Restriction map of the hepatitis B virus DNA cloned in Escherichia coli.

A plasmid carrying the complete genome of hepatitis B virus (HBV) inserted into the BamHI site of the pBR322 plasmid vector has been constructed. The physical map of HBV DNA established for 13 restriction endonucleases allows to conclude that the cloned DNA is similar, but not identical to the HBV DNA of ayw subtype.

Expression of hepatitis B virus surface antigen gene in Escherichia coli.

Direct expression of hepatitis B virus (HBV) surface antigen (HBsAg) gene under the control of the Escherichia coli tryptophan operon (trp) promoter has been achieved. Synthesis of HBsAg (both complete and lacking its N-terminal segment) as a part of hybrid proteins with the N-terminal portion coded by genes cat, kan or bla is controlled by the appropriate promoters, as well as by the trp promoter. The highest levels of expression, including those for direct synthesis of HBsAg, provide the accumulation of about 10(5) polypeptide molecules per bacterial cell.

Subtype ayw variant of hepatitis B virus. DNA primary structure analysis.

The entire genome of human hepatitis B virus (HBV) occurring in Latvia was sequenced. This sequence, which is 3182 nucleotides long, was compared with the other previously published HBV genomes and was shown to share maximum homology with HBV subtype ayw DNA. The coordinates of 4 main open reading frames as well as hairpin structures are very well conserved in the two genomes. The distribution of nucleotide substitutions among different HBV genomes suggest that the open reading frames P and X can fulfil a coding function. On the basis of primary structure comparison for hepadnaviral DNAs several evolutionary conclusions can be drawn.

Recombinant core particles of hepatitis B virus exposing foreign antigenic determinants on their surface.

Insertion of foreign oligopeptide sequences (40-50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells. These capsids are morphologically and immunologically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acid-long C terminus of HBcAg.

Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

[Structure and expression of human hepatitis B virus surface antigen]. / Struktura i ékspressiia gena poverkhnostnogo antigena virusa gepatita B cheloveka.

In view of the growing occurrence rate of the virus-induced hepatitis B and also of the special role played by this particular virus (HBV) in the application of recombinant genetic techniques to the study of complex biological systems, an attempt was made to survey the available evidence concerning the widely investigated and practically the most important part of the viral genome, viz. the gene coding for the surface antigen (HBsAg) and the protein itself. The possible antigenic structure of the protein was investigated using data on the primary structure of 11 cloned HBsAg gene variants and on the synthesis of peptides simulating its immunological properties. Special emphasis was placed on quantitative assessment of antigenicity and immunogenicity. Expression of the gene in homologous systems was studied using cultures of eukaryotic tissues: both as part of HBV nucleotide sequences incorporated into the chromosome and as part of extrachromosomal DNA. The latest findings on HBsAg gene expression in yeast and bacteria are reviewed.

[The comparative mapping of virion and cloned DNA of the hepatitis B virus]. / Sravnitel'noe kartirovanie virionnoi i klonirovannoi DNK virusa gepatita B.

The entire hepatitis B virus (HBV) genome and its fragments have been cloned into the BamHI site of the plasmid pBR322 vector. The identity of physical maps of cloned and authentic virion DNAs was demonstrated by restriction enzyme analysis. The location of restriction sites is suggestive of a certain similarity between the studied HBV DNA and HBV DNA, subtype ayw (Galibert et al., 1979). From the restriction enzyme analysis of virion DNA repaired and 32P-labeled by the endogenous DNA-polymerase reaction, the new information concerning the location and maximal length (approximately 1500 nucleotides) of the single-stranded region of HBV DNA has been established.

[Subjection of tetracycline resistance genes to cat promotor gene in plasmid pBR325]. / Podstanovka genov, opredeliaiushchikh ustoichivost' k tetratsiklinu, pod promotor gena cat v plazmide pBR325.

A series of plasmids with tetracycline resistance genes (Tcr-operon) subjected to transcription from chloramphenicol acetyl transferase promoter (Cmr-promoter) have been constructed on the basis of plasmid pBR325, AprCmrTcr. For this purpose, a 0.8 Md fragment in pBR325 DNA bordered by unique EcoRI and HindIII restriction sites was cut out and structural genes of Tcr-operon were fused to the cat gene nucleotides corresponding to Cmr-promoter and first 72 amino acids of cat (alton, Vapnek, 1979; Marcoli et al., 1980). These plasmids with molecular weight amounting to 3 Md confer AprTcr phenotype to host cells. Tetracycline resistance can be eliminated completely by the deletion of a) Cmr-promoter; b) part of the first Tcr-operon gene.

[Exposure of the major immunodominant epitope of the gp51 envelope protein of bovine leukosis virus on the surface of the hepatitis B core antigen capsid]. / Eksponirovanie mazhornogo immunodominantnogo épitopa obolochechnogo belka gp51 virusa leikoza krupnogo rogatogo skota na poverkhnosti kapsid kor-antigena virusa gepatita B.

Insertion of 48 amino acid long sequence of envelope protein gp51 of bovine leukemia virus (BLV), located from position 56 till 103 of mature protein, into Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids. These capsids preserve morphology of intact HBcAg but expose on their outer surface BLV epitopes which are localised in the inserted gp51 fragment and responsible for the recognition of chimeras by monoclonal anti-gp51 antibodies MAK14. The anti-genicity of gp51 epitopes within chimeric capsids is not disturbed after shortening of C terminal part of inserted gp51 fragment by deletion of amino acids 73-103. The resulting chimeras show the same capsid-forming ability as well as HBcAg and gp51 antigenic properties.

[Bacterial synthesis of immunogenic epitopes of foot-and-mouth disease virus fused either to human necrosis factor or to hepatitis B core antigen]. / Bakterial'nyi sintez immunogennykh épitopov virusa iashchura v sostave gibridov s faktorom nekroza opukholei i kor-antigenom virusa gepatita B.

Using recombinant DNA technology, construction and bacterial expression of genes was carried out which code for hybrid proteins, human tumor necrosis factor and hepatitis B core protein fused to immunogenic epitopes of foot-and-mouth disease virus, strains A22 and O1-194. Hybrids of tumor necrosis factor with foot-and-mouth disease antigenic determinants protected laboratory animals against the experimental challenge with a homologous strain of foot-and-mouth disease virus. Hybrid protein that contained immunogenic regions of two strains, A22 and O1-194, protected animals against infection with both A and O serotypes. Hybrid proteins based on hepatitis B virus core antigen retained the ability to assemble into core-like particles.

[Study of the antigenic structure of hepatitis B virus proteins. I. Synthesis of pre-S fragments of hepatitis B virus proteins and their immunogenicity]. / Izuchenie antigennoi struktury belkov virusa gepatitaa B. I. Sintez fragmentov pre-S belkov virusa gepatita B i izuchenie ikh immunogennosti.

Fragments of hepatitis B virus envelope proteins corresponding to the parts of the pre-S domain were synthesised and immobilized on the carriers with low own immunogenicity. The highest stimulation of the antibody production was observed for the antigens immobilized on microspherical carriers or gelatin modified by H-Gly-Tyr-OH. Among peptides used for immunization, pre-S fragment 134-144, conjugated with microspherical carrier, proved to be the most active.

[PreS-sequence of the hepatitis B virus envelope: molecular biology and immunology]. / PreS-posledovatel'nosti obolochki virusa gepatita B: molekuliarnaia biologiia i immunologiia.

Current data on molecular biology and immunology of the proteins of hepatitis B viral capsid encoded by the preS-region of HBV genome are reviewed. Main attention is paid to study of the immune properties of the capsid proteins, to search of immune dominant epitopes encoded in preS-sequences. The medical and biological significance of the research is shown as exemplified by search of preS-antigens and anti-preS-antibodies in serum from patients suffering hepatitis B. The use of preS-sequences as components of vaccine preparations is discussed.

[Bacteriophage MS2 mutants with disrupted phage replicase repressor activity]. / Mutanty bakteriofaga MS2 a narusheniem repressornoi aktivnosti fagovoi replicazy.

A number of ts-mutants with altered characteristics of RNA synthesis in non-suppressor cells at elevated temperatures have been obtained after HNO2-treatment of phage MS2am623 (amber mutation in site 50). The mutant ts130 is studied in detail: its peak of RNA synthesis is displaced 10 min. later and the total amount of RNA is reduced in 5-10 times. No RNA synthesis is observed in ts130-infected cell at 46 degrees C. Changes in the character of RNA synthesis are accompanied by the over-production of phage subunit of replicase: at 42 degrees C the replicase/RNA ratio in ts130 is 20 times higher than that of the original phage MS2am623. It is assumed that there is a decrease in the activity of replicase--RNA repressor complex in ts130 resulting in the loss of replicase control over its own synthesis while the control of coat protein synthesis remains unaffected.

[Genetic transformation of somatic cells. XIV. Expression of gene coding for the human hepatitis B virus surface antigen in mammalian cells]. / Geneticheskaia transformatsiia somaticheskikh kletok. XIV. Ekspressiia gena, kodiruiushchego poverkhnostnyi antigen virusa gepatita B cheloveka, v kletkakh mlekopitaiushchikh.

A set of recombinant plasmids with a gene encoding surface antigen of hepatitis B virus (HBsAg) is constructed. The plasmids were transfected by DEAE-dextran method into different lines of cultured cells and transient expression of the HBsAg gene was studied. The results indicate that: transcriptional enhancer of hepatitis B virus situated downstream from HBsAg gene is active in green monkey kidney cells (CVI), promoter of 5 LTR of bovine leukemia virus is trans-activated in the goat or calf cells infected with BLV. The results are discussed in the light of hypothesis on the role of transcriptional enhancers in determination of tissue-specificity of hepatitis B virus.

[Regulation of RNA replication in RNA-containing bacteriphages. RNA synthesis in coat protein polar mutants]. / Reguliatsiia replikatsii RNK u RNK-soderzhashchikh bakteriofagov. Sintez RNK u poliarnykh mutantov po belku obolochki.

The synthesis of RNA by polar coat protein mutants f2sus3 and Qbetaam12 under suppressor (Escherichia coli S26R1E, Su+-1; H12R8a Su+-3) and non-suppressor (E. coli AB259; S26) conditions was examined. It was demonstrated that the synthesis of viral RNA under non-suppressor conditions in the presence of rifamycin produced the same gaussian pattern of rates as the synthesis of RNA by wild type phage or non-polar coat protein mutants. However, the total amount of RNA was decreased approximately 10-fold and the peak of RNA synthesis was displaced 7--10 min later. The number of infective centers was reduced also 10-fold indicating that a certain time-lapse was required to overcome the polarity of the parental RNA, this process being of single occurrence, exclusively on the parental RNA, but not on the progeny strains. As a consequence, it was concluded that the initiation of translation at the replicase cistron starts on the nascent RNA chains within the replicative complexes and not on the fully-synthesized templates with their complete secondary structure. The data obtained are not in contradiction with the hypothesis concerning the role of the repressor complex II (replicase-RNA) to slow down the synthesis of replicase and RNA in the coat protein mutants. The polarity can not be responsible probably for the blocking of the replicase cistron on the nascent chain following the block of coat protein cistron. Therefore, it appears appropriate to assume the existence of two binding sites for the replicase as repressor which is in keeping with the conclusions of Weissmann and co-workers.

Replication of RNA bacteriophages in the presence of rifamycin.

Replication of RNA bacteriophages in the presence of rifamycin was studied in different Escherichia coli strains that vary in RNase content but are not isogenic: AB259 RNase+, Q13 RNase I- PNPase-, AB105 RNase I- RNase III-. It was found that rifamycin did not affect characteristics of phage replication such as the general pattern of viral RNA synthesis and intracellular development of the phage. These characteristics are strain specific and independent of the cell growth rate, which defines only phage release. The inhibition of cell division by rifamycin interfered with the release of the phage and thus produced an apparent effect of rifamycin on phage replication.

Control of replication in RNA bacteriophages.

The rates of viral RNA and protein syntheses for wild-type RNA bacteriophages and their nonpolar, coat protein amber mutants were determined in amber suppressor (S26R1E, Su-1 and H12R8a, Su-3) and nonsuppressor (AB259, S26, and Q13) strains of Escherichia coli in the presence of rifamycin. It was demonstrated that the rates of synthesis of phage-specific replicase and RNA minus strands drop off concurrently in both wild-type and coat protein mutant-infected Su(-) and Su(+) cells after 10 and 15 min postinfection, respectively. The rate of synthesis of RNA plus strands started to decline 5 to 10 min later in both cases. Excessive synthesis of replicase in the coat protein mutant-infected cells was accompanied by a similar overproduction of RNA minus strands, but not of plus strands. Partial suppression of protein synthesis in wild-type phage-infected cells abolishing coat protein control over replicase accumulation led to prolongation of replicase synthesis. Such an effect was observed also in coat protein mutant-infected cells, indicating that the excess of replicase itself may be capable of suppression of replicase synthesis in the absence of coat protein. The prolongation of replicase synthesis was followed by the prolonged synthesis of RNA minus strands in both cases. Moreover, replicase and minus strands were formed in nearly equal amounts when protein synthesis was partially inhibited. Assuming functional instability of phage RNAs, the observed coupling of replicase and minus-strand RNA synthesis offers a possibility for control of viral RNA replication by means of control of replicase synthesis on the translational level. A hypothesis is put forward to explain the molecular mechanism of such coupling between the syntheses of replicase and RNA minus strands.

Determination of the minimal length of preS1 epitope recognized by a monoclonal antibody which inhibits attachment of hepatitis B virus to hepatocytes.

The minimal amino acid sequence sufficient to be recognized efficiently by virus-attachment inhibiting murine monoclonal anti-preS1 antibody MA18/7 has been determined. We have constructed a recombinant gene library using the cloned coat protein gene of Escherichia coli RNA bacteriophage fr as a carrier. Different fragments of preS1 region from cloned hepatitis B virus (HBV) genomes, subtype ayw and adw, were inserted at position 2 of the 129 amino acid-long fr coat protein gene in the appropriate E. coli expression vectors. Fine mapping of preS1 epitope recognized by MA18/7 was accomplished by bidirectional shortening of the preS1 within original recombinant preS-fr coat protein genes with Bal31 exonuclease. Immunoblot analysis of the obtained recombinant protein library revealed that the tetrapeptide Asp-Pro-Ala-Phe (DPAF), located at the position preS(31-34) and conserved in all known HBV genomes, is sufficient to bind MA18/7 antibody. Recognition of the preS1 region by MA18/7 occurred irrespective of the amino acid context surrounding this DPAF tetrapeptide. Further shortening of this minimal epitope from the left or from the right side completely prevented antibody binding in immunoblots.