Enhancement of Hepatitis B Virus Replication by the Regulatory X Protein In Vitro and In Vivo

Abstract

The 3.2-kb hepatitis B virus (HBV) genome encodes a single regulatory protein termed HBx. While multiple functions have been identified for HBx in cell culture, its role in virus replication remains undefined. In the present study, we combined an HBV plasmid-based replication assay with the hydrodynamic tail vein injection model to investigate the function(s) of HBx in vivo. Using a greater-than-unit-length HBV plasmid DNA construct (payw1.2) and a similar construct with a stop codon at position 7 of the HBx open reading frame (payw1.2*7), we showed that HBV replication in transfected HepG2 cells was reduced 65% in the absence of HBx. These plasmids were next introduced into the livers of outbred ICR mice via hydrodynamic tail vein injection. At the peak of virus replication, at 4 days postinjection, intrahepatic markers of HBV replication were reduced 72% to 83% in mice injected with HBx-deficient payw1.2*7 compared to those measured in mice receiving wild-type payw1.2. A second plasmid encoding HBx was able to restore virus replication from payw1.2*7 to wild-type levels. Finally, viremia was monitored over the course of acute virus replication, and at 4 days postinjection, it was reduced by nearly 2 logs in the absence of HBx. These studies establish that the role for HBx in virus replication previously shown in transfected HepG2 cells is also apparent in the mouse liver within the context of acute hepatitis. Importantly, the function of HBx can now be studied in an in vivo setting that more closely approximates the cellular environment for HBV replication.Hepatitis B virus (HBV) infection is a major health problem worldwide, with more than 350 million chronically infected individuals who are at risk for developing severe liver disease, including hepatocellular carcinoma (12). The 3.2-kb HBV genome encodes a single regulatory protein termed HBx, but progress towards understanding the role of HBx in virus replication has been hindered by the lack of either a cell culture system or a convenient small animal model. Although initial studies suggested that HBx was not required for virus replication in cell culture (2), experiments with the highly related woodchuck hepatitis virus (WHV) system indicate that the WHV X protein (WHx) is required for virus replication in vivo (23). Other studies have demonstrated that WHVs carrying mutant WHx grow with attenuated kinetics in vivo, depending on the particular mutation (17,22).While many functions have been ascribed to HBx (reviewed in reference 5), most studies have been performed in cell culture and under conditions in which HBx is not required for virus replication. Two recent advances provide new approaches to investigate a role for HBx in the context of virus replication in vivo. The first is a plasmid-based replication assay that utilizes a greater-than-unit-length HBV genome of the ayw subtype (payw1.2) (15) and an identical plasmid containing a stop codon affecting HBx amino acid position 7 (payw1.2*7) (10). These plasmids were used to demonstrate that capsidassoc...

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

“…The HBV regulatory protein HBx has been shown to potentiate viral replication in 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 (23)(24)(25)(26)(27). We have recently shown that transfection of plasmid free linear HBV DNA into Huh7 and HepG2 HCC cell lines (32) allows to fully recapitulate the HBV replication cycle, including the nuclear generation of viable cccDNA (30) (Fig.…”

“…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).…”

Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model

“…It has been demonstrated that HBx transactivates the transcription of the major HBV genes, including HBcAg (4,18). Furthermore, some patients in the inactive viral replication phase of chronic hepatitis B infections do not express HBcAg in their liver (19), suggesting that the lack of HBcAg may contribute to HBV persistence.…”

A zebrafish model of intrahepatic cholangiocarcinoma by dual expression of hepatitis B virus X and hepatitis C virus core protein in liver

“…From the 1 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; 2 Department of Pathology, Chang Gung Memorial Hospital, Keelung, Taiwan; 3 College of Medicine, Chang Gung Univeristy, Taoyuan, Taiwan; 4 Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan; 5 Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan; 6 Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan; 7 Department of Aquaculture, and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan; 8 Division of Molecular and Developmental Biology, National Institute of Genetics, and Department of Genetics, Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan.…”

“…7 HBV X (HBx) is a 16-kDa protein that has been implicated in the enhancement of viral replication, cell death, lipid metabolism, cell cycle regulation, DNA repair, and epigenetic modification. [8][9][10][11][12][13] HBx is frequently expressed in the tissues surrounding the tumor and may be involved in ICC formation. 14 It remains unclear whether the expression of HBx plays a functional role in the pathogenesis of ICC.…”