Proteasome Activator PA28γ-Dependent Nuclear Retention and Degradation of Hepatitis C Virus Core Protein

Abstract

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28␥ (11S regulator ␥) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28␥ with other Flavivirus core proteins was detected. Deletion of the PA28␥-binding region from the HCV core protein or knockout of the PA28␥ gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28␥ enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28␥-dependent pathway through which HCV pathogenesis may be exerted.Hepatitis C virus (HCV) is the causative agent in most cases of acute and chronic non-A, non-B hepatitis (16, 51). Over 50% of patients with acute infection evolve into a chronic carrier state (26), and persistent infection frequently results in chronic hepatitis. Chronic HCV infection may lead to the development of cirrhosis and eventually hepatocellular carcinoma (21, 51). HCV belongs to the Flaviviridae family, a family that also includes Japanese encephalitis virus (JEV) and Dengue fever virus (DEN), and possesses a viral genome consisting of a single positive-strand RNA of approximately 9.6 kb and encoding approximately 3,000 amino acids in a single polypeptide (9, 58). HCV proteins are produced as a single polypeptide that is posttranslationally cleaved by host cellular peptidases and viral proteases to yield at least 10 viral proteins (7,10,12,54).A comparison of the genome structure of HCV with other flaviviruses, as well as the observation of a specific interaction of viral sense RNA with HCV core protein in cells (53, 68), suggests that the HCV core protein forms the nucleocapsid with viral genome RNA. An HCV core protein consisting of the N-terminal 191 amino acids is generated by protein cleavage by host signal peptidase(s) (37, 52). The HCV core protein is further processed into a mature core protein lacking its C-terminal hydrophobic region by either an unknown host protease (52, 65) or by a signal peptide peptidase (36). The matured core protein is retained on the endoplasmic reticulum (ER) either by an interaction with immature core protein on the ER membrane (29) or via E1 envelope protein (32). The C-terminal hydrophobic region between amino acids 174 and 191 is essential for HCV core protein anchoring on the ER membrane and for the signal sequence of E1 protein to translocate into the ER lumen. Core proteins truncated at the C termini are mainly localized in the nucleus and, to lesser extent, in the cytoplasm...

Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein

“…EGFP-Core 191 colocalized with ERDsRed to the ER (Fig. 2), whereas EGFP-Core 179 was localized primarily to the nucleus as reported previously (3,33,43,47), suggesting that the C-terminal signal sequence is essential for anchoring HCV core protein to the ER membrane. However, EGFP-Core 174-191 exhibited diffuse staining similar to that of EGFP, suggesting that the signal sequence alone is not sufficient for ER localization.…”

“…Furthermore, we demonstrated that nuclear localization and degradation of HCV core protein is regulated by PA28␥-dependent proteolysis (33). These findings suggest that HCV core protein plays a pivotal role in the development of hepatocellular carcinoma and that intramembrane proteolysis may regulate the subcellular localization of HCV core protein.…”

“…This is then cleaved by SPP into the mature core protein and localizes primarily to the ER. The mature core protein, further processed by an unidentified protease, is composed of amino acids 151 to 153 and is detected in the nucleus (33,47). Actually, HCV core protein is observed in the cytoplasm, nucleus, and nucleoli in transgenic mice expressing HCV core protein (35).…”

The HCV ARFP/F/core+1 protein: Production and functional analysis of an unconventional viral product

“…Indeed, it was recently reported that the core protein of HBV stimulates the proteasome-mediated degradation of the HBV X protein (HBX), when the HBV viral proteins, which are transcriptionally transactivated by the X protein, reach a sufficiently high levels for viral replication (68)(69)(70)(71)(72). The HCV core protein has also been shown to interact directly with the activator of the IFN-c-inducible immunoproteasome PA28c in the regulation of core nuclear retention and stability (73). The physiological implications of these findings remain unclear.…”

Critical role of PA28γ in hepatitis C virus-associated steatogenesis and hepatocarcinogenesis

“…However, HCV core protein accumulated in the nucleus because knockout of PA28␥ gene abrogated the ability to cause liver pathology, suggesting that interaction of HCV core protein with PA28␥ in the nucleus is prerequisite for the liver pathology induced by HCV core protein. We have previously shown (18) that HCV core protein is degraded through a PA28␥-dependent pathway, and Minami et al (28) reported that PA28␥ has a cochaperone activity with Hsp90. Therefore, degradation products of HCV core protein by means of PA28␥-dependent processing or correct folding of HCV core protein through cochaperone activity of PA28␥ might be involved in the development of liver pathology.…”