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Rolling circular DNA associated with Dane particles in hepatitis B virus
Abstract
PLASMA of hepatitis B antigen (HBsAg) carriers consists
mostly of spherical particles of diameter 20 nm, together with a much
smaller number of filamentous structures with the same diameter. In
addition, larger spherical particles, 42 nm in diameter with a 28 nm
core, first described by Dane et al.1, are routinely found in
crude plasma or purified preparations of HBsAg. Dane
particles also contain a primed DNA polymerase activity2
capable,of synthesising new DNA. Robinson et al.2 examined
DNA from Dane particles by electron microscopy and found open,
double-stranded circular configurations approximately 0.78 µm
long. We have examined DNA isolated from Dane particles directly and
also after formation of radioactive products by the endogenous DNA
polymerase reaction. The DNA structures appeared in electron micrographs
as double-stranded linear strands and closed circles, with examples of
completely and partially open circles, and circles with attached linear
segments in different lengths. The open circles with attached linear
segments are suggestive of a replicative form, and this is consistent
with a rolling circle model4 for replication. This model for
replication is strengthened by the comparison of the types of DNA found
in particles before and after reaction of the polymerase. The number of
rolling circles and the length of the tails were greatly increased after
DNA synthesis. This type of replication has not been reported for animal
viruses.
... It has been widely accepted that there are three morphologically distinct entities of hepatitis B antigen (HBAg) 2 in antigen-positive plasma, i.e., 42-nm double-shelled spherical "Dane" particles with a 28-nm internal core (1), 20-nm spherical, and tubular structures (2). Lately attention has been focused on Dane particles, since they apparently represent the putative hepatitis B virus (HBV); DNA polymerase activity was demonstrated in them (3), and a double-stranded circular DNA molecule was reproducibly visualized in the sample containing Dane particles by electron microscopic studies with an estimated m.w. of 1.6 × 10 s daltons (4,5). ...
... Furthermore, a circular DNA strand extruding directly from the core of Dane particles was demonstrated by electron microscopic observations. The circular DNA strand in full length had the same dimension as the ones reported by Robinson et al. (4) and Overby et al. (5). The physical association of the circular DNA strand to the core of Dane particles has made it unambiguous that the 0.78 gm DNA circles reported by them are in fact the DNA of hepatitis B virus. ...
A method was developed to isolate, on a large scale, Dane particles from five liters of pooled plasma of asymptomatic carriers of hepatitis B antigen. It involved three successive ultracentrifugation procedures; the final preparation was more than 98% pure in Dane particles. Purified Dane particles had the density of 1.23 to 1.24 g/cm3. The concentration of nucleic acids in the preparation containing purified Dane particles was too low to be detected either by chemical or by spectrophotometric method. However, a circular double-stranded DNA molecule extruding directly from the core of Dane particles was clearly demonstrated by elelctron microscopic observations. The cores of Dane particles (hepatitis B core antigen; HBCAg) were prepared by treating the Dane particle preparation with mercaptoethanol and Nonidet P-40. They were sufficient both in purity and amounts to allow the determination of antibody to HBCAg by an immune adherence hamagglutination method.
... DNA polymerase activity has been shown to be associated with the cores of Dane particles that have been exposed by detergent treatment (2). A circular, double-stranded DNA with an estimated m.w. of 1.6 × 106 Daltons has been reproducibly derived from the core of the Dane particle (3,4). ...
Asymptomatic carriers of hepatitis B surface antigen (HBsAg) were surveyed for serum e antigen (e Ag) and antibody to e (anti-e) by immunodiffusion. Seventeen serum samples containing e Ag and 17 containing anti-e, but with similar HBsAg titers, were tested for the presence of hepatitis B core antigen (HBcAg) by means of immune adherence hemagglutination method. HBcAg activity was detected in all of the samples containing e Ag, and the titer of HBcAg paralleled that of HBsAg in each serum. In remarkable contrast, no HBcAg activity was found in any of the serum samples containing anti-e. Dane particles were demonstrated in 14 of 17 serum samples with e Ag by electron microscopy, but in none of 17 with anti-e. Although both e Ag and HBcAg were closely associated with each other, the non-identity of e Ag and HBcAg was clearly demonstrated by a two-dimensional immunodiffusion test.
... It is possible that under conditions which do not permit a high level of virus production, the presence of hepadnaviruses in cells may be maintained by a low level of semiconservative replication, especially for these multimeric forms. Intermediates consistent with a rolling circle model of DNA replication have been reported for HBV (19). ...
The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen. Viral DNA in the PBL and bone marrow was episomal, primarily existing as multimers with some monomeric forms. Integrated HBV DNA was detected in the PBL of one chronically infected chimpanzee, but only for a brief period. Viral RNA was also detected in the PBL, although less frequently than was DNA. HBV RNA in chimpanzee PBL existed as 3.8- and 7.5-kilobase species, while 2.3- and 3.8-kilobase WHV RNA was found in woodchuck PBL. Subfractionation of PBL isolated from the chronically infected chimpanzees demonstrated that HBV DNA and RNA were located in B and T cells. No HBV DNA was detected in the macrophages. These results, along with the recent reports of HBV nucleic acids in the PBL of human patients, suggest that infection of PBL may be a general phenomenon associated with the pathology of hepadnaviruses.
The story of hepatitis B antigen began with an investigation of genetic differences in circulating low density lipoproteins. In the early 1960s, Dr B. S. Blumberg, a geneticist, began to evaluate the antigenic specificities of serum lipoprotein, which could be detected with sera of multiply transfused patients, e.g. in haemophiliacs who develop antibodies against certain lipoproteins (Blumberg et al., 1962; Blumberg et al., 1964). In 1963, Blumberg (1964) tested sera from such patients for the presence of these antibodies by the Ouchterlony immunodiffusion technique. The panel of sera tested for the reaction with haemophilia sera included some obtained from foreign populations, one of which was from an Australian aborigine. This serum produced a precipitation reaction with sera from two haemophiliac patients which is shown diagramatically in Figure 0.1. Further study of the precipitin line showed that it differed from lipoproteins in that it stained only faintly with lipid stain but gave a strong reaction with protein stain. (Blumberg et al., 1965). Although the antigen was later found not to be especially common in Australian aborigines, it was given the tentative name ‘Australia antigen’.
Untersuchungen und Spekulationen über die Pathogenese der Virus-Hepatitis findet man bereits in den medizinischen Schriften des Altertums, doch die Infektiosität und das Auftreten in Epidemien wurde sicher erstmals genauer in einem Brief von Papst Zacharias an St. Bonifacius, Erzbischof zu Mainz im 8. Jahrhundert A.D. beschrieben (Migne, 1850). Papst Zacharias schlägt in diesem Brief vor, daß Personen mit MORBUS REGIO aus der Stadt herausgebracht werden, und daß Gelbsuchtskranke in der Kirche die heilige Kommunion erst am Schluß nach allen anderen Gemeindemitgliedern erhalten sollten: „De his qui regio morbo vexantur inquisisti sive homines, sive equi sint, quid faciendum sit de illis. Si homines ex navitate, aut genere hujus morbi sunt, hi extra civitatem commanere debebunt...“ und „At tamen in ecclesia, dum ad communionem venerit post omnium impletionem erit ingressurus ad participandum munus“. Es dauerte aber noch mehr als 1000 Jahre bis McDonald (1908, 1918) als erster am Anfang des 20. Jahrhunderts an eine Virusätiologie der Gelbsucht dachte und die Bemühungen, die Erreger der Hepatitis darzustellen, waren bis vor kurzem durch eine lange und oft hoffnungslos erscheinende Kette von Untersuchungen gekennzeichnet. Versuche, die Hepatitiserreger in Zell- oder Gewebekulturen zu isolieren, sie mit immunologischen Methoden darzustellen oder die Erkrankung auf Versuchstiere zu übertragen, waren erfolglos, anfänglich positive Resultate konnten später nicht wiederholt werden oder waren durch nicht-spezifische Ursachen bedingt (für generelle Übersichten siehe Deinhardt und Holmes, 1965; Deinhardt, 1970, 1971; Deinhardt et al., 1975; Regamey et al., 1975; Zuckerman, 1975; Krugman et al., 1975; Deinhardt, 1976; Deinhardt und Deinhardt, 1976). Die Entwicklung, die in den letzten Jahren zumindest zu einer teilweisen Charakterisierung der Erreger der Virus-Hepatitis geführt hat, wurde hauptsächlich durch sieben Entdeckungen oder Beobachtungen ermöglicht:
1.
Unterscheidung zwischen Hepatitis A(HA) und B(HB) durch epidemiologische Beobachtungen;
2.
Übertragung der Hepatitis auf menschliche Freiwillige;
3.
Erkenntnis, daß Affen Hepatitis auf den Menschen übertragen können (Primatenassoziierte menschliche Hepatitis);
4.
Experimentelle Übertragung menschlicher Hepatitis auf Affen;
5.
Entdeckung des sogenannten Australia- oder Hepatitis-B-Antigens und Dane-Virusteilchens;
6.
Entdeckung von virusähnlichen Teilchen in Faeces, Leberzellen oder Seren von Kranken mit HA oder experimentell infizierten Affen und Nachweis von Antikörpern gegen diese virusähnlichen Teilchen in Konvaleszentenseren durch Immunelektronenmikroskopie;
7.
Entwicklung serologischer Untersuchungsmethoden (Komplementbindungsreaktion, Immunadherenz und Radioimmunoassays für HA-Virus-Antigene und -Antikörper (HA-Ag, anti-HA).
A hypothetical model for the replication of hepatitis-B virus is presented. It is suggested that the D.N.A. polymerase associated with Dane particles facilitates integration of viral D.N.A. into the genome of the liver cell. The viral D.N.A. is then replicated with the host genome. The hypothesis accounts for certain curious experimental and clinical observations and makes several predictions which are amenable to laboratory investigation.
The discovery of hepatitis B antigen has led to studies of the physical and chemical nature of the antigen and to the discovery of different structural forms of the antigen, some of which have properties of an incomplete virus and at least one having properties consistent with those of a complete virus. Here, we review some of the more recently acquired knowledge of the morphologic, antigenic and macromolecular structure of the hepatitis B antigen forms and compare hepatitis B virus (HBV) with better studied viruses. Some of the clinical and biologic implications of the recent findings are described. The earlier work on the discovery of the hepatitis B surface antigen (HBs Ag) and its association with hepatitis B and other diseases has been extensively reviewed and is not considered in detail here.
The nucleocapsid of Dane particles (= hepatitis B core antigen; HBcAg) was isolated from human sera either positive or negative for e-antigen (HBeAg)--an apparent marker for the level of infectious hepatitis B virus in serum. HBcAg from the HBeAg-positive serum pool consisted of two distinct populations of particles, one with a buoyant density (d) of 1.358 g/ml and a sedimentation coefficient (S20, w) of approximately 110, and another with d = 1.25 to 1.30 g/ml and S20, w approximately 70. Only the latter type of particles was isolated from an HBeAg-negative serum pool. HBcAg was labelled with 125I-p-hydroxyphenylpropionic acid N-hydroxysuccinimide ester, dissociated and analysed by polyacrylamide gel electrophoresis. One major and one minor polypeptide with apparent mol. wt. of 16000 +/- 500 and 68000, respectively, were detected. Another component have the properties of a glycolipid with a mol. wt. in the order of 10(3) was observed. After isoelectric focusing, HBcAg was recovered in fractions with a pH between 4.0 and 5.8, suggesting heterogeneity in isoelectric points.
The hepatitis B virus (HBV), the causal agent of serum hepatitis, has a diameter of 42 nm and is comprised of an outer surface coat and a 27 nm core. A unique DNA-dependent DNA polymerase is associated with the core of the virus. The core also houses a circular DNA that contains both double-stranded and single-stranded regions. In the endogenous reaction, the DNA polymerase repairs the single-stranded gaps of the viral DNA. The surface protein of the virus, called hepatitis B surface antigen, contains both lipid and carbohydrate, and is often present in particulate form in the blood of infected patients. In Asia and Africa HBV infection is associated with subsequent development of primary hepatocellular carcinoma. Although most patients recover completely from acute illness, the hepatitis B virus may cause chronic infection. Recently, a virus similar to human HBV was discovered in woodchucks. HBV has not yet been propagated in a cell culture system and the mode of replication of this unusual virus in hepatocytes is still moot. Although reliable therapy has not yet been provided, the problem of this world-wide infection has led to many interesting approaches to both vaccine production and anti-viral chemotherapy.
Hepatitis B core antigen (HBcAg) particles, approximately 27-28 nm in diameter and rho = 1.30-1.35 g/cm3, were purified from the liver of a chimpanzee experimentally infected with hepatitis B virus (HBV) while under cyclophosphamide treatment. The purified HBcAg particles incorporated radioactive deoxythymidine triphosphate. The product was precipitable by trichloroacetic acid and sensitive to DNase, but resistant to digestion by RNase. The reaction required four deoxyribonucleosise triphosphates- dATP, dCTP, dGTP and dTTP. Exogenous template did not enhance the reaction. From these findings, it was suggested that HBcAg particles purified from the HBV-infected chimpanzee liver contained DNA polymerase and endogenous DNA.
Three patterns of activity were evident when the differential activation of the DNA polymerase associated with serum Dane particles by nonionic detergent and salt was investigated. The patterns were obtained by plotting the increase in enzyme activity mediated by the detergent Nonidet P-40 (NP-40) in increasing concentrations of KCl compared to the activity observed in the absence of detergent. The pattern of differential activity of hepatitis B (HB) DNA polymerase in detergent and salt was altered by subjecting the HBAg preparations to shearing forces. Hepatitis B DNA polymerase activity was stable even in NP-40 concentrations as high as 10%. In addition to hepatitis B DNA polymerase, DNA polymerase activated by calf thymus DNA was found in pellets containing Dane particles. The latter DNA polymerase activity was also activated by NP-40 and was not decreased by DNAse; this DNA polymerase coprecipitated with hepatitis B antigen (HBAg) upon addition of anti-HBs. However, the DNA polymerase activated by calf thymus DNA was inhibited by 0.4 M KCl. Electron microscopic observations of serum Dane particles in 0.4 M KCl showed no alterations of morphology of these particles when compared to particles in low-salt buffer. The data indicated that KCl activated HB DNA polymerase by a different mechanism from that of shear or NP-40, which removed the surface antigen coat from the Dane particles.
Covalently closed small circular DNA isolated from Drosophila melanogaster is described. The small circular DNA is found in blastema stage eggs and in Schneider's cell culture line 2 and a cloned subline of line 2. It is heterogeneous in size, although the size distributions and mean sizes differ for each source. The small circular DNA from Schneider's line 2 cells ranges from 0.09-7.3 mum, with a mean contour length of 1.1 mum. This DNA has a buoyant density of 1.703 g/cc and appears to be present predominantly in the nuclear fraction of detergent-disrupted cells. the restriction enzyme EcoRl cleaves approximately 40% of the small circular DNA with a bias toward the larger size classes. Both logarithmic and stationary phase cells contain approximately 3-40 average sized small circular DNA molecules per cell, representing a maximum of 0.03% of the total cellular DNA. Exposure to cycloheximide or puromycin for 14 hr results in a 30 fold increase in the number of small circles per cell, but reduces the mean length of the circular DNA to 0.3 mum. The drug-amplified DNA has a buoyant density in the range of 1.698-1.703 g/cc. No amplification was seen in cells treated with either inhibitor for 3.5 hr. Ethidium bromide, cytosine arabinoside, beta-ecdysone, and insulin all had no significant effect on the amount per cell of either small circular DNA or mitochondrial DNA.
DNA isolated from the hepatitis B antigen form known as the Dane particle was examined by electron microscopy before and after the endogenous Dane particle DNA polymerase reaction. The most frequently occurring form was an untwisted circular double-stranded DNA molecule approximately 1 mum in length. Less frequently occurring forms included circular DNA of approximately unit length and having one or more small single-stranded regions, similar circular molecules with one or more tails either shorter or longer than 1 mum in length, and very small circular molecules with tails. There was no increase in frequency or length of tails after a DNA polymerase reaction, suggesting that tails were not formed during this reaction. The mean length of circular molecules increased by 23% when DNA was spread in formamide compared with aqueous spreading, suggesting that single-stranded regions are present in most of the molecules. The mean length of circular molecules obtained from aqueous spreading increased by 27% after a Dane particle DNA polymerase reaction. This indicates that single-stranded regions were converted to double-stranded DNA during the reaction.
Hepatitis B core antigen (HBcAg) was purified from Dane particles and from infected hepatocytes. An identical isoelectric pH of 4.0 was determined for labeled preparations of both Dane-derived and liver-derived HBcAg. Unlabeled liver-derived HBcAg demonstrated a lower isoelectric pH of 3.7. Molecular weight determinations by Sepharose 4B column chromatogrpahy revealed that liver-derived HBcAg had a molecular weight of 8.5-9.0 X 10(6) daltons. The sedimentation coeficient of both Dane- and liver-derived HBcAg was found to be 124S. PAGE revealed that iodinated HBcAg derived from Dane particles was very similar in polypeptide structure to HBcAg derived from infected liver tissue. Twelve polypeptides were resolved from Dane core particles, and seven to nine were resolved from liver core particles. Several of the polypeptides in both preparations co-migrated with iodinated hepatitis B surface antigen (HBsAg). However, three polypeptides (mol. wt. 88,000, 79,000 and 59,000) were found in both Dane- and liver-derived HBcAg but not in HBsAg, which suggests that these polypeptides are HBcAg-specific. Endogenous DNA polymerase activity was observed in both Dane- and liver-derived core particles.
Serum samples of 403 asymptomatic blood donors carrying hepatitis B surface antigen (HB5Ag) were concentrated threefold and tested for e antigen and antibody to e antigen (anti-e) by immunodiffusion. Hepatitis B antigen (HBAg)-associated deoxyribonucleic acid (DNA) polymerase activity was specifically determined by the difference in incorporation of [methyl-3H]thymidine 5' -triphosphate into DNA by an aliquot of centrifuged serum samples after it had been treated either with normal rabbit serum or with rabbit antibody to HBSAg. All of 58 serum samples containing e antigen revealed HBAg-associated DNA polymerase activity, whereas none of 96 samples containing anti-e did. In the remaining 249 samples in which neither e antigen nor anti-e was found, 62 showed specific DNA polymerase activity, although at lower levels than the samples containing e antigen.
To determine the relation between the presence of donor DNA polymerase and e antigen, and recipient hepatitis, we tested, under code, serums from a controlled trial of hepatitis B immune globulin used to treat individuals accidentally inoculated with HBs Ag-positive blood. All recipients lacked antibody to HBs Ag. In 29 of 31 donors, both polymerase and e were in perfect agreement; both demonstrated a highly significant correlation with recipient hepatitis (P less than 0.001). DNA polymerase/e-negative blood did not cause hepatitis. Blood containing polymerase or e antigen did not cause hepatitis in six of 31 and four of 18 recipients, respectively. Hepatitis did not correlate with transaminase or duration of antigenemia in the donor. Polymerase and e appear to be indicators of the relative infectivity of HBs Ag-positive serum, particularly after small-volume exposure. They may be important determinants in assessing infectivity of chronic carriers of HBs Ag and in evaluating efficacy of hepatitis B immune globulin and hepatitis B vaccines.
When the polypeptides of hepatitis B surface antigen were examined by SDS-polyacrylamide gel electrophoresis under a variety of conditions, anomalous results were found to be due to (i) variable and at times incomplete dissociation of polypeptides after boiling with 1% SDS and reducing agent, (ii) reaggregation of solubilized material under certain electrophoretic conditions and during laboratory manipulations, and (iii) the variable presence of additional components in hepatitis B surface antigen prepared from certain individual donors. When these factors were taken into account, two major components were consistently identified by discontinuous buffer polyacrylamide gel electrophoresis, of apparent mol. wt. 60000 to 70000 and 12000 to 14000. However, in view of the demonstrated limitations of this technique in examining HBsAg polypeptides, alternative methods are necessary to confirm the true mol. wt. of the unique virus-specified amino acid sequence present.
DNA extracted from Dane particles has been characterized by gel electrophoresis and restriction enzyme cleavage with endonuclease R-HaeIII (from Hemophilus aegyptius). Dane particle DNA is proposed to be a double-stranded circular DNA approximately 3600 nucleotides in length containing a single-stranded gap of 600-2100 nucleotides. The endogenous DNA polymerase (DNA nucleotidyl-transferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) reaction appears to repair this single-stranded gap.
The open circular genome of human hepatitis B virus (HBV) is known to contain a partially double-stranded DNA with a single-stranded gap region of variable length. This circular structure of the genome is maintained by base-pairing of the 5' ends of the two DNA stands, the long or L(-) strand and the short or S(+) strand. By cloning, mapping, and sequencing studies, we have localized three recombinational junctions of the integrated HBV in two hepatoma samples, HT14 and FOCUS. Breakpoints of recombination derived from these results and those of others appear to be clustered and coincidental with the identified 5' or the deduced 3' end of the long-strand DNA, respectively. Statistical analysis of these results supports the hypothesis that integration preferentially occurs in an extremely narrow region on the HBV genome. This site-specific recombinational mechanism appears to be conserved among different HBV subtypes. No extensive sequence homology was found between each pair of the recombining parental molecules; however, at the site of crossover, 2- to 3-base-pair junctional homology was consistently observed. Examination of the patterns of the integrated HBV DNAs allowed us to categorize these various patterns into four different groups according to their end specificity and strand polarity. The molecular form of relaxed circle is proposed to be one major substrate for HBV integration. The effect of free strand in the integration of HBV is emphasized in this model. Unlike any other known DNA animal viruses, the site specificity of HBV integration appears to be similar to that of the retroviruses.
Human liver tissues obtained at autopsy from two patients chronically infected with hepatitis B virus (HBV) were found to contain several distinct species of HBV DNA. Southern blot analysis using a nick-translated HBV [32P]DNA probe identified specific DNA bands migrating at the positions expected for linear double-stranded DNA of 3.6 and 2.0 kb. These DNA bands were shown to represent relaxed circular and closed circular (supercoiled) HBV DNA, respectively. In addition to these distinct bands several minor bands as well as a heterogeneous population of HBV DNA molecules were present. When infected cell nuclei were isolated, and the nuclear and cytoplasmic nucleic acid separately analyzed, the nuclear fraction contained the 2.0-kb DNA species. This species was shown to be supercoiled 3.2-kb HBV DNA by electron microscopy, restriction endonuclease digestion, and thermal denaturation. The cytoplasmic fraction contained DNA forms similar to those found in virions isolated from plasma (i.e., migration in the position of linear double-stranded molecules of 3.6 and 3.2 kb) and no supercoiled DNA was detected. Particles isolated from the cytoplasmic fraction were able to incorporate dNTPs into viral DNA sequences. Southern blot analysis of the nucleic acid isolated from the particles revealed the presence of HBV DNA forms migrating in positions expected for 3.6- and 3.2-kb linear double-stranded molecules as well as a heterogeneous population of HBV molecules. The 3.6- and 3.2-kb species were identified as relaxed circular and double-stranded linear genome-length HBV DNA. Digestion of the viral nucleic acid with pancreatic ribonuclease increased the electrophoretic mobility of a portion of the heterogeneous HBV molecules and resulted in the appearance of a distinct 1.9-kb DNA band suggesting the same viral DNA was complexed with RNA. Experiments to be reported elsewhere showed this DNA species to be genome-length minus-strand HBV DNA which was released from DNA-RNA hybrid molecules by RNase digestion. Thus, supercoiled HBV DNA exists free in the nucleus of infected liver cells and cytoplasmic particles contain relaxed circular and linear HBV DNA as well as a heterogeneous population of HBV DNA and DNA-RNA hybrid molecules, and a DNA polymerase reaction in the particles results in incorporation of dNTP into DNA strands of these molecules.
A modified and improved technique for the detection of hepatitis B virus-specific DNA polymerase activity is described. DNA polymerase is released from Dane particles by mixing samples with the detergent Nonidet P-40 and beta-mercaptoethanol. After incubation of pretreated samples with a reaction mixture containing tritiated thymidine-methyl-5'-triphosphate (3H-TTP), DNA is precipitated onto a trichloroacetic acid (TCA)-treated paper. Unincorporated 3H-TTP is then chromatographically eluted with a 5% TCA solution and precipitated counts are determined. A sample is considered positive for DNA polymerase if the incorporated counts are significantly higher than the counts of a group of negative control samples. The modifications include pretreatment of the paper with TCA, chromatographic elution of unincorporated 3H-TTP with TCA solution, prefiltration of the sample through bacteriological filters, and use of sound statistical methods for evaluation of data. These changes have led to a highly reproducible, reliable and sensitive technique. The coefficient of variation of negative control samples from various test runs was in the range of 2.7-8.5%. A linear relationship between incorporated counts and DNA polymerase concentration was shown. A total of 419 serum samples from asymptomatic HBsAg-carrying blood donors were tested. Twenty-three (5.5%) of these were found to contain detectable DNA polymerase activity. All 23 samples also contained HBeAg.
Conclusion Hepatitis B research until recently was confined to a further understanding of virus immunopathology and sequelae. The application of molecular biology techniques to the hepatitis B virus genome has added a new and important dimension to the investigation of the processes accompanying replication of the virus, as has been outlined in this review. It is anticipated that the availability of monoclonal antibodies to hepatitis B antigens will complement this newly acquired information allowing a clearer picture of hepatitis B virus replication to emerge whereby accurate predictions from the genome sequence will be combined with the identification of virus specific products using sera of single specificity. It has often been stressed that the virology of hepatitis B is unique; this concept has now been swept aside to reveal the hepatitis B virus as one of a family of variously related viruses with similar morphology, proteins, and genome structure. These newly described agents are pathogens of widely differing animal species including American woodchucks [88], prairie dogs [78], ducks [95] and squirrels [96]. There is also a preliminary report that one agent of non-A, non-B hepatitis of man is related to hepatitis B virus [19] but this remains to be confirmed. An expansion of research into the pathogenesis and properties of these similar viruses will inevitably further our understanding of human hepatitis B virus infection.
Spherical hepatitis B surface antigen particles (HbsAg) of 22-nm diameter were treated with sodium dodecylsulphate in the absence of reducing agents, and their nuclei were exposed. Factors that interact with the nucleus of HbsAg were detected in the serum of HBsAg carriers who were seropositive for hepatitis B e antigen and identified as tubular forms of HBsAg. The other categories of hepatitis B antigen, Dane particles, and 22-nm spherical HBsAg did not bind with the nucleus of HBsAg. When tubular forms of HBsAg had been treated with a proteolytic enzyme, they lost the reactivity to bind with the nucleus of HBsAg. On the basis of the results obtained, tubular forms of HBsAg bear the receptor of protein nature for the nucleus of 22-nm spherical HBsAg. The receptor allows rapid determination of tubular forms by a haemagglutination method for the evaluation of their clinical and epidemiological implications.
A hypothesis is presented which suggests that hepatitis B DNA in the Dane particles is only a partial viral genome which becomes integrated into the hepatocyte cellular DNA. The Dane particle DNA must enter a liver cell containing an active e gene, in order to become functional. It is suggested that the partial genome of hepatitis B virus is released from the cellular DNA by the mechanism of “escaping genes”.
DNA polymerase activity was detected in each of eight preparations of concentrated human hepatitis B antigen (HBAg) rich in Dane particles prepared by high-speed centrifugation of antigen-positive human plasma and in none of seven control preparations prepared in the same way from HBAg-negative plasma. The incorporation of (3)H-thymidine-methyl-5'-triphosphate into DNA was dependent on four deoxyribonucleoside triphosphates and MgCl(2). Treatment of the concentrated HBAg preparations with the nonionic detergent Nonidet P-40 (NP40) more than doubled the enzyme activity. Fractionation of the concentrated HBAg preparation in sucrose density gradients after treatment with NP40 revealed that the enzyme activity appeared within the density range of Dane core antigen but at a slightly higher density than the average for core antigen. The only particles observed by electron microscopy in this region of the gradient were typical 28-nm cores, suggesting that the DNA polymerase activity was associated with a subpopulation of cores. No DNA polymerase activity was found in purified 20-nm HBAg particles. The DNA product of the reaction remained associated with the 110S core and was not susceptible to DNase digestion when associated with the core. Inhibition of the reaction by actinomycin D and daunomycin suggested that the reaction was dependent on a DNA template associated with the core.
Virus-like particles about 42 nm. in diameter have been found in multiple serum specimens from three Australia-antigen-positive hepatitis patients. It is suggested that these particles may be complete virus and that the much more numerous 22 nm. particles and long forms of Australia antigen are surplus virus-coat material.
Particles containing DNA polymerase (Dane particles) were purified from the plasma of chronic carriers of hepatitis B antigen. After a DNA polymerase reaction with purified Dane particle preparations treated with Nonidet P-40 detergent, Dane particle core structures containing radioactive DNA product were isolated by sedimentation in a sucrose density gradient. The radioactive DNA was extracted with sodium dodecyl sulfate and isolated by band sedimentation in a preformed CsCl gradient. Examination of the radioactive DNA band by electron microscopy revealed exclusively circular double-stranded DNA molecules approximately 0.78 mum in length. Identical circular molecules were observed when DNA was isolated by a similar procedure from particles that had not undergone a DNA polymerase reaction. The molecules were completely degraded by DNase 1. When Dane particle core structures were treated with DNase 1 before DNA extraction, only 0.78-mum circular DNA molecules were detected. Without DNase treatment of core structures, linear molecules with lengths between 0.5 and 12 mum, in addition to the 0.78-mum circles were found. These results suggest that the 0.78-mum circular molecules were in a protected position within Dane particle cores and the linear molecules were not within core structures. Length measurements on 225 circular molecules revealed a mean length of 0.78 +/- 0.09 mum which would correspond to a molecular weight of around 1.6 x 10(6). The circular molecules probably serve as primer-template for the DNA polymerase reaction carried out by Dane particle cores. Thermal denaturation and buoyant density measurements on the Dane particle DNA polymerase reaction product revealed a guanosine plus cytosine content of 48 to 49%.
Excerpt
Why do DNA circles occur? The rolling circle model for DNA replication suggests that DNA must be circular in order to be copied completely; the basic mode of reproducing an entire genome is to copy it from a circular template, using the circularity in an intrinsic way to guarantee that all of the genetic information is preserved. The guarantee is enforced by copying always more than one full genome's worth: copying the circle plus a bit. The model uses an asymmetric mode of replication and thus can employ the E. coli DNA polymerase or analogous enzymes. The synthesis begins by opening one strand of the original circle at a specific point. We imagine that the positive strand is opened, that the newly exposed 5′ end is attached to the ‘membrane,’ and that a new copy of this strand is synthesized by chain elongation of the 3′ end of the old...