Technical standards for hepatitis B virus X protein (HBx) research

Abstract

Chronic infection with hepatitis B virus (HBV) is a risk factor for developing hepatocellular carcinoma (HCC). The life cycle of HBV is complex and has been difficult to study because HBV does not infect cultured cells. The HBV regulatory X protein (HBx) controls the level of HBV replication and possesses an HCC cofactor role. Attempts to understand the mechanism(s) that underlie HBx effects on HBV replication and HBV-associated carcinogenesis have led to many reported HBx activities that are likely influenced by the assays used. This review summarizes experimental systems commonly used to study HBx functions, describes limitations of these experimental systems that should be considered, and suggests approaches for ensuring the biological relevance of HBx studies.

Figure 1. Organization of the HBV genome

The HBV genome is a partially double-stranded DNA. The central circle depicts the conventional numbering of the circular genome where positions 1 and 3182 usually represent a unique EcoR1 restriction site. Small differences in genome length among genotypes (and serotypes) exist; the ayw serotype is shown. The black circular lines represent the DNA genome with a completed negative-strand DNA (-strand) and a partially completed (dashed lines) positive-strand DNA (+ strand). Also indicated are: direct repeats (DR1, DR2), enhancers (EN1, EN2) and polymerase (orange circle labeled "pol"). Colored arrows indicate the ORFs for preCore, core, polymerase, envelope (preS1, pres2, and S), and HBx proteins. Outer black arrows depict genomic and subgenomic polyadenylated transcripts transcribed from cccDNA, also depicted at the bottom of the figure highlighting overlapping regions. Colored boxes below the circular genome indicate ORFs for HBV proteins, highlighting their overlapping regions.

Figure 2. The HBV lifecycle

HBV infects susceptible cells by interacting with its cell-surface receptor, the sodium taurocholate cotransporting polypeptide (NTCP), also known as SLC10A1. HBV enters the cell by an incompletely understood process, loses its envelope, and the capsid-enclosed genome is transported to the nucleus and releases the genome into the nucleus. In the nucleus, the partially double-stranded DNA genome (dsDNA) is repaired and converted to covalently closed, circular DNA (cccDNA), which is the template for HBV transcripts. HBV transcripts are transported to the cytoplasm and translated to produce HBV proteins. One of the transcripts, the pregenomic RNA is encapsidated and reverse-transcribed to the HBV DNA genome. Envelopment of genome-containing capsids occurs, and HBV is then released from the cell. Some of the encapsidated genome does not acquire an envelope and can be recycled back to the nucleus to contribute to the pool of nuclear cccDNA.

Figure 3. Schematic of the HBx protein (box)

HBx is a 154 amino acid, 17kD protein. Lines below the HBx schematic represent identified functional regions of HBx. A region that inhibits some HBx activities is located at the N-terminus of HBx (Negative). Regions required for binding to transcription factors (Transcription factor binding) and Damage DNA Binding Protein 1 (DDB1 binding) are indicated. A nuclear export signal (nuclear export) is also indicated. Comparative sequence analysis of HBx from multiple genotypes has identified a hypervariable region (Hypervariable). Asterisks (*) indicate conserved cysteines.