As part of the defense mechanism of host organisms, cells infected by viruses are induced to initiate apoptotic cell death by signals delivered from CTL (25
). On the other hand, a number of viruses have been reported to cause infected cells to escape from this apoptosis to maintain persistent infection (29
). In this study, we investigated the effects of whole HCV proteins produced in HepG2 cells on Fas-mediated apoptotic cell death and found that these cells became resistant to apoptosis. These collected cells are likely to be a good model of HCV-infected cells because all HCV proteins were authentically produced from a precursor polyprotein encoded in a single open reading frame. This antiapoptotic effect of HCV proteins turned out to be a contribution of the core and was seen for different cell lines treated with anti-Fas or TNF-α. Furthermore, we demonstrated that the antiapoptotic effect of the core was exerted through enhanced activation of NF-κB, especially in HepG2 and MCF-7 cells.
Discrepancies regarding the effects of the core on the cellular apoptotic responses have been reported previously: the core functions antiapoptotically according to some papers (32
) and proapoptotically according to others (34
). The reason for the discrepancy among these reports is still unclear. It may be that the core has bipotential roles in the apoptotic signaling. This discrepancy may be, however, explained by the possibility that it was caused by use of clonally selected permanent transfectant cells in the previous studies. As cultured cell lines are likely to be mixed populations of certain cells, a clonally selected cell population cannot be certified to have characteristic features of the parental mixed population. To decrease the chance of selecting particular cells from mixed populations, we enriched the transfected cell population magnetically. Under these conditions, the cell populations which were originally sensitive to the apoptosis mediated by Fas or TNF-α gained the ability to resist such stimuli from the HCV core protein production. During preparation of the manuscript, Shrivastava et al. reported that the core suppressed TNF-mediated NF-κB activation in MCF-7 cells (37
). The discrepancies between our findings and theirs might be derived from the difference in cells used in the experiments as mentioned above. The other difference between transient-transfection and permanent transfectant systems seems to be the expression levels of exogenous genes: that is, a relatively higher level of expression would be expected in the former case. Furthermore, the production of exogenous proteins, not only by transient but also by permanent transfection, may cause a kind of nonspecific stress in the cells. Therefore, to try to reduce the production level we chose the transfection method that enabled us to produce a relatively small amount of exogenous protein in a single cell but in many cells. Moreover, as shown in Fig. A, HepG2 cells transfected with pCMV-3010, in which only less than 1/20 of the core production was seen compared with that for pCMV-Core transfection, also showed the effects on both suppression of apoptosis and NF-κB activation. In addition, we found that the production of FLAG-core fusion protein did not show the above-reported biological effects at all despite the similarity in expression patterns to that of the wild-type core protein, including production level. Taken together, the biological effects of the core reported here should be attributable to the core-specific function irrespective of its expression levels.
We showed here that one of the antiapoptotic effects of the core was exerted through the activation of NF-κB in certain cells. The fine structure of the core required for its ability to activate NF-κB is not clear at this time. However, deletion analysis indicated that at least the C-terminal region of the core is important for that function. Although a simple explanation for this is that the C-terminal portion of the core forms the NF-κB activation domain, the real reason seems to be more complicated. Deletions of the C-terminal hydrophobic region of the core caused changes of subcellular localization of those products (Fig. ). As this region was suggested to act as a signal peptide for E1 protein of HCV during processing of the precursor polyprotein (13
), this region is likely to function as a primary topogenic signal of the core for the cytoplasmic surface of the endoplasmic reticulum. From the nuclear localization of the C-terminal deletion mutants, ΔCore173 and ΔCore151, it is assumed that the decrease in and the loss of NF-κB activation abilities of these mutants, respectively, are due to the isolation of these products from cytoplasm by translocation into the nucleus. It is well known that the regulation of NF-κB activation is based on its localization in the cell: NF-κB is present as an inactive form in the cytoplasm as a complex with IκB, but when the degradation of IκB is induced via activation of several protein kinases, for example, IKK-α and -β, NF-κB translocates into the nucleus as an active form (3
). Therefore, it seems reasonable that the core modulates the pathway for NF-κB activation in the cytoplasm, as do several other viral proteins (16
It is still unknown how NF-κB activation by the core leads to suppression of Fas- and TNF-α-mediated apoptosis. However, it was recently reported that NF-κB induces a group of gene products such as TNF receptor-associated factors 1 and 2 and inhibitor-of-apoptosis proteins 1 and 2, which suppress TNF-α-mediated apoptosis, and blocks the activation of caspase-8 (48
). We have not observed the induction of those factors in the core-producing cells treated with anti-Fas or TNF-α. However, it may be possible that the core activating NF-κB acts on not only the TNF-α- but also the Fas-mediated apoptotic pathway by a similar mechanism, since we found that the activation of caspase-8 in anti-Fas- and TNF-α-treated HepG2 and MCF-7 cells, respectively, was diminished by production of the core. In contrast to HepG2 and MCF-7 cells, the mechanism of the suppressive effect on Fas-mediated apoptosis introduced by the core in Jurkat cells is unknown so far, because this effect was revealed to be independent of NF-κB activation.
We concluded from our results that HCV core protein inhibits the onset of apoptotic cell death, and at least one of the important pathways for this includes NF-κB activation by the core. This antiapoptotic effect introduced by the core might be advantageous for HCV by allowing the host hepatocytes to survive apoptosis, resulting in sustained infection. Further studies are necessary to determine the molecular mechanism by which the core enhances NF-κB activity and to find the other antiapoptotic pathway mediated by the core independently of NF-κB, because this might allow development of effective strategies for the prevention of chronic sustained viral infection.