23-26 Thus, we used TNF-α selleck kinase inhibitor to mimic natural immunomediated apoptosis and found that the JFH-1/S2–replicating cells have lower susceptibility to the apoptosis induced by these cytokines. In JFH-1/S2–transfected cells, TNF-α–induced apoptosis detected by TUNEL assay was substantially lower than that of JFH-1/wt–transfected cells (Fig. 4). We confirmed it by staining with anticleaved PARP. In complete agreement with the results produced by way of TUNEL assay, the number of anticleaved PARP stained cells among JFH-1/S2–infected cells was significantly lower than that among JFH-1/wt–infected cells (Fig. 5). In our previous study,

we reported that HCV-specific immune responses with T cell proliferation and interferon-γ production were maintained until the disappearance of viremia in the patient serum–infected chimpanzee.11 This finding indicates that continuous selection pressure in the infected chimpanzee might have contributed to the emergence of a clone with an ability to escape the cytokine-induced apoptosis. We are not sure whether this phenotype of JFH-1/S2 is due to its lower replication VX-770 efficiency and thus lower production of HCV proteins. The accumulation of viral proteins might predispose cells to the apoptosis induced by TNF-α. To answer this question, it will be necessary

to investigate the genomic regions of JFH-1/S2 and cellular host factors responsible for the ability of this strain to escape the apoptosis. Fenbendazole By way of mapping analysis for JFH-1/S2, we could determine responsible regions;

NS5B was for lower replication efficiency (Supporting Fig. 1B), and P7 and NS2 were for enhanced viral particle assembly (Supporting Table 2). For the evasion of apoptosis, we could not specify the responsible region, because both chimeric constructs, JFH-1/S2-wt and JFH-1/wt-S2, showed less susceptibility to cytokine-induced apoptosis to a certain extent. These data indicate that both structural and nonstructural regions might have contributed to the acquisition of this phenotype. Previously, a potent antiapoptotic effect of the HCV NS5A protein was described.27 NS5A interacts with Bin1, which is a nucleocytoplasmic c-Myc–interacting protein with tumor suppressor and apoptotic properties, thus inhibiting Bin1-associated apoptosis. Because JFH-1/S2 contains several mutations in the NS5A region (Supporting Table 1), one or more mutations in this protein may be associated with antiapoptotic effects. In conclusion, we demonstrated that the JHF-1/S2 strain acquired phenotypes of lower replication, higher virus production, and less susceptibility to cytokine-induced apoptosis. These phenotypes were associated with mutations that emerged 23 weeks after infection in a chimpanzee, and might have contributed to long-term infection in vivo. Such control of viral functions by specific mutations may be a key viral strategy to establish persistent infection. We are grateful to Francis V. Chisari for providing the Huh-7.5.