Structural and Functional Characterization of Interaction between Hepatitis B Virus X Protein and the Proteasome Complex

Abstract

Hepatitis B virus (HBV) has a unique fourth openreading frame coding for a 16.5-kDa protein known as hepatitis B virus X protein (HBX). The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial. We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator. HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX. In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-␤-galactosidase, two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX.Human hepatitis B virus (HBV) 1 belongs to a group of hepadnaviruses that includes the hepatitis viruses of the woodchuck, ground squirrel, tree squirrel, Pekin duck, and heron. HBV has a unique fourth open reading frame, termed the hepatitis B virus X (HBX) gene. HBX gene is well conserved among the mammalian hepadnaviruses and codes for a 16.5-kDa protein (1, 2). The protein can activate the transcription of a variety of viral and cellular genes (3-5) and induce liver cancer in certain transgenic mouse model (6). Since HBX does not bind to DNA directly, its activity is thought to be mediated via protein-protein interactions. HBX has been shown to enhance transcription through AP-1 and AP-2 (7-9) and to activate various signal transduction pathways (10, 11). Several recent studies have also identified possible cellular targets of HBX, including members of the CREB/ATF family (12, 13), the TATA-binding protein (14), RNA polymerase subunit RPB5 (15, 16), the UV-damaged DNA-binding protein (17), and the replicative senescence p55 sen (18). HBX has also been shown to interact with p53 and inhibit its function (19,20). Furthermore, HBX possesses amino acid sequence homology to the functionally essential domains of Kunitz-type serine proteases inhibitors and mutation of this putative motif inactivates the transactivation function of HBX (21).Using the Saccharomyces cerevisiae two-hybrid syste...

Identification and analysis of tumour-associated antigens in hepatocellular carcinoma

“…These observations suggest that HDAC inhibitors may be candidate drugs in therapy for human cancer (Cress and Seto, 2000). PSMA7, which was found to be overexpressed in seven of 16 HCC patients, is a subunit of proteasome, and has been shown to interact specifically with the hepatitis B virus x protein, a protein critical to viral replication (Zhang et al, 2000). It is also involved in regulating hepatitis virus C internal ribosome entry site activity, which is essential for viral replication (Kruger et al, 2001).…”

“…Of these 30 antigens, one is the CT antigen CAGE protein, and the other 13 antigens, which had previously been reported to be involved in tumorigenesis or suspected to be involved in tumour progression, including CDC37 (Stepanova et al, 2000), MIF (Li et al, 2004), galectin 4 (Huflejt and Leffler, 2004), galectin 8 (Bidon-Wagner and Le Pennec, 2004), PINCH (WangRodriguez et al, 2002), SPRY2 (Lee et al, 2004a), HSPCA (Becker et al, 2004;Huang et al, 2004b), transgelin 2 (Shields et al, 2002), HDAC2 , H factor (Junnikkala et al, 2000), AAT (Huang et al, 2004a), B factor (Perou et al, 1999), PIBF (Lachmann et al, 2004). Three other antigens (SR140 protein, SFRS2IP, RNPC2) may be involved in the regulation of alternative mRNA splicing, PSMA7 is related to hepatitis B and hepatitis C viral replication (Zhang et al, 2000;Kruger et al, 2001), and the remaining 12 antigens have no known association with cancer or hepatitis B or hepatitis C.…”

The Proteasome α-Subunit XAPC7 Interacts Specifically with Rab7 and Late Endosomes

“…XAPC7 or PSMA7 interacts with both viral and cellular proteins. Liang and co-workers (45,44,61) were the first to report and map an interaction between XAPC7 and the Hepatitis B virus X (HBX) protein. Functionally, the interaction of HBX with XAPC7 was shown to be crucial for virus replication in that it blocks proteasome activity and stimulates transcriptional transactivation by HBX.…”

“…In human and mouse tissue, XAPC7 is a bona fide component of the 26 S proteasome (43). XAPC7 also has regulatory functions in hepatitis and HIV viral infection (44,45,46,47) and some disease states including cancer and glaucoma (48,49). The 26 S proteasome consists of two 19 S regulatory units, flanking a central 20 S degradative unit.…”

Genetic mechanisms of hepatocarcinogenesis

“…The ®nding that HBxAg relieves the p53 suppression of the alpha-fetoprotein gene (Ogden et al, 2000), which is associated with the nuclear co-localization of HBxAg and p53 (Greenblatt et al, 1997), may provide an explanation for the upregulation of alpha-fetoprotein in up to 80% of HCCs. In these and other studies (Boix-Ferrero et al, 1999;Bourdon et al, 1995;Buetow et al, 1992), high levels of wild type p53 in HCC cells are likely to be tolerated because it is inactivated by direct binding to HBxAg, and because HBxAg has been shown to inhibit the proteasome (Sirma et al, 1998;Zhang et al, 2000), which is responsible for the normal degradation of p53 (Maki et al, 1996). HBxAg also transcriptionally down-regulates the expression of the translation initiation factor, sui1, as well as the senescence factor and cyclin dependent kinase inhibitor, p21 WAF1/CIP1/SDI1 , both of which inhibit hepatocellular growth (Feitelson et al, 1999).…”