Three envelope proteins of hepatitis B virus: large S, middle S, and major S proteins needed for the formation of Dane particles.

The infectious particles of hepatitis B virus are called Dane particles and consist of viral nucleic acid encapsulated within a core particle that is enveloped by virus-coded surface proteins. The major S protein constitutes a significant fraction of these surface proteins. In addition, there are two other related proteins (large S and middle S), but their role in envelope formation has not yet been elucidated. We modified the translation initiation codon ATG of each of the envelope proteins by site-directed mutagenesis and found that mutant genomes that did not produce one or two of these proteins were unable to form Dane particles. The particles released into the culture medium by such mutants did not carry DNA. Synthesis of virus-coded RNA still occurred normally, and core particles carrying DNA accumulated intracellularly. The DNA in such core particles was mostly in the double-stranded open circular form, in contrast to the normal situation in which the particles contain mostly RNA and its complementary single-stranded DNA or else contain linear DNA that is partially single stranded and otherwise duplex. The role of the large S and middle S proteins in the formation of Dane particles is discussed.     

Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.     

The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly.

Assembly of replication-competent hepatitis B virus (HBV) nucleocapsids requires the interaction of the core protein, the P protein, and the RNA pregenome. The core protein contains an arginine-rich C-terminal domain which is dispensable for particle formation in heterologous expression systems. Using transient expression in HuH7 cells of a series of C-terminally truncated core proteins, I examined the functional role of this basic region in the context of a complete HBV genome. All variants containing at least the 144 N-terminal amino acids were assembly competent, but efficient pregenome encapsidation was observed only with variants consisting of 164 or more amino acids. These data indicate that one function of the arginine-rich region is to provide the interactions between core protein and RNA pregenome. However, in cores from the variant ending with amino acid 164, the production of complete positive-strand DNA was drastically reduced. Moreover, almost all positive-strand DNA originated from in situ priming, whereas in wild-type particles, this type of priming not supporting the formation of relaxed circular DNA (RC-DNA) accounted for about one half of the positive strands. Further C-terminal residues to position 173 restored RC-DNA formation, and the corresponding variant did not differ from the full-length core protein in all assays used. The observation that RNA encapsidation and formation of RC-DNA can be genetically separated suggests that the core protein, via its basic C-terminal region, also acts as an essential auxiliary component in HBV replication, possibly like a histone, or like a single-stranded-DNA-binding protein. In contrast to their importance for HBV replication, sequences beyond amino acid 164 were not required for the formation of enveloped virions. Since particles from variant 164 did not contain mature DNA genomes, a genome maturation signal is apparently not required for HBV nucleocapsid envelopment.     

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 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 Mechanism of an Immature Secretion Phenotype of a Highly Frequent Naturally Occurring Missense Mutation at Codon 97 of Human Hepatitis B Virus Core Antigen

A very frequent missense mutation at codon 97 of human hepatitis B virus (HBV) core antigen (HBcAg) has been found in chronic carriers worldwide. Functional characterization of this mutant revealed one intracellular and two extracellular phenotypes in contrast to wild-type HBV: (i) a 6- to 12-fold decrease in the level of the full-length relaxed circular DNA, a 4- to 5-fold decrease in the plus-strand DNA, and an approximately 1.8-fold decrease in the minus-strand and overall DNA levels in the intracellular viral core particles; (ii) a 5.7-fold increase in the immature secretion of Dane particles, containing minus-strand, single-stranded virion DNA; and (iii) a significant reduction of nonenveloped core particles in the medium. The steady-state levels of mutant and wild-type core proteins expressed from the same vector appeared to be similar. Using a complementation assay and gradient centrifugation analysis, we demonstrated that this mutant core protein alone is necessary and sufficient for immature secretion. The decreased level of intracellular HBV DNA is caused by both the cis defect of the mutant genome and the trans defect of the mutant core protein. We have dissected further the relationship between the intracellular and extracellular phenotypes of mutant F97L. The pleiotropic effects of the HBcAg codon 97 mutation were observed consistently in several different experimental settings. The mechanism and biological significance of these findings are discussed.     

血源乙肝疫苗的电镜制样方法

用作样品支持物喷碳的铜网,磷钨酸染色制备血源乙肝疫苗电镜检测样品。４９３批血源乙肝疫苗的镜检结果显示：经溴化钾和蔗糖离心后,原料血浆中的Ｄａｎｅ颗粒和管形颗粒已去除,只观察到２２－２４纳米的ＨＢｓＡｇ球形颗粒。     

C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich (167)RRRSQSPRR(175) Domain Is Critical for HBV Replication

To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221-262 amino acids of DHBV C protein, in place of 146-185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221-241 and 251-262 amino acids of DHBV C, in place of HBV C 146-166 and 176-185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242-250 of DHBV C ((242)RAGSPLPRS(250)) introduced in place of 167-175 of HBV C ((167)RRRSQSPRR(175)) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich (167)RRRSQSPRR(175) domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important.     

Native hepatitis B virions and capsids visualized by electron cryomicroscopy

Hepatitis B virus (HBV) infects more than 350 million people, of which one million will die every year. The infectious virion is an enveloped capsid containing the viral polymerase and double-stranded DNA genome. The structure of the capsid assembled in vitro from expressed core protein has been studied intensively. However, little is known about the structure and assembly of native capsids present in infected cells, and even less is known about the structure of mature virions. We used electron cryomicroscopy (cryo-EM) and image analysis to examine HBV virions (Dane particles) isolated from patient serum and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice. Both types of capsids assembled as icosahedral particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope were attributed to surface glycoproteins. Their packing was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.     

Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus.

Phosphorylation of core particles derived either from hepatitis B viruses or from livers of hepatitis B-infected individuals has been long recognized, but the nature and function of the phosphorylating enzyme remained unknown. By immunoblotting with a monoclonal antibody, we have now detected protein kinase C within the liver-derived core particles. To study the significance of the encapsidated protein kinase C for the viral life cycle, we established an in vitro assembly system consisting of Escherichia coli-expressed core protein, protein kinase C, and in vitro-synthesized hepatitis B virus RNA. Phosphorylation of the core protein led to a reduced RNA encapsidation capacity of the core particles. Furthermore, RNA and protein kinase C competed for their target sequence, which is the carboxy-terminal arginine-rich domain of the core protein. This finding implies that phosphorylation of the nucleic acid binding site in the core protein occurs within the particles after encapsidation of protein kinase C, pregenomic RNA, and viral polymerase at a later step during viral genome maturation.     

Cytokine induction by the hepatitis B virus capsid in macrophages is facilitated by membrane heparan sulfate and involves TLR2

The hepatitis B virus (HBV) core Ag (HBcAg) serves as the structural subunit of the highly immunogenic capsid shell. HBcAg harbors a unique arginine-rich C terminus that was implicated in immune responses induced by the capsid. In this study, we examined the capacity of the HBV capsid to induce proinflammatory and regulatory cytokines in human THP-1 macrophages and the possible underlying mechanism. Full-length HBc capsids, but not HBc-144 capsids lacking the arginine-rich domain of HBcAg, efficiently bound differentiated THP-1 macrophages and strongly induced TNF-alpha, IL-6, and IL-12p40. Capsid binding to macrophages and cytokine induction were independent of the RNA associated with the arginine-rich domain. Soluble heparin and heparan sulfate but not chondroitin sulfates greatly diminished cytokine induction through inhibition of capsid binding to THP-1 macrophages. Furthermore, serine phosphorylation in the arginine-rich domain modulates capsid binding to macrophages and the cytokine response. Induction of cytokines by the capsid involved activation of NF-kappaB, ERK-1/2, and p38 MAPK and did not require endosomal acidification. Finally, NF-kappaB activation by the capsid in HEK 293 cells specifically required expression of TLR2 and was compromised by soluble heparin. Thus, cytokine induction by the HBV capsid in macrophages is facilitated by interaction of its arginine-rich domain with membrane heparan sulfate and involves signaling through TLR2.     

Immune tolerance split between hepatitis B virus precore and core proteins

The function of the hepatitis B virus (HBV) precore or HBeAg is largely unknown because it is not required for viral assembly, infection, or replication. However, the HBeAg does appear to play a role in viral persistence. It has been suggested that the HBeAg may promote HBV chronicity by functioning as an immunoregulatory protein. As a model of chronic HBeAg exposure and to examine the tolerogenic potential of the HBV precore and core (HBcAg) proteins, HBc/HBeAg-transgenic (Tg) mice crossed with T cell receptor (TCR)-Tg mice expressing receptors for the HBc/HBeAgs (i.e., TCR-antigen double-Tg pairs) were produced. This study revealed three phenotypes of HBe/HBcAg-specific T-cell tolerance: (i) profound T-cell tolerance most likely mediated by clonal deletion, (ii) T-cell clonal ignorance, and (iii) nondeletional T-cell tolerance mediated by clonal anergy and dependent on the structure, location, and concentration of the tolerogen. The secreted HBeAg is significantly more efficient than the intracellular HBcAg at eliciting T-cell tolerance. The split T-cell tolerance between the HBeAg and the HBcAg and the clonal heterogeneity of HBc/HBeAg-specific T-cell tolerance may have significant implications for natural HBV infection and especially for precore-negative chronic hepatitis.     

Identification of two separable modules in the duck hepatitis B virus core protein.

Hepadnavirus replication requires the concerted action of the polymerase and core proteins to ensure packaging of the RNA pregenome and DNA maturation. The arginine-rich C terminus of the core protein plays an essential role in both of these steps while being dispensable for nucleocapsid formation. In an attempt to identify other functional domains of the core protein, we performed a series of trans-complementation experiments analyzing the ability of duck and human hepatitis B virus (DHBV and HBV) core protein subunits to support the replication of a core-defective DHBV genome. Plasmids expressing the N-terminal amino acids 1 to 67 or the remaining C-terminal portion, amino acids 67 to 262, of the DHBV core protein were cotransfected into LMH cells along with a replication-deficient construct coding for the DHBV pregenome and polymerase. Neither the N nor the C terminus alone yielded replication-competent core particles. However, cotransfection of plasmids that separately expressed both regions restored a normal replication pattern. Furthermore, the DHBV C terminus but not the N terminus could be replaced by the corresponding domain of the HBV core protein in this assay. Finally, coexpression of the complete HBV core protein and the N terminus from DHBV resulted in DHBV replication, while the HBV core protein alone was not functional. Taken together, these findings suggest a modular organization of the DHBV core protein in which the C terminus is functionally conserved among different hepadnaviruses.     

The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice.

In this study, we examined the ability of the hepatitis B virus (HBV) precore, envelope, and X gene products to modulate HBV replication in the livers of transgenic mice that replicate the virus. Hepatic HBV replication was not affected by overexpression of the envelope or X gene products when these animals were crossed with transgenic mice that express the corresponding viral genes in the hepatocyte. Overexpression of the precore protein, however, eliminated nucleocapsid particles from the cytoplasm of the hepatocytes and abolished HBV replication without affecting the hepatic steady-state content of pregenomic HBV RNA. These observations suggest that the precore protein can exert a dominant negative effect on HBV replication, presumably at the level of nucleocapsid particle maturation or stability, suggesting an important role for this enigmatic viral protein in the HBV life cycle.     

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