Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection

Background & Aims

The molecular biology of hepatitis B virus (HBV) has been extensively studied but the exact role of the hepatitis B X protein (HBx) in the context of natural HBV infections remains unknown.

Methods

Primary human hepatocytes and differentiated HepaRG cells allowing conditional trans complementation of HBx were infected with wild type (HBV(wt)) or HBx deficient (HBV(x−)) HBV particles and establishment of HBV replication was followed.

Results

We observed that cells inoculated with HBx-deficient HBV particles (HBV(x−)) did not lead to productive HBV infection contrary to cells inoculated with wild type HBV particles (HBV(wt)). Although equal amounts of nuclear covalently closed circular HBV-DNA (cccDNA) demonstrated comparable uptake and nuclear import, active transcription was only observed from HBV(wt) genomes. Trans-complementation of HBx was able to rescue transcription from the HBV(x−) genome and led to antigen and virion secretion, even weeks after infection. Constant expression of HBx was necessary to maintain HBV antigen expression and replication. Finally, we demonstrated that HBx is not packaged into virions during assembly but is expressed after infection within the new host cell to allow epigenetic control of HBV transcription from cccDNA.

Conclusions

Our results demonstrate that HBx is required to initiate and maintain HBV replication and highlight HBx as the key regulator during the natural infection process.

Introduction

HBV is a small, enveloped DNA virus which replicates its genome via an RNA intermediate. The encapsidated viral genome consists of a 3.2 kb partially double stranded relaxed circular DNA (rcDNA) molecule. Upon translocation to the nucleus, the rc genome is converted into cccDNA which serves as template for viral transcription. The 3.5 kb pre-genomic RNA (pgRNA) is the mRNA for the synthesis of polymerase and core proteins but also the template for the reverse transcription. The 2.4/2.1 kb subgenomic RNAs encode for three viral envelope proteins. In the cytoplasm, pgRNA is encapsidated and reverse transcribed within the viral capsid into the rcDNA genome. Mature viral capsids are then either directed to the secretory pathway for envelopment or redirected toward the nucleus to establish a cccDNA pool.

Beside structural proteins, HBV encodes for two non-structural proteins – HBe and HBx – of less defined functions. The secreted HBe (also named HBeAg) is supposed to be immunoregulatory [8], whereas HBx seems to interact with various cellular partners and modify diverse cellular processes [2]. Its exact role in the viral life cycle, however, has not been defined yet.

It has been shown in the woodchuck model of HBV infection that woodchuck hepatitis B virus (WHV) X protein is needed to establish productive infection in the animal [7], [38]. Low level replication in vivo allowed genotypic reversions to wild type WHV [37] pointing at the importance of WHV X. The importance of the human HBx in the context of HBV infection was demonstrated recently using human hepatocyte chimeric mice. HBx deficient HBV developed measurable viremia only in HBx-expressing livers [31]. In addition, it was shown that HBx-deficient HBV genomes are strongly attenuated for HBV replication using HBV transgenic mice [35], hydrodynamically-injected mice [15], [16], or cell culture models [1], [3], [15], [18]. Whereas HBx-deficiency had little effect on HBV replication in human hepatoma Huh7 cells, replication was impaired in HepG2 cells [3], [15], [16], [18].

Different functions have been attributed to HBx regarding HBV replication. Several studies indicated that HBx stimulates HBV replication by activating viral transcription [18], [30], [36], [37] and enhancing viral polymerase activity via calcium signaling pathways [5], [6], [17]. HBx may also enhance pgRNA encapsidation by increasing phosphorylation of the viral core protein [20]. In primary rat hepatocytes, HBx seems to regulate cell cycle progression and thereby stimulate HBV replication [10]. Although these studies suggested pleiotropic functions of HBx using transfection or transduction of HBV genomes, none of them assessed the role for HBx in the setting of a complete infection cycle. Moreover, contradictory results obtained in surrogate models highlight the importance of performing experiments in the context of an HBV infection that allows studying the complete life cycle.

For years, freshly prepared primary human hepatocytes (PHH) were the only cells known to support HBV infection [11]. However, the access to human livers is restricted and the quality of cells varies. More recently, the discovery of the HepaRG cell line, which supports HBV infection and replication, has opened new perspectives. Specificity of in vitro HBV infection has been demonstrated using neutralizing antibodies and HBV envelope-derived peptides [12], [13]. HepaRG cells are bi-potent liver progenitor cells that may be differentiated into both biliary epithelial- and hepatocyte-like cells [21]. HepaRG cells are capable of long-term stable expression of liver-specific metabolizing enzymes and membrane transporters – both indicators of highly differentiated hepatocytes closely correlating with susceptibility to HBV infection [12], [25].

Using the two relevant in vitro models of HBV infection, we showed here that HBx is essential for productive HBV infection when the natural HBV transcription template – cccDNA – has been established.