Polypeptides of hepatitis B virus surface antigen produced by a hepatoma cell line.

The PLC/PRF/5 cell line derived from a human hepatoma produces hepatitis B surface antigen (HBsAg) in 22-nm particles of the same buoyant density as those found in the serum of infected patients. The HBsAg particles from this cell line were labeled with [35S]methionine and purified, and the polypeptides were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with those of serum-derived particles. The two major polypeptides of serum-derived HBsAg particles (p20 and p23) were found in the same relative amounts in the particles from the cell line. The three smallest of the five minor components observed in HBsAg particles from serum were present in particles from the cell line. These polypeptides (p31, p36, and p43), as well as p20 and p23, were precipitated with anti-HBs-containing serum. The two largest polypeptides of serum particles (p49 and p66) were not detected in particles from these cells. When the PLC/PRF/5 HBsAg particles were radiolabeled with tritiated sugars, p23, and not p20, was found to contain radioactivity, indicating that the pattern of polypeptide glycosylation is similar to that of serum HBsAg. None of the other possible gene products of hepatitis B virus was detected in the PLC/PRF/5-derived HBsAg particles, in the cells, or in the cell supernatants.     

Structural relationships between the surface antigens of ground squirrel hepatitis virus and human hepatitis B virus.

Several physical, chemical, and serological properties of surface antigen particles from ground squirrel hepatitis virus (GSHsAg) and human hepatitis B virus (HBsAg) were compared. GSHsAg and HBsAg particles were purified from positive sera by gel chromatography and isopycnic centrifugation. Both antigens consisted mainly of spherical particles with an average diameter of approximately 20 nm and a buoyant density in CsCl of approximately 1.19 g/ml. Their UV absorption spectra indicated the presence of more tryptophane than tyrosine and the absence of detectable nucleic acid. GSHsAg was found to contain two major polypeptides of approximately 23,000 and 27,000 daltons, with electrophoretic migration rates distinctly faster than those of the two major polypeptides of HBsAg particles. After radiolabeling of purified antigen preparations with Bolton-Hunter reagent, the two major polypeptides of GSHsAg showed almost identical tryptic peptide maps. The tryptic peptide map of the major polypeptide from GSHsAg contained 13 of 37 spots also present in the map of the major HBsAg polypeptide, and 13 of 27 spots in the map of the major HBsAg polypeptide were also present in the map of the major GSHsAg polypeptide. This suggests considerable sequence homology between the major surface antigen polypeptides of the two viruses. However, there was only a weak serological cross-reactivity between antigens of the two viruses. Using an anti-HBs-containing serum with a relatively strong cross-reactivity, GSHsAg was found to consist of at least two antigenically different subspecies. The more strongly cross-reacting from had a slightly higher buoyant density than the other antigenic form.     

Antigenicity of the major polypeptides of hepatitis B surface antigen (HBsAg).

The major polypeptides (P-1, P-2, and P-6) of HBsAg were isolated from purified preparations of 22-nm HBsAg particles, iodinated, and analyzed by double-antibody radioimmunoprecipitation assays for the presence of hepatitis B virus (HBV)-specific antigens. Each polypeptide fraction contained both group (a) and subtype (d) specific determinants in common by virtue of their immunoreaction with antiserum to native HBsAg and antisera to the other structural polypeptides. The antigenic and structural similarities of the HBsAg polypeptides establish that they are not each unique gene products of the HBV genome.     

Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame.

The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for beta-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinants of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42- and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.     

Mutational analysis of hepatitis B surface antigen particle assembly and secretion.

Cells infected with hepatitis B virus produce both virions and 20-nm subviral (surface antigen or HBsAg) particles; the latter are composed of viral envelope proteins and host-derived lipid. Although hepatitis B virus encodes three envelope proteins (L, M, and S), all of the information required to produce an HBsAg particle resides within the S protein. This polypeptide spans the bilayer at least twice and contains three hydrophobic regions, two of which are known to harbor topogenic signal sequences that direct this transmembrane orientation. We have examined the effects of mutations in these and other regions of the S protein on particle assembly and export. Lesions in the N terminal signal sequence (signal I) can still insert into the endoplasmic reticulum bilayer but do not participate in any of the subsequent steps in assembly. Deletion of the major internal signal (signal II) completely destabilizes the chain. Deletion of the C-terminal hydrophobic domain results in a stable, glycosylated, but nonsecreted chain. However, when coexpressed with wild-type S protein this mutant polypeptide can be incorporated into particles and secreted, indicating that the chain is still competent for some of the distal steps in particle assembly. The correct transmembrane disposition of the N terminus of the molecule is important for particle formation: addition of a heterologous (globin) domain to this region impairs secretion, but the defect can be corrected by provision of an N-terminal signal sequence that restores the proper topology of this region. The resulting chimeric chain is assembled into subviral particles that are secreted with normal efficiency.     

Structure of the Mouse Mammary Tumor Virus: Characterization of Bald Particles

The polypeptide, antigenic, and morphological structure of the mouse mammary tumor virus was studied following protease digestion of intact virions. Intact, untreated virions (rho = 1.17 g/ml) had characteristic envelope spikes, five major polypeptides, and were precipitated by antisera against gp52. Two of the major polypeptides, with molecular weights of 52,000 (gp52) and 36,000 (gp36), had carbohydrate moieties. Protease treatment resulted in spikeless, "bald" particles (rho = 1.14 g/ml), which had altered surface antigenicity and which contained neither gp52 nor gp36. These data indicated that gp52 and gp36 were on the viral envelope. Bald particles retained a 28,000 dalton polypeptide (p28) which was proposed as the major internal polypeptide.     

Major polypeptide of duck hepatitis B surface antigen particles

The 40- to 50-nm pleomorphic particles found in the sera of domestic Pekin ducks infected with duck hepatitis B virus were purified by rate zonal and isopycnic centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide analysis of these particles, called duck hepatitis B surface antigen particles, revealed the major component to be a single 17,500-dalton polypeptide. This result is in contrast to polypeptide analyses of the surface antigens of related mammalian viruses, including hepatitis B, in which a major doublet of polypeptides is seen with molecular weights ranging from 23,000 to 29,000. Tryptic maps of 17,500-dalton polypeptide resembled that of the major non-glycosylated polypeptide of the adw subtype of hepatitis B surface antigen. A serological assay for antibody to the purified duck virus particles is also described.     

Insertions in the hepatitis B surface antigen. Effect on assembly and secretion of 22-nm particles from mammalian cells

The envelope protein of hepatitis B virus carrying the surface antigen, HBsAg, has the unique property of mobilizing cellular lipids into spherical or elongated particles, about 22 nm in diameter, which are secreted from mammalian cells. We have created mutant envelope proteins by insertion of various sequences of different lengths into two regions of the S gene encoding the major envelope protein. S genes carrying inserts in phase with HBsAg were expressed in mouse L cells from the simian virus 40 early promoter. Various single or double inserts in the two major hydrophilic domains of HBsAg were compatible with secretion of 22-nm particles. In all mutant envelope proteins studied, the HBsAg domains required for intracellular aggregation appeared to be intact. However, assembly into particles was not sufficient to assure transport into the extracellular space. The 22-nm HBsAg particle may be a useful vehicle for the export and presentation of foreign peptide sequences.     

Antigenic and structural relationships of the surface antigens of hepatitis B virus, ground squirrel hepatitis virus, and woodchuck hepatitis virus.

The surface antigens of human hepatitis B (HBsAg), ground squirrel hepatitis (GSHsAg), and woodchuck hepatitis (WHsAg) viruses were compared serologically, and their major polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping. Results showed that both GSHsAg and WHsAg are antigenically cross-reactive, that their major pairs of polypeptides have identical mobilities on sodium dodecyl sulfate gels, and that the major polypeptides of GSHsAg and WHsAg migrate faster in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than do the corresponding bands of HBsAg. The peptide maps of the major (P-22) surface antigen polypeptides of GSHsAg and WHsAg showed that they shared over half of their spots. Peptide mapping of HBsAg subtypes indicated a close relationship between the major polypeptides (P-24) of adw and adr and a more distal relationship to ayw. Only about 25% of the spots shared by the combined HBsAg subtypes were also found in the peptide maps of GSHsAg and WHsAg, indicating at least some structural homology among the major polypeptides of the human and animal virus surface antigen particles. This is also reflected in the serological cross-reactivity among HBsAg, GSHsAg, and WHsAg. Further, the detection of ground squirrel and woodchuck antigens by Ausria II radioimmunoassay, combined with peptide mapping data indicating the common origin of these viruses, suggests that the common a determinant is shared by each and is restricted to approximately 25% of the sequences in their major polypeptides.     

A 49,000-dalton polypeptide bearing all antigenic determinants and full immunogenicity of 22-nm hepatitis B surface antigen particles

Spherical 22-nm hepatitis B surface antigen (HBsAg) particles with a subtype adr were purified from plasma of asymptomatic carriers of hepatitis B virus. When purified HBsAg preparation was treated with sodium dodecylsulfate in the absence of reducing agents, it yielded spherical particles with a diameter smaller than 22 nm, and in addition, a polypeptide with a molecular size of 49,000 daltons, which seemed to constitute the outer coat of HBsAg particles. The recovery of the polypeptide on the basis of optical density at 280 nm was 2%, starting from 22-nm HBsAg particles. The 49,000-dalton polypeptide apparently represented a structural unit of the surface of HBsAg particles, since it bore all common (a, Re) and subtypic (d, r) determinants with essentially the same antigenic titers as intact HBsAg particles. Furthermore, this polypeptide was equally immunogenic as 22-nm HBsAg particles in raising corresponding antibodies in mice. When the 49,000-dalton polypeptide was reduced in the presence of 2-mercaptoethanol, it cleaved into 22,000- and 27,000-dalton polypeptides with a drastic decrease in both antigenicity and immunogenicity. These results indicate the different molecular arrangements between outer coat and inner portion of HBsAg particles, and a potential application of the 49,000-dalton polypeptide as a component vaccine, owing to its strong antigenicity both in vitro and in vivo.     

Membrane structure of the hepatitis B virus surface antigen particle

Expression of S protein, an envelope protein of hepatitis B virus, in the absence of other viral proteins, leads to the secretion of hepatitis B virus surface antigen (HBsAg) particles that are formed by budding from the endoplasmic reticulum membranes. The HBsAg particles produced by mouse fibroblast cells show a unique lipid composition, with 1,2-diacyl glycerophosphocholine being the dominant component. The lipid organization of the HBsAg particles was studied by measuring electron spin resonance (ESR) using various spin-labeled fatty acids, and the results were compared with a parallel study on HVJ (Sendai virus) and vesicles reconstituted with total lipids of the HBsAg particles (HBs-lipid vesicles). HVJ and the HBs-lipid vesicles showed typical ESR spectra of lipids arranged in a lipid bilayer structure. In contrast, the ESR spectra obtained with the HBsAg particles showed that the movement of lipids in the particle is severely restricted and a typical immobilized signal characteristic of tight lipid-protein interactions was also evident. Phosphatidylcholine (PC) in the HBsAg particles was not exchangeable by a PC-specific exchange protein purified from bovine liver, while phospholipase A(2) from Naja naja vemon was able to hydrolyze all the PC in the particles. These analyses suggest that the lipids in the HBsAg particles are not organized in a typical lipid bilayer structure, but are located at the surface of the particles and are in a highly immobilized state. Based on these observations we propose a unique lipid assembly and membrane structure model for HBsAg particles.     

Intracellular assembly and packaging of hepatitis B surface antigen particles occur in the endoplasmic reticulum.

Hepatitis B surface antigen (HBsAg) particles are secreted by Chinese hamster ovary cells that are stably transfected with the S gene of hepatitis B virus. The assembly of HBsAg into cylindrical and spherical particles occurred intracellularly within the endoplasmic reticulum. HBsAg particles accumulated within large dilated areas of the endoplasmic reticulum and remained within these structures for most of the time prior to secretion from the cells. Once the particles were formed, the HBsAg polypeptides did not appear to become associated with subsequent intracellular organelle membranes or the plasma membrane. HBsAg within the dilated structures did not bind wheat germ agglutinin, indicating that its oligosaccharide chains had not yet been processed to the complex form (containing terminal sialic acid-N-acetylglucosamine residues). The oligosaccharide chains of HBsAg are processed to the complex form and can be detected on the HBsAg after secretion, but this event was not detected within cells. In addition, HBsAg was not observed on the cell surface by indirect immunofluorescence or immunoprecipitation, although immunoelectron microscopy revealed some staining at or near the cell surface. These results suggested that HBsAg was either secreted from cells without being incorporated into the plasma membrane, or that the levels of HBsAg in the plasma membrane were below the limits of detection.     

Core particles of hepatitis B virus and ground squirrel hepatitis virus. II. Characterization of the protein kinase reaction associated with ground squirrel hepatitis virus and hepatitis B virus.

The recently described protein kinase activity in hepatitis B virus core antigen particles (Albin and Robinson, J. Virol. 34:297-302, 1980) has been demonstrated here in the liver-derived core particles of ground squirrel hepatitis virus. Both protein kinase activities were initially associated with DNA polymerase-positive heavy core particles in CsCl density equilibrium gradients and shifted to polymerase-negative cores during the course of purification. The major core-associated polypeptide of each virus was the dominant species labeled. A variable number of other polypeptide species were also labeled by this reaction. Tryptic peptide mapping of both major and minor phosphorylated polypeptides of each virus resulted in similar patterns, suggesting that many of the sites of phosphorylation were the same in the components of each core particle. Hydrolysis of these phosphorylated core particles revealed a major phosphoamino acid as serine and a minor phosphoamino acid as threonine. The products of the protein kinase reaction in both human hepatitis B and ground squirrel hepatitis virus core particles, then, share many characteristics. The possible function(s) of this protein kinase activity is discussed in the light of similarly characterized activities in other animal viruses.     

Myristylation is involved in intracellular retention of hepatitis B virus envelope proteins.

The envelope of hepatitis B virus contains three related proteins, one of which is myristylated. The nonmyristylated small and middle protein are assembled into empty envelope particles which are secreted from cells, whereas the myristylated large envelope protein is mainly found in complete virions and is not secreted in the absence of the nucleocapsid. The block to secretion can be partially overcome by mutation or deletion of the myristylation site. Creation of a myristyl attachment site in the small protein impairs the secretion of empty envelope particles but not their intracellular assembly. Myristylation may therefore play a crucial role in hepatitis B virus replication by channeling the envelope proteins into complete viral particles.     

The essential region for assembly and particle formation in hepatitis B virus surface antigen produced in yeast cells

The hepatitis B virus (HBV) genome carries a HBV surface antigen (HBsAg) gene that can encode a polypeptide of 226 amino acids (aa). This gene can be expressed in the yeast, Saccharomyces cerevisiae, and the products can be assembled into 22-nm particles indistinguishable from those recovered from a patient's serum. We constructed a set of deletion derivatives of the HBsAg gene, and examined the particle-forming ability of the resulting polypeptides by expressing the gene in yeast. Elimination of 9 aa from the N terminus had no effect, whereas the elimination of 21-80 aa decreased the ability to form particles, and the particles formed were correspondingly smaller. Elimination of 100 aa that delete the major hydrophobic domain of the molecule abolished the ability to form particles completely. Deletion of 53 aa from the C terminus showed little effect. However, deletions proceeding further toward the center of the molecule rendered the polypeptides unstable.     

Construction and characterization of hybrid hepatitis B antigen particles carrying a poliovirus immunogen

The hepatitis B surface antigen (HBsAg) has the unique property of assembling with cellular lipids into spherical or elongated particles of 22 nm diameter which are secreted by mammalian cells expressing HBsAg. We have studied the structural requirements for particle formation and secretion by creating in-phase insertions into different regions of the S gene of the hepatitis B virus, coding for HBsAg. Modified genes were integrated into an appropriate vector and expressed in mouse L cells. Various single and double inserts in the two major hydrophilic domains of HBsAg were compatible with particle synthesis and secretion. The level of secretion was influenced by the length of the insert, its primary structure, and the site of insertion into the HBsAg molecule. One of the inserted sequences was a synthetic DNA fragment encoding a continuous type 1 poliovirus neutralization epitope (the C3 epitope). Mammalian cells expressing the modified hepatitis B virus S gene secreted hybrid particles carrying the poliovirus antigen. The hybrid polio-HBsAg particles reacted with a monoclonal antibody specific for the C3 epitope and induced poliovirus neutralizing antibodies at low, but significant, titers in mice and at high titers in rabbits. However, the immune response to HBsAg was weaker to hybrid particles than to unmodified HBsAg particles. By cotransfection with two different plasmids carrying either modified or unmodified genes, we obtained phenotypically mixed particles containing both polio-HBsAg and HBsAg molecules. Inoculated into rabbits, the mixed particles induced high antibody titers against both poliovirus and HBsAg.     

Impaired Maturation of Mouse Mammary Tumor Virus Precursor Polypeptides in Lymphoid Leukemia Cells, Producing Intracytoplasmic A Particles and No Extracellular B-Type Virions

Processing of polypeptides of the mouse mammary tumor virus, a type B retrovirus, was investigated in a transplanted thymic lymphoma cell line of the GR strain (GRSL). This cell line was maintained in vivo in ascites form and in vitro as a suspension culture. GRSL cells produce clusters of intracytoplasmic A particles and are virtually deficient in the production of mature extracellular B-type particles. As control, a mammary tumor cell line of the same mouse strain capable of complete virion synthesis was used. The kinetics of viral polypeptide synthesis were studied by pulse labeling with various isotopes (including (35)S and (32)P), followed by immunoprecipitation of cell lysates with monospecific antisera to the major mouse mammary tumor virus gag and env proteins, p27 and gp52, respectively. Both the primary gag and env precursor polypeptides were synthesized in the GRSL cells, but their conversion into viral proteins was impaired. The major gag precursor, Pr73(gag), was stable over a period of 8 h, and mature viral core polypeptides could not be detected. Also, the highly phosphorylated intermediates in the proteolytic processing of Pr73(gag) in virus-producing cells were absent in GRSL cells. By immunoprecipitation, Pr73(gag) was detected in a GRSL particle fraction with the density of intracytoplasmic A particles. The precursor for envelope proteins, Pr73(env), was turned over without the generation of mature viral envelope components gp52 and gp36. The in vivo-transplanted ascites GRSL cells, however, were shown to express gp52 on the cell surface together with a 73,000-dalton polypeptide, as indicated by cell surface iodination and immunoprecipitation.     

Distinctive properties of the hepatitis B virus envelope proteins.

Using recombinant adenoviral vectors, we expressed and characterized the large, middle, and major envelope proteins of hepatitis B virus (HBV). Cells infected with the recombinant adenovirus which contained the large envelope gene (HS1.HP) expressed predominantly large envelope and small but detectable quantities of middle (4%) and major (6%) envelope proteins in the cell lysate. No HBV envelope proteins were detected in the culture medium from HS1.HP-infected cells. Cells infected with recombinant adenovirus which contained the middle envelope gene (HS2.HP) expressed and secreted the middle and major envelope proteins in a molar ratio of 3:1. Cells infected with the recombinant adenovirus which contained the major envelope gene (HS.HP) expressed and secreted major envelope proteins. The HBV envelope proteins secreted by cells infected with either HS2.HP or HS.HP were assembled in 22-nm particles, as shown by velocity sedimentation rate determination, buoyant densities, and electron microscopy. Cells coinfected with a recombinant adenovirus which contained the large envelope gene and with either HS2.HP or HS.HP expressed similar quantities of the large, middle, and major envelope proteins in the cell lysates. Secretion of the major and middle envelope proteins was inhibited more than 95% by the presence of the large envelope proteins. These results suggest that differential biosynthesis, transport, and processing of the envelope proteins occur during HBV infection, allowing efficient assembly and secretion of virions and hepatitis B surface antigen particles.