Cloning and characterization of a novel hepatitis B virus x binding protein that inhibits viral replication.

M Melegari,J R Wands,P P Scaglioni

doi:10.1128/jvi.72.3.1737-1743.1998

Abstract

The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. Expression of the protein product (HBx) appears necessary for establishment of natural infection. However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters. Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements. Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus. In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression. We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.

Highlights

The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x
The mammalian hepadnaviruses encode an HBV x protein (HBx) protein that may function as a weak transactivator of a variety of viral and cellular promoter and enhancer elements
The ability of HBx to alter signaling pathways involved in cellular growth and differentiation as well as apoptosis has led to the hypothesis that continuous low-level HBx expression due to persistent viral infection of the liver may contribute to the development of hepatocellular carcinoma (HCC)

Summary

Introduction

The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. Regarding the viral life cycle, in the related woodchuck hepatitis virus (WHV), ablation of the WHx start codon or creation of C-terminal truncations has been found to decrease viral replication in vitro [34] and to inhibit the establishment of productive infection in vivo [7, 34] It is not known whether such cellular pathways influenced by HBx expression are needed or sufficient to establish an environment required for viral replication. The function of XIP was further investigated in the context of the HBx transactivation properties and effect on viral replication

Methods

Results

Conclusion