The proteins of hepatitis B Dane particle cores.

Although several studies have been done to analyze the peptides of purified 22-nm HbsAg particles, no information has been published about the peptides of the core of the Dane particle which bears the other hepatitis B viral antigen. HbcAg. Dane particles and Dane particle cores (produced by NP-40 treatment of Dane particles) were purified by equilibrium centrifugation in CsCl density gradients. Two populations of Dane particles were observed at densities 1.27 and 1.24 g/ml, respectively. The higher buoyant density Dane particles yielded exclusively cores of buoyant density 1.38 g/ml in CsCl, and the lower buoyant density Dane particles yielded two kinds of cores with buoyant densities of 1.38 and 1.325 g/ml, respectively. Only the higher density Dane particles and cores manifested endogenously primed DNA polymerase activity. The peptides of density 1.38 g/ml Dane particle cores purified by equilibrium CsCl density gradient centrifugation and HBcAg particles from HBV infected chimpanzee liver purified in the same way were analyzed by SDS-polyacrylamide gel electrophoresis. Both kinds of particles were found to consistently contain 3 Coomassie blue staining peptides with approximate molecular weights of 19,000, 70,000 and 80,000 daltons (designated P-19, P-70 and P-80 respectively). In addition, the HBcAg particles from infected liver regularly yielded a protein component with molecular weight greater than 200,000 daltons. This component was occasionally present in electrophoresis runs of core peptides from only one of two patients. Its irregular appearance after gel electrophoresis suggests it may have been an aggregate not completely dissociated under the conditions used. The lower density core component consistently contained P-19, P-70, and P-80, and infrequently additional minor peptides of uncertain origin. The irregular occurrence of the minor peptides in varying amounts suggests they were not intrinsic core proteins.     

Demonstration and partial characterization of an intermediate HBcAg (dane particle) population

Hepatitis-B core antigen (HBcAg) was released from Dane particles previously separated from anti-HBc by repeated pelleting through sucrose gradients separated into three HBcAg populations when analysed by cesium chloride density gradient centrifugation. Heavy HBcAg particles banded at a density of 1.355 gm/ml, intermediate HBcAg particles at a density of 1.33 gm/ml, and light HBcAg particles at a density of 1.30 gm/ml. Like heavy HBcAg particles, intermediate HBcAg particles contained DNA polymerase activity, but the ratio of HBcAg to DNA polymerase activity was significantly different in both populations. Intermediate HBcAg particles could not be separated from heavy HBcAg particles by rate sedimentation centrifugation. The size of the HBV-DNA and the size of its single-stranded gaps were not significantly different in heavy and and intermediate HBcAg populations. Data accumulated in this paper suggest that the intermediate HBcAg particle differs from the heavy HBcAg particle by the amount of HBcAg polypeptides and the number of HBcAg determinants exhibited.     

Expression of hepatitis B virus-specific markers in asymptomatic hepatitis B surface antigen carriers.

A study was undertaken to assess the state of hepatitis B virus infection in a group of asymptomatic hepatitis B surface antigen (HBsAg) carriers. This study confirmed that the presence of hepatitis B e antigen (HBeAg) in serum was closely associated with serum HBsAg-specific deoxyribonucleic acid polymerase activity, hepatitis B core antigen (HBcAg) in serum and liver cell nuclei, and a histological picture of chronic hepatitis. No HBsAg-specific deoxyribonucleic acid polymerase activity or HBcAg was detected in highly concentrated anti-HBe-positive sera. In addition, liver biopsy specimens from carriers with anti-HBe were negative for HbcAg by immunofluorescence, and the liver histology was either normal or revealed only fatty changes. These data indicate that the anti-HBe-positive sera contained either no Dane particles or, if present, at least a 500-fold-lower concentration of Dane particles than that found in HBeAg-positive sera.     

Epstein-Barr virus-induced proteins. II. Analysis of surface polypeptides from EBV-producing and -superinfected cells by immunoprecipitation

Cells of the Epstein-Barr virus (EBV)-producing line P3HR-1 induced by the tumor promoter TPA and NC37 cells Superinfected with P3HR-1 EBV were surface labeled with ¹²⁵I by the lactoperoxidase method and analyzed by immunoprecipitation with human VCA⁺ MA⁺ sera for virus-induced cell surface polypeptides. Two dominant polypeptides with molecular weights of 80,000 and 250,000 were specifically precipitated. In addition, only traces of polypeptides with 130,000 and 140,000 MW were identified on P3HR-1-EBV-producing cells. The surface of superinfected NC37 cells contained two polypeptides of 80,000 and 140,000 molecular weight. Our experiments demonstrated that the synthesis of the 140,000 polypeptide is Ara C sensitive, while the 80,000 polypeptide is Ara-C insensitive. Both polypeptides were found to be identical in size with [³⁵S]-methionine- and ¹²⁵I-labeled 80,000 and 140,000 envelope polypeptides from purified virus particles. These results may indicate that the identified polypeptides carry antigenic determinants of the EBV-induced membrane antigen (MA) complex.     

Analysis of early and late Epstein-Barr virus associated polypeptides by immunoprecipitation.

The Epstein-Barr virus-producing cell lines P3HR-1 and B95-8 and the nonproducer cell lines Raji clone No. 7 and NC37 were induced to viral antigen synthesis by the tumor promoter TPA and then analyzed by immunoprecipitation with human sera for early and late virus-associated polypeptides. After labeling of producer cells for a 4-day period with [³⁵S]methionine, two polypeptides with molecular weights of 140,000 and 150,000 were identified reacting predominately with virus capsid antigen (VCA⁺) sera. Analysis of purified Epstein-Barr virus demonstrated that the 140,000 polypeptide presumably represents an envelope protein while the 150,000 polypeptide is a nucleocapsid protein. In 4-hr radioactively labeled producer cells an additional polypeptide with a molecular weight of 130,000 was found to be immunoreactive with VCA⁺ sera. Immunoprecipitation of [³⁵S]methionine-labeled cell extracts from nonproducer cells resulted in the specific precipitation of two polypeptides with molecular weights of 85,000 and 35,000 which most likely represent early EBV-associated proteins. Producer cells exhibit three additional apparently early EBV-associated polypeptides with molecular weights of 120,000, 18,000, and 16,000. None of these polypeptides could be detected in EBV genome-negative Ramos cells after TPA treatment.     

Epstein-Barr virus-induced proteins. III. Analysis of polypeptides from P3HR-I-EBV-superinfected NC37 cells by immunoprecipitation

P3HR-I-EBV induces in superinfected nonproducer NC37 cells at least 13 [³⁵S]methionine-labeled polypeptides with molecular weights between 18,000 and 150,000, which were identified by immunoprecipitation using human sera. Application of Ara-C inhibited the synthesis of the two structural polypeptides p150 and p140 while the synthesis of the other polypeptides was not affected.     

Solid-phase radioimmunoassay for hepatitis be antigen (HBeAg)

Summary A solid-phase radioimmunoassay was developed for the detection of HBeAg and anti-HBe in sera or serum fractions. HBe/sAg positive sera, partially purified HBeAg, partially purified HBsAg, and HBe/sAg negative sera were polymerized in polyacrylamide and compared for their ability to bind¹²⁵I-IgG (anti-HBe). Only gels containing HBeAg reacted specifically with the iodinated antibody. The specificity of the binding was confirmed by blocking and inhibition tests using anti-HBe, HBeAg, HBsAg, and negative control sera. The radioimmunoassay allows the specific and quantitative detection of HBeAg and anti-HBe even in the presence of detergents and high salt concentrations.

---

Abbreviations HBsAg hepatitis B surface antigen - HBeAg hepatitis Be antigen - HBe/sAg hepatitis Be antigen and surface antigen - anti-HBe antibody to hepatitis Be antigen     

Morphologic, immunohistochemical, and ultrastructural studies of the production of hepatitis B virus in vitro

Well differentiated human hepatoblastoma Hep G2 cells after transfection with cloned hepatitis B virus (HBV) genomes produce replicative HBV DNA intermediates, high levels of HBsAg, HBeAg and HBcAg as well as mature Dane particles. To analyze the replication cycle of HBV, we studied the expression of HBV antigens with monoclonal antibodies by immunomorphologic methods in the transfected cells at various time intervals after plating. HBcAg and HBeAg were detected in the cytoplasm and less frequently in the nuclei of transfected cells. The percentage of positive cells increased with time after plating and reached a plateau of about 50% positive cells at 10 days. HBsAg and the large and middle HBsAg polypeptides were observed in the cytoplasm of transfected cells and a maximum of 20 to 30% positive cells was reached during the 3rd week after plating. Examination of viable cells in suspension revealed HBcAg/HBeAg and HBsAg expression on the cell surface. Electron microscopy demonstrated characteristic core particles in the nuclei and cytoplasm and Dane particles in cytoplasmic vesicles and culture media of transfected cells. The HBV producing cells did not show any evidence of a cytopathic effect. These observations demonstrate significant similarities between the HBV DNA transfected cells and infected human hepatocytes which support active HBV replication in vivo. Taken together, the results suggest that the cultured cells may serve as a model to elucidate a number of unsolved problems of the molecular and cellular pathobiology of hepatitis B.     

The generation of defective interfering rubella virus particles

Rubella virus (RV) particles produced under varying degrees of autointerference have been studied. RV has been purified from these stocks and studied by isopycnic centrifugation. RNA was extracted from the purified virions, labeled with ¹²⁵I, electrophoresed on 5% polyacrylamide slab gels, and subjected to autoradiography. The viral particles varied considerably with respect to their density and their RNA content. Virions present in low-interference stocks were contained in one band at ? = 1.19 g/ml³. Two single-stranded RNA species could be extracted from this band with molecular weights of 2.95 and 2.80 × 10⁶. Virions present in high-interference stocks were contained in at least three bands with densities of 1.19, 1.17, and 1.15 g/ml³. The molecular weights of the RNA molecules extracted from these three bands were 2.95, 2.80, 1.25, and 1.05 × 10⁶. Therefore, defective RV particles have been detected which possessed a density less than 1.19 g/ml³, contained a smaller RNA, and elicited autointerference.     

Distribution of HBcAg in hepatitis B detected by immunoperoxidase staining with three different preparations of anti-HBc antibodies.

To evaluate the role of the expression of hepatitis B core antigen (HBcAg) in liver cell damage the immunoperoxidase staining pattern of cryostat liver biopsy specimens from 16 chronic carriers of hepatitis B surface antigen (HBsAg) was investigated using three different kinds of anti-HBc antibodies. Polyclonal antibody prepared from recombinant HBcAg seemed to be more sensitive in detecting HBcAg than did monoclonal antibody from the same antigen. The topographical distribution of HBcAg detected by these two antibodies was similar, showing a close correlation to the histological activity of disease. Furthermore, the predominant localisation of cytoplasmic HBcAg usually reflected an active and severe ongoing hepatitis. On the other hand, monoclonal antibody prepared from purified Dane particles resulted in the prominent cytoplasmic staining for HBcAg regardless of histological severity of the hepatitis. The quantitative expression and topographical distribution of HBcAg depended on the type of anti-HBc antibodies used.     

Chick embryo lethal orphan (CELO) virus-induced early and late polypeptides.

Polypeptides of chick embryo lethal orphan virus (an avian adenovirus) were analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Virions (p = 1.338 g/cm³) and virion-like particles having lower density (light particles, p = 1.289 g/cm³) were composed of at least 11 and 14 types of polypeptides, respectively. At least 23 virus-induced polypeptides in infected cells (primary monolayer culture of chicken kidney cells) were detected; eight polypeptides (early polypeptides) were synthesized even in the presence of 1-β-d-arabinofuranosyl cytosine, but the others (late polypeptides) were not. On the basis of migration in SDS-PAGE, 5 polypeptides were found to be common to virions and infected cells; 5, to light particles and infected cells; 10, to virions and light particles; and 4, to all three. Scarcely any of the early polypeptides were found in the soluble fraction when extracted with the low salt buffer. Some of the early polypeptides were solubilized with the high salt buffer. Other early polypeptides were solubilized by sodium deoxycholate; three of these were found in the so-called “M-band,” as are early polypeptides of human adenoviruses. An experiment with radioactive glucosamine indicated that one of early polypeptides found in the M-band was a glycoprotein.     

Cell-mediated immunity to HBcAg in chronic HBV infection

The role of hepatitis B core antigen (HBcAg) as a possible target of cell-mediated immune response in chronic hepatitis B virus (HBV) infection has been recently emphasized. Peripheral blood leukocytes (PBLs) from 35 chronic carriers of hepatitis B surface antigen (HBsAg) were studied in vitro for their immune response to a purified preparation of HBcAg isolated from circulating Dane particles. PBLs from all the studied HBsAg-positive patients yielded a stimulation index above 3, with values ranging from 3.1 to 38.1. None of the healthy seronegative subjects, taken as control group, had a stimulation index above 2, with a mean value +/- SD of 1.28 +/- 0.35. Levels of PBL stimulation correlated with the histologic activity of liver disease, and the differences reached statistical significance. These results indicate that lymphocyte response to HBcAg may be relevant in determining liver cell damage.     

Structural polypeptides of the enteropathogenic bovine coronavirus strain LY-138

Summary The bovine coronavirus strain LY-138 was purified by differential as well as velocity and isopycnic centrifugation in sucrose or CsCl gradients. The substrate for purification was contents of the small intestine of experimentally inoculated calves. This strain is highly enteropathogenic, but it could not yet be propagated in cultured cells. Intact virions had a density of 1.245 g/cm³ in CsCl and 1.185 g/cm³ in sucrose. A spherical core-like structure with an average diameter of 82 nm remaining after treatment with chloroform had a density of 1.299 g/cm³ in CsCl and 1.201 g/cm³ in sucrose.Seven distinct bands of polypeptides and 4 shoulders were detected after electrophoresis of SDS-solubilized virions in polyacrylamide gels. The approximate molecular weights ranged from 110,000 to 36,000. Four of the bands gave a PAS positive reaction. These 4 glycoproteins and an additional protein with an approximate molecular weight of 70,000 were removed by chloroform treatment. The remaining core-like structure contained the 2 polypeptides VP3 and VP7.With 5 Figures     

Electron microscopy of serum of healthy hepatitis B antigen carriers.

The sera of 36 blood donors who are established HBsAg carriers were examined with the electron microscope. The findings were correlated with the histological and electronoptic appearances of the liver and the titre and subtype of the antigen. Antigen-antibody complexes could not be detected. Dane particles constituted 2 percent or more of the total particle count in five of the 36 sera, including three sera from five carriers with chronic aggressive hepatitis and two sera from 11 carriers with chronic persistent hepatitis. In sera from carriers with normal histology or the minimal histological lesion of focal parenchymal necrosis they were detected very infrequently or not at all. Three biopsies revealed intranuclear inclusions when examined electronoptically and the corresponding sera all contained greater than 2 percent Dane particles. Where greater than 2 percent Dane particles were seen the antigen titre tended to be high. The predominant subtype was ad. There was no correlation between the number of Dane particles and the antigen subtype nor between subtype and histology.     

Cryptic association of e antigen with different morphologic forms of hepatitis B surface antigen

When highly purified HBsAg particles, separated by rate zonal centrifugation into populations differing in predominant size, were tested for HBeAg, the e1 specificity was detected preferentially in association with particle fractions containing large filaments and Dane particles. These results were obtained both by agar gel diffusion and by radioimmunoassay for e antigen. The e antigen activity present in these fractions was potentiated by prior treatment of particles with Tween 80, suggesting cryptic localization of e1 specificity within or under the outer membrane. The HBeAg released by detergent treatment from a purified preparation composed predominantly of small-particle forms of HBsAg was separated by electrofocusing into a peak of nonparticulate e antigen in the pH range of 5.7--6.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major polypeptides in this preparation with approximate molecular weights of 25,000, 55,000, and 70,000. Furthermore, two additional peaks of e antigen activity were detected which migrated in association with HBsAg particles at isoelectric points of 4.4 and 5.5--5.6. The major portion of e antigen remained in association with particles after further purification by rate zonal centrifugation.     

Intra- and extracellular distribution and immunochemical characterization of hepatitis B virus nucleocapsid proteins produced by a human hepatoma cell line transfected with cloned viral DNA

Hepatitis B virus (HBV)-related antigens produced by the human hepatoma cell line (HB611 cell), which had been transfected with a cloned HBV DNA and established as a stable producer of HBV (T. Tsurimoto, A. Fujiyama, and K. Matsubara, 1987, Proc. Natl. Acad. Sci. USA 84, 444-448), were investigated immunochemically and morphologically. All HBV-related antigens, HBV surface (HBsAg), e (HBeAg), and core (HBcAg), were semiquantitatively examined by the respective reversed passive hemagglutination assay (RPHA). RPHAs for HBcAg and for HBeAg were characterized as reacting only to the core particles and to the free form of nucleocapsid proteins, respectively. The amounts of HBsAg and nucleocapsid protein in culture medium were roughly related to the number of viable cells. The amount of core particles was, instead, proportional to the number of dead cells. Relative amounts of HBsAg, core particles, and nucleocapsid proteins in culture medium, cell surface, and cell lysate were determined and it was found that HBsAg and nucleocapsid proteins were effectively secreted into culture medium but core particles were not. Molecular species of nucleocapsid proteins were identified to be p17 and p18 (HBeAg polypeptides) in the culture medium and HBeAg polypeptides and p21.5 (HBcAg polypeptide) in the cytosol fraction. The p21.5 was preferentially found in the nuclear fraction.     

Epstein-Barr Virus induced proteins V: Comparison of EBV-specific polypeptides from different virus strains

EBV-associated polypeptides induced in different Epstein-Barr Virus (EBV)-producing cell lines by the tumor promotor TPA, and from purified EBV particles derived from the same lines were radioactively labeled and analyzed by immunoprecipitation with human VCA⁺MA⁺ sera. In virus-producing cells no significant differences in the molecular weight of³⁵S-methionine-labeled EBV-associated polypeptide patterns could be observed. The analysis of¹²⁵I-labeled polypeptides from purified virus particles of four different strains revealed that, in addition to common polypeptides, individual EBV strains contain strain-specific high molecular weight glycopolypeptides. These polypeptides, constituting part of the membrane antigen complex, are present in varying amounts. While P3HR-1 virus particles contain a major component of 250 000 and small amounts of 340 000 molecular weight polypeptides, QIMR-WIL virus particles have more 340 000 than 240 000 molecular weight polypeptides. Furthermore, in B95-8 particles and in particles from an EBV strain isolated from an African green monkey (AGM-EBV) respectively, large amounts of 360 000 and 250 000 polypeptides could be observed. Since these glycopolypeptides carry strain-, subgroup- and group-specific antigenic determinants, also found in virus strains produced in human and marmoset cells, it should be further investigated whether these differences in molecular weight are virus-strain- or cell-specific.     

Detection of Thy-1.2 membrane complexes shed from thymocytes and lymphoma cells by an immunoradiometric assay

A direct binding immunoradiometric assay (IRA) for Thy-1 antigen was developed to study the properties of membranous complexes shed from murine thymocytes and lymphoma cell lines. Monoclonal anti-Thy-1.2 antiserum was iodinated and used to study the shedding from AKR (Thy-1.1) and C3H(Thy-1.2) thymocytes, and S49.1(Thy-1.2), S49-Thy-1^? and BW5147(Thy-1.1) continuous lymphoma cell lines. Culture supernatant fluids or purified shed complexes were allowed to bind to microtiter plates followed by measurement of the binding of iodinated anti-Thy-1.2. The assay was found to be completely specific for the Thy-1.2 allotype, and in conjunction with antibody coated wells could detect Thy-1 solubilized from cells with N-P40 detergent. Shed complexes containing Thy-1 from thymocytes and lymphoma cell lines were analysed by isopycnic centrifugation with continuous potassium tartrate gradients (5–40%). Shed complexes had a buoyant density of 1.06–1.10 g/cm³ as compared to 1.15–1.17 g/cm³ expected for murine leukemia virus or 1.20–1.24 g/cm³ expected for mycoplasma. We concluded that the shed membranous complexes had a buoyant density similar to plasma membrane and the complexes were similar from both thymocytes and lymphoma cell lines. Thy-1 was not associated with virus, mycoplasma or other particles found in the gradients and Thy-1 was not found in the unsedimented fraction. The release of Thy-1 from thymocytes and cultured cell lines results in only one defined density of particles which may participate in cellular communication or in the survival of malignant cells.     

Genetic analysis of adenovirus type 2: VII. Cleavage-modified affinity for DNA of internal virion proteins

The core structures derived from wild-type (wt) Ad2 and ts1 virions, by disintegration with N-laury sarcosine and pyridine, were analyzed biochemically and by electron microscopy. Disintegration of wt virus with Sarkosyl yielded cores of density 1.58 g/ml (in CsCl) whereas the particles synthesized by ts1 at 39° (ts1-39°) generated subviral structures ranging in density from 1.58 to 1.72, indicating the increased lability of the mutant virions. The wt core (1.58 g cm^?3) contained predominantly polypeptide VII, while the ts1-39° cores only contained traces of this polypeptide. Significantly, Pre-VII, which is the principal form of this polypeptide in the ts1-39° virions, was completely absent from the cores. Electron microscopy of wt and ts1-Sarkosyl cores revealed identical types of structures on negative staining, although the ts1-39° cores were found to unfold more rapidly in the presence of high salt. The polypeptide analysis of the pyridine cores showed significant differences between wt and ts1. Wild-type core contained polypeptides V, VII, and 14K; ts1-33° core contained V, Pre-VII, VII, traces of IVa2, Pre-VI, VI, and possibly IX and X; while ts1-39° core contained V, Pre-VI, and Pre-VII. These findings clearly establish structural differences between wt and ts1 cores, most important the differential affinity of precursor polypeptides for DNA in ts1 virions under different conditions of disintegration. It is suggested that the loss of infectivity by the mutant is due to the altered composition and structure of the viral core.