Hepatitis B Virus X Protein Stimulates Viral Genome Replication via a DDB1-Dependent Pathway Distinct from That Leading to Cell Death

Abstract

The hepatitis B virus (HBV) X protein (HBx) is essential for virus infection and has been implicated in the development of liver cancer associated with chronic infection. HBx can interact with a number of cellular proteins, and in cell culture, it exhibits pleiotropic activities, among which is its ability to interfere with cell viability and stimulate HBV replication. Previous work has demonstrated that HBx affects cell viability by a mechanism that requires its binding to DDB1, a highly conserved protein implicated in DNA repair and cell cycle regulation. We now show that an interaction with DDB1 is also needed for HBx to stimulate HBV genome replication. Thus, HBx point mutants defective for DDB1 binding fail to complement the low level of replication of an HBx-deficient HBV genome when provided in trans, and one such mutant regains activity when directly fused to DDB1. Furthermore, DDB1 depletion by RNA interference specifically compromises replication of wild-type HBV, indicating that HBx produced from the viral genome also functions in a DDB1-dependent fashion. We also show that HBx in association with DDB1 acts in the nucleus and stimulates HBV replication mainly by enhancing viral mRNA levels, regardless of whether the protein is expressed from the HBV genome itself or supplied in trans. Interestingly, whereas HBx induces cell death in both HepG2 and Huh-7 hepatoma cell lines, it enhances HBV replication only in HepG2 cells, suggesting that the two activities involve distinct DDB1-dependent pathways. Chronic infection by hepatitis B virus (HBV) affects 350million people worldwide and is a major causative agent of liver diseases, including cirrhosis and hepatocellular carcinoma, one of the most common cancers in humans (27). HBV belongs to the Hepadnaviridae family of small enveloped DNA viruses that replicate primarily in the livers of their hosts and exhibit similarities to retroviruses. This family also contains rodent viruses, such as woodchuck hepatitis virus and ground squirrel hepatitis virus, as well as more distantly related members infecting avian species. While all mammalian hepadnaviruses cause liver cancer in their hosts, avian viruses do not (13).The main features of the hepadnavirus replication cycle are quite well understood (for a review, see reference 14). Upon infection, the 3.2-kb circular, partially double-stranded viral genome is transported into the cell nucleus, where it is converted into a covalently closed circular DNA. The covalently closed circular DNA serves as a template for transcription by host cell RNA polymerase II of four major viral RNA species, including the more than full-length pregenomic RNA. The pregenomic RNA is then reverse transcribed into DNA replicative intermediates in the cytoplasm within immature viral core particles by the virally encoded polymerase.

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

“…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). In addition, HBx interactions with the proteasome complex or DDB1 and HBx activation of Src tyrosine kinases have also been reported to participate in the regulation of HBV replication (Klein et al, 1999;Zhang et al, 2004;Leupin et al, 2005).…”

Hepatitis B virus X protein affects S phase progression leading to chromosome segregation defects by binding to damaged DNA binding protein 1

“…Green fluorescent protein (GFP), GFP-HBx, and GFP-HBx(R96E) expressed from EBS-PL 7 and from the lentiviral vector pWPTS, 6 and HA-DDB1 7 and myc-HBx 9 produced from pSRS have been described. Myc-HBx(R96E) was constructed by replacing the region encoding wild-type HBx in pSRS.…”

“…The HBx(R96E)-DDB1 and HBx(R96E)-DDB1(i947) fusions were expressed from EBS-PL. 9 The replicationcompetent, greater-than-unit-length wild-type HBV genomic DNA (payw1.2) and the HBx-deficient derivative (payw*7) 6 were recloned into pBluescript (Stratagene) upstream of a GFP gene derived from pEGFP-N1 (Clontech). Details of the constructs will be provided on request.…”

“…5 Previous work demonstrated that HBx stimulates HBV genome replication by binding to DDB1 in the nuclear compartment of cells. 6 A nuclear interaction with DDB1 is also required for HBx to interfere with cell viability. 7 A similar DDB1-binding dependent cytotoxic activity has been re-ported for the woodchuck HBx protein, 8 suggesting that this conserved feature is of potential physiological significance.…”

The G1 phase E3 ubiquitin ligase TRUSS that gets deregulated in human cancers is a novel substrate of the S-phase E3 ubiquitin ligase Skp2

“…[15][16][17] The HBx protein of Hepatitis B virus (HBV) is also known to bind DDB1 possibly to facilitate viral replication by extending the S phase of cell cycle. 18,19 Further, HBx is shown to stabilize c-Myc by disrupting the SCF Skp2 complex and interfering with its ubiquitination. 20 Although both TRUSS and Skp2 regulate the intracellular levels of c-Myc, it is not clear if both regulators work in synergy or act sequentially during different phases of the cell cycle.…”