The mechanism of hepatocellular carcinoma development in patients with chronic hepatitis C remains unclear. It has been demonstrated that expression of the HCV core protein alone is sufficient for the induction of hepatic steatosis and hepatocellular carcinoma in transgenic mice (28
). These findings suggest that the HCV core protein plays a pivotal role in the development of hepatocellular carcinoma. In this study, we isolated PA28γ from a human fetal brain library as a host protein that specifically binds to the HCV core protein. We further suggest that HCV core protein interaction with PA28γ correlates with the retention of HCV core protein in the nucleus and regulates the stability of the HCV core protein in a proteasome-dependent manner. There are two isoforms of PA28γ in humans, a major form and a splicing variant that contains an additional 13 amino acids in the second helix domain. The second isoform is detected only in the human fetal brain and is not found in other human tissues or other mammals (3
). In this screen, we did not obtain the splicing variant of PA28γ from the human fetal brain library; it is, therefore, still unknown whether the human-specific isoform of PA28γ binds to the HCV core protein.
The C-terminal hydrophobic region of the HCV core protein is processed by host proteases such as signal peptidase and/or intramembrane proteases. The processed, mature HCV core protein transferred into lipid droplets when a full length of core protein was expressed by an alphavirus expression system (14
). However, the mature core protein remained in the ER when the full length of core protein was expressed by transfection in this study (Fig. ). This discrepancy might be due to the difference in expression systems, cell lines, and genotypes of the HCV clone.
When fused to EGFP, the PA28γ-binding region of the HCV core protein (EGFP-Core44-71) migrated into the nucleus, indicating that this region may function as an NLS. Deletion of the PA28γ-binding region from the HCV core protein (EGFP-Core151Δ44-71) or depletion of PA28γ from cells, however, did not eliminate nuclear transport of the HCV core protein, suggesting the presence of an alternative mechanism for the nuclear transport of the HCV core protein other than its interaction with PA28γ. Within the C-terminally truncated HCV core protein there exist three putative NLSs consisting of a cluster of basic amino acids (8
). β-Galactosidase-fused C-terminal truncated core protein lacking one of these clusters (β-gal-Core123Δ38-43) was localized primarily in the cytoplasm rather than the nucleus in COS cells (55
); an EGFP-fused mutant, EGFP-Core151Δ38-43, however, was localized in the nucleus in the HeLa and 293T cell lines (data not shown). These results suggest that there are at least two possible mechanisms, PA28γ dependent and PA28γ independent, leading to nuclear transport of the HCV core protein. EGFP-Core151Δ38-43 and EGFP-Core151Δ44-71 are translocated into the nucleus by the PA28γ-dependent and -independent pathways, respectively. Both pathways may be mediated through importin or importin-like molecules because PA28γ has a c-Myc-like NLS in its homolog-specific region. Furthermore, the interaction with PA28γ was shown by time-lapse microscopy to play an important role in the retention of the HCV core protein in the nucleus. HCV core proteins lacking the PA28γ-binding region, EGFP-Core151Δ44-71 and EGFP-Core151, were exported from the nucleus to the cytoplasm in HeLa cells and embryonic fibroblasts derived from PA28γ knockout mice, respectively. The nuclear exporting signal was found in the C-terminal half of the HCV core protein and plays a role in the export of the HCV core protein from the nucleus to the cytoplasm (R. Suzuki, S. Sakamoto, T. Tsutsumi, A. Rikimaru, T. Shimoike, S. Machida, Y. Matsuura, T. Miyamura, and T. Suzuki, unpublished data). The putative PA28γ-dependent and -independent translocation of the HCV core protein from the cytoplasm to the nucleus, as well as the possible functions and fates of the HCV core protein in the nucleus, are illustrated in Fig. .
FIG. 10. Putative PA28γ-dependent and -independent translocation of the HCV core protein from the cytoplasm into the nucleus and possible function and fate. A precursor HCV core protein (Core191) is processed from a polyprotein in the ER by signal peptidase (more ...)
Although many host proteins have been reported to interact with the HCV core protein in relation to carcinogenesis (18
), this is the first report demonstrating the interaction of the HCV core protein with an endogenously expressed host protein. In the livers of HCV core transgenic mice, the HCV core protein was primarily detected in the cytoplasm but some protein was found in the nucleus, albeit to a lesser extent (40
). PA28γ was shown to coimmunoprecipitate with HCV core proteins irrespective of their intracellular localization (Fig. and ), suggesting that the core proteins bind to PA28γ after cell disruption. HCV core proteins truncated at the C terminus (HCV Core151 and 173) migrated into the nucleus and were degraded by ubiquitin-mediated proteolysis (57
). In this study, overexpression of PA28γ led to the degradation of the HCV core protein; this degradation was able to be partially blocked by the proteasome inhibitor MG132. Additionally, HCV core protein was detected in the nucleus of a HeLa cell expressing the full-length HCV core protein in the presence of MG132 (Fig. ). These results suggest that the HCV core protein migrates into the nucleus and is then promptly degraded by the nuclear proteasome.
The F protein generated by ribosomal frameshift in the gene encoding the core protein was mainly localized in the cytoplasm and degraded by the proteasome (63
). Although the expected mass of 14 kDa of the F protein from strain J1 was not detected in HeLa cells expressing HA-Core151 even in the presence of MG132 (Fig. ), we examined the interaction of the protein of −2/+1 frame of the gene encoding the HCV core protein with PA28γ. Lack of interaction of endogenous PA28γ with the F protein (Fig. ) suggests that PA28γ specifically interacts with the HCV core protein but not with the F protein.
Hepatitis B virus X factor (HBx) alone induces hepatocellular carcinoma in mice (20
), suggesting that HBx plays an important role in hepatocellular carcinoma. HBx bound to PSMA7 and PSMC1, subunits of PA700 and the 20S proteasome, respectively, leads to the enhancement of the transcription activities of AP-1 and VP-16 (69
). Like HBx, the HCV core protein is processed by the proteasome in a PA28γ-dependent manner. An HCV core protein with the same molecular mass as HCV Core151 was detected in cells in the presence of MG132 (57
). The proteasome is well known to regulate many transcription factors such as NF-κB, p53, and c-Myc, etc. (4
). For example, NF-κB and its inhibitor IκB are degraded by the proteasome, resulting in translocation of active NF-κB into the nucleus (19
). Upon processing, the active form of NF-κB acquires transcription activity that regulates many biological functions such as cell proliferation (43
). The HCV core protein is known as a regulatory factor that modulates some signaling pathways as well as affecting expression levels of a variety of proteins under the control of different promoters (reviewed in reference 56
). The short-lived, C-terminally truncated HCV core protein may acquire an as yet undetermined biological function in the nucleus. Additionally, peptides derived from the HCV core protein that has been processed by the PA28γ-activated proteasome may play some role in the transcriptional regulation that is involved in hepatocellular carcinogenesis.
The PA28γ homopolymer is able to associate with the 20S proteasome (60
) and strongly activates the peptidase activity of the latent proteasome (48
). The PA28α/β heteropolymer forms a hybrid proteasome with the 20S proteasome and PA700; this complex efficiently enhances antigen processing in an ATP-dependent manner (59
). The PA28γ homopolymer, PA700, and the 20S proteasome may also form a hybrid proteasome that may be responsible for the proteolysis of the HCV core protein in the nucleus. PA28γ knockout mice demonstrate no abnormality other than growth retardation; this suggests that PA28γ is either dispensable for host physiological function or that suitable compensation mechanisms exist within the organism (42
). Translocation and degradation of the HCV core protein by the PA28γ-activated proteasome in the nucleus may also contribute to the establishment and maintenance of persistent infection of HCV through the down regulation of viral assembly.
Although the biological significance of PA28γ is not well understood, in this study we have demonstrated new mechanisms by which PA28γ translocates and retains the HCV core protein in the nucleus; PA28γ is also involved in the proteolysis of the HCV core protein. Another nuclear proteasome activator, PA200, was recently purified from bovine testis and was demonstrated to enhance the peptidase activity but not the protease activity of the 20S proteasome (61
). This report suggests that PA200 may be the functional homologue of PA28 in the nucleus. PA200 is predominantly localized to the nucleus and demonstrates homology to yeast and worm proteins that are implicated in the repair of DNA double-strand breaks. Thus, nuclear proteasome activity may be associated with DNA repair. Therefore, it may be possible that the interaction of PA28γ with the HCV core protein results in a perturbation of DNA repair activity through the nuclear proteasome, and these changes may subsequently induce hepatocellular carcinoma in humans and mice.
In conclusion, we have demonstrated that PA28γ specifically interacts with the HCV core protein in cell culture as well as in the livers of both HCV core transgenic mice and a patient with chronic hepatitis C. This interaction correlates to the nuclear retention and degradation of C-terminally truncated HCV core proteins. Understanding the precise function of PA28γ may give us new insight into virus-cell interactions and lead to a greater understanding of the pathogenicity of HCV infection. Establishment of HCV core transgenic mice deficient in PA28γ gene expression will allow the direct assessment of the involvement of PA28γ in the development of hepatocellular carcinoma induced by HCV core protein; these experiments are under way.