HCV develops chronic infection in the vast majority of infected patients (1
), however, the mechanisms of its persistence are still under investigation. Many viruses have evolved different strategies to cope with host immune systems, thus causing the development of persistent infection. For example, some viruses interfere with the major histocompatibility complex (MHC) class I presentation of viral antigens, while others modulate lymphocyte and macrophage functions, including cytokine production (12
). In our previous study, we detected increasing number of mutations in HCV genome isolated from JFH-1 patient serum-infected chimpanzees. Thus we reasoned that these detected mutations might have imparted some advantage to this virus for long-time survival. To examine this hypothesis, we compared the phenotypes of JFH-1 variant strains emerged at early and late stages of infection in JFH-1 patient serum- and JFH-1cc- infected chimpanzees, and found that the JFH-1/S2 strain isolated from the patient serum-infected chimpanzee at a later time-point of infection replicated slowly, produced more infectious viruses, and displayed reduced susceptibility to cytokine-induced apoptosis.
The JFH-1 variant strains JFH-1/C, which contains 7 non-synonymous mutations identified in JFH-1cc infected chimpanzee at week 7, showed comparatively slower replication kinetics and slightly enhanced infectious virus production in cell culture. The intracellular specific infectivity of this strain in Huh7–25 cells was 3.9 times higher than that of JFH-1/wt (Table 2). These characteristics might have imparted some advantage to this strain for establishing productive infection in the chimpanzee. The other JFH-1 variant strains, JFH-1/S1 and S2, contain 6 and 17 non-synonymous mutations identified in JFH-1 patient serum infected chimpanzee at weeks 2 and 23 post-infection, respectively. Replication kinetics and infectious virus production of the JFH-1/S1 strain were comparable to that of JFH-1/wt in cultured cells (, Table 2). In contrast, the JFH-1/S2 strain showed lower replication efficiency. Although the intracellular HCV RNA level of this strain in Huh7–25 cells was lower than that of JFH-1/wt and S1, and almost the same as that of JFH-1/C (Table 2), intracellular specific infectivity was 18.0 and 12.9 times higher than that of JFH-1/wt and S1, respectively, suggesting a significant increase in the assembly of infectious virus particles (p < 0.005, Table 2). The enhanced capacity of this strain to assemble infectious virus particles resulted in a higher extracellular infectivity titer that contributed to the rapid spread of virus to surrounding cells. Flow cytometry analyses of cells transfected with JFH-1/wt and variant strains revealed that the percentage of the HCV NS5a-positive population in JFH-1/S2 transfected cells was higher, but the MFI of the anti-NS5a signal was lower than that in JFH-1/wt transfected cells, thus confirming higher spread and lower replication of this strain. Taken together, both JFH-1/C and JFH-1/S2 exhibited a tendency towards decreased replication and increased infectious virus production. However, the extent of enhanced virus production was substantially lower in JFH-1/C than JFH-1/S2 strain, which might have led to the earlier elimination of infection in the JFH-1cc-infected chimpanzee. In other words, the potency of infectious virus production and spread seems to correspond to the duration of infection in infected animals.
The association between a lower replication efficiency and persistent infection is still unclear. It has been reported that an escape mutant with an amino acid substitution at the CTL epitope in the NS3 region exhibits lower NS3/4 protease activity and replication capacity in vitro
). The JFH-1/S2 strain contains the T1077A mutation in the NS3 region (), and this mutation is located close to mutations reported to be associated with immune evasion and lower replication (17
). Thus, the lower replication efficiency of the JFH-1/S2 strain may be a result of an immune escape mutation at the expense of viral fitness. Meanwhile, we cannot deny the advantage of lower replication in establishing persistent infection. Lower replication may contribute to the avoidance of MHC class I mediated antigen presentation and to escape from the host immune system. Either way, by acquiring the ability to produce more viral particles, the JFH-1/S2 strain could rapidly spread to surrounding cells, irrespective of its lower replication efficiency. Importantly, these emerged mutations did not attenuate in vivo
infectivity, unlike cell culture adaptive mutations reported to cause attenuated infection in vivo
). Upon inoculation into human hepatocyte-transplanted mice, JFH-1/S1, S2 and C strains could establish infection without any mutations, produced levels of viremia similar to JFH-1/wt, and persisted for a similar observed period of infection (). This observation is different from that in chimpanzees, where JFH-1/wt and JFH-1/C strains were eliminated earlier than JFH-1/S2. In contrast to chimpanzees, human hepatocyte-transplanted mice lack cytotoxic T lymphocytes (CTL) and the natural killer (NK) cell-mediated immune system, which could be responsible for this difference (6
). Taken together, our results suggest that along with efficient infectious virus production, the JFH-1/S2 strain might have acquired an advantage that helps it evade the CTL and NK cell-mediated immune system.
Apoptosis of virus-infected cells by the immune system is crucial as a general mechanism of clearing infections (20
). The J6/JFH-1 chimeric virus has been reported to exhibit pro-apoptotic characteristics in cell culture (22
). However, because HCV needs to escape the host immune system in order to establish chronic infection, immune cell-mediated apoptosis may be inhibited in infected hepatocytes. In the liver, HCV-infected hepatocytes are eliminated by targeted apoptosis induced by NK cells, macrophages, and CTL with ligand- and receptor-mediated signals such as TNF-α, FasL, and TNF-related apoptosis inducing ligand (TRAIL) (23
). Thus, we used TNF-α to mimic natural immune-mediated apoptosis, and found that the JFH-1/S2 strain 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 (). We confirmed it by staining with anti cleaved PARP. In complete agreement with the results produced by TUNEL assay, number of anti cleaved PARP stained cells among JFH-1/S2-infected cells was significantly lower than that among JFH-1/wt infected cells (). In our previous study, we reported that HCV-specific immune responses with T-cell proliferation and interferon gamma production were maintained until the disappearance of viremia in the patient serum-infected chimpanzee (11
). This 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 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 region(s) of JFH-1/S2 and cellular host factors responsible for the ability of this strain to escape the apoptosis.
By mapping analysis for JFH-1/S2 strain, 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 wt-S2, showed less susceptibility to cytokine-induced apoptosis to a certain extent. These data indicated that both structural and non-structural regions might have contributed to the acquisition of this phenotype. Previously, a potent anti-apoptotic 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. As JFH-1/S2 contains several mutations in the NS5a region (Supporting Table 1
), one or more mutations in this protein may be associated with anti-apoptotic 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.