Virus Precore Protein Research Articles

Conserved aromatic residues of the hepatitis B virus Precore propeptide are involved in a switch between distinct dimeric conformations and essential in the formation of heterocapsids

The Hepatitis B virus Precore protein, present in the secretory pathway as the HBeAg precursor, can associate in the cytoplasm with the Core protein to form heterocapsids, likely to favor viral persistence. Core and Precore proteins share their primary sequence except for ten additional aminoacids at the N-terminus of Precore. To address the role of this propeptide sequence in the formation of Precore heterocapsids, we designed a Precore mutant in which the two propeptide tryptophans are replaced by glycines. This mutant retains the properties of the wild-type Precore, notably cell trafficking and ability to interact with Core. However, it is not incorporated into heterocapsids and forms stable dimers distinct from the labile HBe dimers and the presumably Core-like dimers assembled into heterocapsids. Our data highlights the essential role of Precore׳s propeptide in switching between different conformations for different functions and pinpoint the propeptide Tryptophan residues as central in these properties.

Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody

The Hepatitis B virus precore protein is processed in the endoplasmic reticulum (ER) into secreted hepatitis B e antigen (HBeAg), which acts as an immune tolerogen to establish chronic infection. Downregulation of secreted HBeAg should improve clinical outcome, as patients who effectively respond to current treatments (IFN-α) have significantly lower serum HBeAg levels. Here, we describe a novel reagent, a single variable domain (VNAR) of the shark immunoglobulin new antigen receptor (IgNAR) antibodies. VNARs possess advantages in stability, size (~14kDa) and cryptic epitope recognition compared to conventional antibodies. The VNAR domain displayed biologically useful affinity for recombinant and native HBeAg, and recognised a unique conformational epitope. To assess therapeutic potential in targeting intracellular precore protein to reduce secreted HBeAg, the VNAR was engineered for ER-targeted in vitro delivery to function as an intracellular antibody (intrabody). In vitro data from HBV/precore hepatocyte cell lines demonstrated effective intrabody regulation of precore/HBeAg.

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.

Hepatitis B virus precore protein augments genetic immunizations of the truncated hepatitis C virus core in BALB/c mice

DNA immunization has been used to induce either humoral or cellular immune responses against many antigens, including hepatitis C virus (HCV). In addition, DNA immunizations can be enhanced or modulated at the nucleotide level. Genetic immunizations were examined in BALB/c mice through the use of plasmids and chimeric DNA constructs encoding HCV core proteins and hepatitis B virus (HBV) precore (preC) regions. Plasmids encoding the truncated HCV core induced potent humoral and cellular responses to HCV; pcDNA3.0A-C154 produced a stronger antibody response than pcDNA3.0A-C191 (P < 0.01) and pcDNA3.0A-C69 (P < 0.05). HBV preC enhanced the humoral and cellular immune responses of BALB/c mice to HCV; however, pcDNA3.0A-C69preC resulted in a weak cytotoxic T lymphocyte (CTL) response. In addition, the humoral and cellular immune responses to HCV of groups immunized with pcDNA3.0A-C154preC and pcDNA3.0A-C191preC plasmids were higher than those of groups immunized with pcDNA3.0A-C154 and pcDNA3.0A-C191. In vivo CTL responses verified that mice immunized with preC core fused DNAs showed significantly high specific lysis compared with mice immunized with HCV cores only (P < 0.01). In our study, pcDNA3.0A-C154preC led to the highest immune response among all DNA constructs. DNA that encodes truncated HCV core proteins may lead to increased immune responses in vivo, and these responses may be enhanced by HBV preC.

A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen.

A unique characteristic of the hepatitis B virus is the production of a secreted form (precore or HBeAg) of the structural nucleocapsid (core or HBcAg). By using T cell receptor (TCR) transgenic (Tg) and TCR x HBc/HBeAg double- and triple-Tg pairs, we demonstrate that HBeAg elicits T cell tolerance, whereas HBcAg is nontolerogenic in this system. In fact, TCR x HBc double-Tg mice spontaneously seroconvert to IgG anti-HBc positivity at an early age. However, the presence of HBeAg in the serum of TCR x HBc x HBe triple-Tg mice prevents anti-HBc seroconversion. HBeAg mediates its immunoregulatory effect by eliciting tolerance in HBc/HBeAg-specific T cells. The results suggest that hepadnaviruses have retained a secretory form of the nucleoprotein because it functions as a T cell tolerogen and regulates the immune response to the intracellular nucleocapsid. This HBeAg-mediated immune regulation may predispose to chronicity during perinatal infections and prevent severe liver injury during adult infections.

Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells*

Abstract A novel tetracydine regulation expression system was used to regulate the expression of enhanced green fluorescent protein EGFP and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

Controlled expression of enhanced green fluorescent protein and hepatitis b virus precore protein in mammalian cells

Controlled expression of enhanced green fluorescent protein and hepatitis b virus precore protein in mammalian cells

Characterization of nuclear localization of a hepatitis B virus precore protein derivative P22.

Both of hepatitis B virus core protein and a precore protein derivative, named P22, have been shown to localize in the nucleus. Although P22 has ten additional amino acid residues at its amino-terminus, both proteins contain the same nuclear localization signal. In order to understand the mechanism that regulates the activity of this signal, we have studied the nuclear localization of P22 and compared it with that of core protein. It was found that both cytosolic and nuclear fractions of P22 were phosphorylated but to a lesser extent when compared with cytosolic core protein. This distinction was likely attributed to different conformations between these two proteins since the density gradient analysis revealed a different particle formation for P22 in the cytosol. When expressed in Vero cells synchronized by serum deprivation, P22 remained in the cytosol during G0 and G1 phases, accumulated gradually in the nucleus during S phase, and largely localized in the nucleus when cells were confluent. On the other hand, the core protein was transported into the nucleus during mid-G1 phase, shuttled back to the cytosol in S phase and again accumulated in the nucleus when cells were confluent. Interestingly, when aphidicolin was used to arrest the cells in late G1 phase, both proteins were found to accumulate in the nuclei. These results indicated that although both P22 and core proteins possessed the same nuclear localization signal, the cellular regulation of their nuclear transport was not identical and might involve different molecular mechanisms.

Importance of the C terminus of the hepatitis B virus precore protein in secretion of HBe antigen.

The hepatitis B virus (HBV) e antigen (HBeAg) is a 15 kDa soluble antigen derived from a precursor protein (precore protein) by two processing events, cleavage of the N-terminal signal peptide and cleavage of the C-terminal 34 amino acids. So far, the role of the C-terminal sequences in secretion has not been analysed in full. In this study deletion of the last 60 amino acids was found to abrogate HBeAg secretion whereas deletions of the last 10, 25 or 39 amino acids decreased its secretion rate. These data demonstrate that C-terminal precore protein sequences are crucial for HBe secretion and determine its secretion rate.

ChemInform Abstract: Synthesis of Hepatitis B Virus Precore Protein (13‐29) Fragment and Evaluation of Its Immunogenicity.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.

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.

Intrahepatic expression of HBcAg in chronic HBV hepatitis: Lessons from molecular biology

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.

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.

Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm.

The major hepatitis B virus (HBV) core protein is a viral structural protein involved in nucleic acid binding. Its coding sequence contains an extension of 29 codons (the "precore" region) at the amino terminus of the protein which is present in a fraction of the viral transcripts. This region is evolutionarily conserved among mammalian and avian HBVs, suggesting it has functional importance, although at least for duck HBV it has been shown to be nonessential for replication of infectious virions. Using in vitro assays for protein translocation across the endoplasmic reticulum membrane, we found that the precore region of the HBV genome encodes a signal sequence. This signal sequence was recognized by signal recognition particle, which targeted the nascent precore protein to the endoplasmic reticulum membrane with efficiencies comparable to those of other mammalian secretory proteins. A 19-amino acid signal peptide was removed by signal peptidase on the lumenal side of the microsomal membrane, generating a protein similar to the HBV major core protein, but containing 10 additional amino acids from the precore region at its amino terminus. Surprisingly, we found that 70-80% of this signal peptidase-cleaved product was localized on the cytoplasmic side of the microsomal vesicles and was not associated with the membranes. We conclude that translocation was aborted by an unknown mechanism, then the protein disengaged from the translocation machinery and was released back into the cytoplasm. Thus, a cytoplasmically disposed protein was created whose amino terminus resulted from signal peptidase cleavage. The remaining 20-30% appeared to be completely translocated into the lumen of the microsomes. A deletion mutant lacking the carboxy-terminal nucleic acid binding domain of the precore protein was similarly partitioned between the lumen of the microsomes and the cytoplasmic compartment, indicating that this highly charged domain is not responsible for the aborted translocation. We discuss the implications of our findings for the protein translocation process and suggest a possible role in the virus life cycle.