The quaternary structure, antigenicity, and aggregational behavior of the secretory core protein of human hepatitis B virus are determined by its signal sequence.

Human hepatitis B virus encodes a secretory core protein, referred to as the HBe protein, whose secretion is mediated by the pre-C signal sequence. Here we examined whether this sequence is important only for translocation of the HBe precursor (the precore protein) or whether it also contributes to the structural and biophysical properties of the mature HBe protein. When a truncated hepatitis B virus precore protein, lacking the basic C-terminal domain which is cleaved from the wild-type protein during its conversion into HBe, was expressed in human hepatoma cells, only a small amount of HBe-like protein was produced. This protein was slightly smaller than the wild-type HBe protein, suggesting that C-terminal cleavage of the precore protein does not occur at the suggested site. When the authentic signal sequence of the precore protein (the pre-C sequence) was replaced by the unrelated signal sequence of an influenza virus hemagglutinin, not only the full-length but also the C-terminally truncated protein was expressed and secreted with high efficiency. Western blot (immunoblot) analyses with nonreducing gels and conformation-specific monoclonal antibodies revealed that the HBe protein secreted under control of the pre-C signal sequence was a monomer with HBe antigenicity, whereas the HBe-like protein secreted under control of the hemagglutinin signal sequence was a disulfide-bridge-linked dimer with both HBe and HBc antigenicity. Electron microscopic examination of gradient-purified particulate core gene products showed that HBe protein secreted under control of the hemagglutinin signal sequence forms core particles, whereas HBe protein secreted under control of the pre-C sequence does not. Thus, the pre-C sequence not only mediates the secretion but also determines the structural and aggregational properties of the HBe protein.     

Hepatitis B virus core protein interacts with the C-terminal region of actin-binding protein

Hepatitis B viral core protein is present in the nucleus and cytoplasm of infected hepatocytes. There is a strong correlation between the intrahepatic distribution of core protein and the viral replication state and disease activity in patients with chronic hepatitis. To understand the role of core protein in the pathogenesis of HBV, we used a yeast two-hybrid system to search for cellular proteins interacting with the carboxyl terminus of core protein, as this region is involved in a number of important functions in the viral replication cycle including RNA packaging and DNA synthesis. A cDNA encoding the extreme C-terminal region of human actin-binding protein, ABP-276/278, was identified. This interaction was further confirmed both in vitro and in vivo. In addition, the extreme C-terminal region of ABP-276/278 interacted with the nearly full-length HBV core protein. Since this region is present in both the core and the precore proteins, it is likely that both core and precore proteins of HBV can interact with the C-terminal region of ABP-276/278. The minimal region of ABP-276/278 which interacted with the HBV core protein was the C-terminal 199 amino acid residues which correspond to part of the 23rd repeat, the entire 24th repeat and the intervening hinge II region in ABPs. The potential functional outcome of ABP interaction in HBV replication and its contribution to the pathological changes seen in patients with chronic HBV infection are discussed.     

The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ER-associated degradation pathway

The hepatitis B virus precore protein is closely related to the nucleocapsid core protein but is processed distinctly in the cell and plays a different role in the viral cycle. Precore is addressed to the endoplasmic reticulum (ER) through a signal peptide, and the form present in the ER is the P22 protein. P22 is then cleaved in its C-terminal part to be secreted as HBe antigen. In addition, a cytosolic form of 22 kDa less characterized has been observed. Precore gene was shown to be implicated in viral persistence, but until now, the actual protein species involved has not been determined. Our work focuses on the cytosolic form of precore. Using human cells expressing precore and a convenient fractionation assay, we demonstrated that the cytosolic form is identical to the ER form and retrotransported in the cytoplasm through the ER-associated degradation pathway. This cellular machinery translocates misfolded proteins to the cytoplasm, where they are ubiquitinated on lysine residues and degraded by proteasome. We showed that precore escapes proteasome due to its low lysine content and accumulates in the cytosol. The role of this retrotransport was investigated. In the presence of precore, we found a specific redistribution of the Grp78/BiP chaperone protein to cytosol and demonstrated a specific interaction between precore and Grp78/BiP. Altogether, these data support the idea that the hepatitis B virus develops a strategy to take advantage of the ER-associated degradation pathway, allowing distinct subcellular localization and probably distinct roles for the viral precore protein.     

Transport of hepatitis B virus precore protein into the nucleus after cleavage of its signal peptide.

The precore and core proteins of hepatitis B virus have identical deduced amino acid sequences other than a 29-residue amino-terminal extension (precore region) on the precore protein. The first 19 of these residues serve as a signal sequence to direct the precore protein to the endoplasmic reticulum, where they are cleaved off with formation of precore protein derivative P22 for secretion. In this report, we show that P22 can alternatively be transported into the nucleus following signal peptide cleavage. Experiments with deletion mutants indicated that this nuclear transport proceeds via the cytosol and is dependent on the amino-terminal portion of P22. Thus, the hepatitis B virus precore protein is a secreted, cytosolic, and nuclear protein.     

The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation.

Analysis of the serum of duck hepatitis B virus (DHBV)-infected ducks has revealed the presence of C-terminally truncated viral core proteins (e antigens). These proteins are glycosylated and therefore were not released from infected cells by lysis but rather by active secretion, indicating that the DHBV core protein can be synthesized alternatively as a cytoplasmic or a secretory protein. Transient expression of cloned wild-type DHBV DNA and of a specifically designed viral mutant in a human hepatoma cell line (Hep-G2) showed that the DHBV core gene promoter is active in differentiated human liver cells and that synthesis and secretion of the processed core proteins are dependent on the expression of the pre-C region, a small open reading frame which precedes the core gene. In addition, these experiments showed that the mechanism of core protein processing and secretion is conserved between DHBV and the human hepatitis B virus and therefore might be important for the hepatitis B virus life cycle in general. In spite of this, intrahepatic injection of the pre-C mutant into uninfected ducks resulted in viremia without concomitant e-antigen synthesis, indicating that virus formation is independent of pre-C expression.     

Relevance of cysteine residues for biosynthesis and antigenicity of human hepatitis B virus e protein.

The mature form of the secretory core protein (HBe protein) of human hepatitis B virus contains four cysteines which are located at amino acid positions -7, 48, 61, and 107 relative to the HBc start methionine. In addition, there is a cysteine, Cys-183, located in the C-terminal domain of the HBe precursor, which is cleaved during HBe maturation. Here, the significance of these cysteines for biosynthesis and antigenicity of the HBe protein was examined. The cysteines at positions -7 and 61 were found to be crucial for HBe biosynthesis. As has already been described, if the Cys at position -7 is mutated, disulfide-linked HBe homodimers which have both HBe antigenicity and HBc antigenicity are expressed. Here we show that these dimers are due to Cys-61-Cys-61 disulfide bridges which are formed only if the Cys at position -7 is not present. In the wild-type protein, this dimerization appears to be inhibited by formation of intramolecular disulfide bridges between the Cys at -7 and one of the internal cysteines. Moreover, Cys-61 is important for HBe biosynthesis in general since mutation of this amino acid results in production of HBe proteins which are either only poorly secreted or possess a different antigenicity.     

Point mutations upstream of hepatitis B virus core gene affect DNA replication at the step of core protein expression

The pregenomic RNA directs replication of the hepatitis B virus (HBV) genome by serving both as the messenger for core protein and polymerase and as the genome precursor following its packaging into the core particle. RNA packaging is mediated by a stem-loop structure present at its 5' end designated the epsilon signal, which includes the core gene initiator AUG. The precore RNA has a slightly extended 5' end to cover the entire precore region and, consequently, directs the translation of a precore/core protein, which is secreted as e antigen (HBeAg) following removal of precore-derived signal peptide and the carboxyl terminus. A naturally occurring G1862T mutation upstream of the core AUG affects the bulge of the epsilon signal and generates a "forbidden" residue at the -3 position of the signal peptide cleavage site. Transfection of this and other mutants into human hepatoma cells failed to prove their inhibition of HBeAg secretion but rather revealed great impairment of genome replication. This replication defect was associated with reduced expression of core protein and could be overcome by a G1899A covariation, or by nonsense or frameshift mutation in the precore region. All these mutations antagonized the G1862T mutation on core protein expression. Cotransfection of the G1862T mutant with a replication-deficient HBV genome that provides core protein in trans also restored genome replication. Consistent with our findings in cell culture, HBV genotype A found in African/Asian patients has T1862 and is associated with much lower viremia titers than the European subgroup of genotype A.     

The secretory core protein of human hepatitis B virus is expressed on the cell surface.

Human hepatitis B virus (HBV) causes acute and chronic liver disease, which can result in tumor formation. An as yet unexplained phenomenon is that virus elimination usually correlates with the development of antibodies directed against the HBeAg, a secretory HBV core gene product which can be detected in the serum of infected patients. Expression of HBeAg in a human hepatoma cell line by using recombinant vaccinia viruses revealed that the HBeAg is not only secreted from HBeAg-producing cells but also incorporated into the outer cell membrane. No membrane-expressed core gene product could be detected when the cytoplasmic core protein (HBcAg) was expressed. Immune sera from patients who developed anti-HBe antibodies efficiently recognized the membrane-bound HBeAg, suggesting that surface-expressed HBeAg can serve as a target for an antibody-mediated elimination of HBV-infected cells.     

The arginine-rich domain of hepatitis B virus precore and core proteins contains a signal for nuclear transport.

Precore and core proteins are two related co-carboxy-terminal proteins of hepatitis B virus. Precore protein contains the entire sequence of core protein plus an amino-terminal extension of 29 amino acid residues. Both proteins can display a common antigenic determinant known as core antigen (HBcAg). Clinically, HBcAg is detected in the nucleus, cytoplasm, or both of hepatitis B virus-infected hepatocytes. In order to understand the mechanism that regulates nuclear transport of HBcAg, various portions of precore and core proteins were linked to a reporter protein, human alpha-globin, and expressed in mammalian cells. Our results indicate that the precore protein-specific sequence, although important for nuclear transport, does not contain a nuclear localization signal. Instead, a signal for nuclear transport is located near the carboxy termini of precore and core proteins in the arginine-rich domain. This signal is made up of a set of two direct PRRRRSQS repeats and is highly conserved among mammalian hepadnaviruses.     

The duck hepatitis B virus core protein contains a highly phosphorylated C terminus that is essential for replication but not for RNA packaging.

In this report, we present biochemical and mutational analyses of the duck hepatitis B virus core protein (DHBcAg). The data show that duck hepatitis B virus core particles consist of at least four different proteins with sizes between 32 and 34 kilodaltons, all of which react with DHBcAg-specific antiserum. Most of the heterogeneity was found to be due to extensive phosphorylation of the DHBcAg C terminus. Bacterially synthesized DHBcAg was not phosphorylated, and mutations within the viral P gene did not influence phosphorylation, suggesting that the kinase activity is not encoded by the viral C or P gene. Removal of the last 12 C-terminal DHBcAg amino acids, which are at least in part located on the core particle surface, had only a minor effect on DHBcAg phosphorylation and did not interfere with packaging of the capsids into viral envelopes or with genome replication. However, an attempt to infect ducklings with this mutant failed. Removal of the last 36 C-terminal DHBcAg amino acids abolished core protein heterogeneity but did not prevent particle formation. Interestingly, these particles were defective in genome replication, although they could still package viral pregenomic RNA.     

Constrained evolution of overlapping genes in viral host adaptation: Acquisition of glycosylation motifs in hepadnaviral precore/core genes

Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (⁷⁵NIT⁷⁷ and ⁹⁰NDT⁹²) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes.     

Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity

Five new hepadnaviruses were cloned from exotic ducks and geese, including the Chiloe wigeon, mandarin duck, puna teal, Orinoco sheldgoose, and ashy-headed sheldgoose. Sequence comparisons revealed that all but the mandarin duck viruses were closely related to existing isolates of duck hepatitis B virus (DHBV), while mandarin duck virus clones were closely related to Ross goose hepatitis B virus. Nonetheless, the S protein, core protein, and functional domains of the Pol protein were highly conserved in all of the new isolates. The Chiloe wigeon and puna teal hepatitis B viruses, the two new isolates most closely related to DHBV, also lacked an AUG start codon at the beginning of their X open reading frame (ORF). But as previously reported for the heron, Ross goose, and stork hepatitis B viruses, an AUG codon was found near the beginning of the X ORF of the mandarin duck, Orinoco, and ashy-headed sheldgoose viruses. In all of the new isolates, the X ORF ended with a stop codon at the same position. All of the cloned viruses replicated when transfected into the LMH line of chicken hepatoma cells. Significant differences between the new isolates and between these and previously reported isolates were detected in the pre-S domain of the viral envelope protein, which is believed to determine viral host range. Despite this, all of the new isolates were infectious for primary cultures of Pekin duck hepatocytes, and infectivity in young Pekin ducks was demonstrated for all but the ashy-headed sheldgoose isolate.     

Preparation of monoclonal antibodies against duck hepatitis B core antigen and analysis of the monoclonal antibodies

Mice were immunized against duck hepatitis B virus core (DHBc) particles isolated from the liver of asymptomatic carrier ducks of duck hepatitis B virus (DHBV) by ultracentrifugation. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 12 clones of hybridoma cells secreting antibodies against DHBc (anti-DHBc) were isolated. According to the reactivity to core particles and core peptide obtained from DHBc particles treated with SDS-2ME, the 12 antibodies were classified into two groups. Two monoclonal antibodies reacted against both core particles and core peptide (B-type), the other ten monoclonal antibodies reacted against core particles but did not react against core peptide obtained from DHBc particles treated with SDS-2 ME. (A-type). Solid phase enzyme immuno assay (EIA) using these two types of antibodies could detect core antigenisity not only in the liver homogenate but also in the DHBV infected serum. Sucrose gradient analysis and gel filtration analysis revealed this DHBc antigenisity in the serum is not carried by core particles but carried by core peptide, equivalent to HBe antigen in the serum of Hepatitis B virus (HBV) carrier. This EIA may provide sensitive test monitoring both serum DHBe antigen levels and DHBc antigen levels in the liver during DHBV infection.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d