagnetic sorting, we established an Huh7OK1/TG-LNGFR cell line and demonstrated that a fifteen-fold concentration of HCV-infected cells could be achieved with a single sorting step. In addition, it would be possible to use this system in research on various cell lines other than Huh7 derivatives and primary hepatocytes in order to isolate novel HCV strains that exhibit no susceptibility to Huh7-derived cell lines. Although a few HCV strains for productive infection in cultured cells have been reported (3, 6, 47), no reliable and robust cell culture system for propagation of serum-derived HCV has thus far been established. We have inoculated sera from the chronic and window periods of patients with hepatitis C, but so far no significant reporter gene expression has been obtained in the indicator cell lines described in this study (data not shown), indicating that more factors are required for efficient propagation of serum-derived, naturally occurring HCV.
While it has been reported that HCV can spread through cell-to-cell pathways even in the presence of neutralizing antibodies (48–51), the precise mechanisms are not known. Although human CD81 and Claudin-1 are known to be major receptor candidates for HCV entry, cell-to-cell transmission between hepatoma cells is dependent on the expression of Claudin-1 but not of human CD81 (49, 50). In addition, peripheral blood B cells, in which JFH-1 virus cannot replicate, are able to transfer JFH-1 virus to hepatoma cells through SR-BI-, DC-SIGN- and L-SIGN-dependent pathways (51). The indicator cell lines established in this study could be used as recipient cells to investigate cell-to-cell transmission.
In conclusion, we have constructed chimeric transcription factors which are specifically and efficiently cleaved by HCV NS3/4A cleavage and established indicator cell lines capable of monitoring infection with JFH-1 virus based on reporter gene activation through cleavage of the transcription factors by HCV protease. By introducing the present system into various cell lines and modifying the reporter gene, it might be possible to establish a cell culture system capable of propagating serum-derived HCV.