The Transcriptional Transactivation Function of HBx Protein Is Important for Its Augmentation Role in Hepatitis B Virus Replication

Abstract

The role and functional domain of hepatitis B virus (HBV) X protein (HBx) in regulating HBV transcription and replication were investigated with a transient transfection system in the human hepatoma cell line HepG2 using wild-type or HBx-minus HBV genome constructs and a series of deletion or mutation HBx expression plasmids. We show here that HBx has augmentation effects on HBV transcription and replication as a HBV mutant genome with defective X gene led to decreased levels of 3.5-kb HBV RNA and HBV replication intermediates and that these decreases can be restored Hepatitis B virus (HBV) is the prototypic member of the Hepadnavirdae family, which contains a group of closely related hepatotropic small DNA viruses that infect their respective animal hosts (13,29). The HBV genome is a 3.2-kb, circular, partially double-stranded DNA molecule with four overlapping open reading frames (ORFs) named PC-C, PS-S, P, and X (35, 38). Upon HBV infection of the hepatocyte, the HBV genome is converted to covalently closed circular DNA in the nucleus. The covalently closed circular DNA serves as the template for transcription by the host RNA polymerase II, generating the 3.5-, 2.4-, 2.1-, and 0.7-kb viral transcripts that encode the HBV core and polymerase polypeptides, the large surface antigen polypeptide, the middle and major surface antigen polypeptides, and the HBx polypeptide. The expression of those transcripts are directed by four HBV promoters (Cp, PS1p, Sp, and Xp), respectively, and influenced by two HBV enhancers (Enh I and Enh II) (14,29,35,38,43). HBV replicates by reverse transcription of the viral pregenomic 3.5-kb RNA (pgRNA) using the HBV polymerase that catalyzes RNA-dependent DNA synthesis and DNA-dependent DNA synthesis (36,40). Besides encoding for the HBV core polypeptide and HBV DNA polymerase that compose the viral capsid, the greater-than-genome-length 3.5-kb pgRNA is also encapsidated and serves as the template for reverse transcription. The encapsidated pgRNA is converted into the 3.2-kb partially double-stranded genomic DNA inside the viral capsid in the cytoplasm of the hepatocytes (35,36,38,40). There are several putative regulatory steps for HBV replication, e.g., pgRNA synthesis, encapsidation of pgRNA, and reverse transcription of pgRNA. As the pgRNA encodes both the HBV polymerase and core polypeptides and serves an additional function as the replication template, regulation of the synthesis of this RNA is therefore a critical step in the viral life cycle (35,38,39).HBV infection is a worldwide health problem and is one of the major causes of hepatocellular carcinoma (HCC). The crucial role of HBV in hepatocarcinogenesis is established, while the mechanism by which HBV causes transformation of hepatocytes remains unclear (1, 2, 6). HBV X protein (HBx) has long been suspected of playing a positive role in hepatocarcinogenesis, as avian hepadnaviruses missing the X ORF seem not to be associated with HCC, and some HBx transgenic mice appear to develop HCC (15, 44) or be more sensitive to a car...

Hepatitis B virus-related hepatocellular carcinoma: paradigms for viral-related human carcinogenesis

“…In transgenic mice, HBx is not essential for HBV replication, but it can enhance viral replication (Reifenberg et al, 2002;Xu et al, 2002). Recently, in vitro experiments suggest that HBx stimulates HBV replication via its transactivation function (Melegari et al, 2005;Tang et al, 2005). It has been demonstrated that HBx action on viral replication may be dependent to modulation of calcium homeostasis, which provides novel clues for HBx biology (Bouchard et al, 2001(Bouchard et al, , 2003Choi et al, 2005).…”

Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-β induction by disrupting the VISA-associated complex

“…25,31,32 Replication of the HBx-deficient HBV replicon in HepG2 cells was reduced by threefold compared with wild-type HBV, suggesting that HBx plays a stimulatory role in HBV replication. 33 The information on HBx protein continues to grow, although many of the roles and functions of HBx remain controversial.…”

Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function

“…Although initial studies suggested that HBx was not required for virus replication in cell culture (18), experiments with the highly related woodchuck hepatitis virus (WHV) system indicate that the WHV X protein (WHx) is required for virus replication in vivo (19)(20)(21)(22). Studies performed using a plasmid-based replication assays that use greater-than-unit-length HBV genomes transfected in HCC cell lines or injected via the mouse tail vein under hydrodynamic conditions have repeatedly confirmed that HBx potentiate HBV replication (23)(24)(25)(26)(27).…”