Immunoelectron microscopic detection of the hepatitis B virus major surface protein in dilated perinuclear membranes of yeast cells.

The major surface protein of hepatitis B virus produced in Saccharomyces cerevisiae can be recovered from cell lysates in the form of 22-mm lipoprotein particles. Immunoelectron microscopy was applied to investigate site and time of particle assembly. Thin sections of yeast cells revealed that production of the S protein provoked a dilation of the endoplasmic reticulum. Dilated areas were specifically labeled with a polyclonal antibody raised against glutaraldehyde-treated yeast-derived HBsAg particles. In contrast to previous postulates of particle formation during cell lysis and extract preparation, these results suggest that particle formation in yeast occurs in the endoplasmic reticulum and that transport of particles along the secretion pathway is blocked.

Subcellular localization of recombinant truncated Gag precursor proteins of HIV expressed in Saccharomyces cerevisiae.

OBJECTIVE: To determine signals contained in the HIV-1 Gag precursor implicated in protein transport. DESIGN: To study the localization of truncated Gag proteins expressed in Saccharomyces cerevisiae. METHODS: Thin-section immunoelectron microscopy studies were performed on S. cerevisiae cells producing myristoylated or non-myristoylated Pr55gag, the core protein (p24) and several truncated Gag proteins. RESULTS: Pr55gag and the carboxy-terminal truncated Gag proteins were myristoylated and localized at the plasma membrane. p24 was localized in the nucleus or perinuclear membrane. However, addition of a myristoyl group to p24 targeted this molecule to the plasma membrane. CONCLUSIONS: The myristoylated amino-terminal 214 amino acids are sufficient to target Pr55gag to the plasma membrane. Subcellular signals implicated in protein transport are present in the core p24 polypeptide which may become dominant or accessible in the absence of the amino-myristoyl group.

Properties of a recombinant yeast-derived hepatitis B surface antigen containing S, preS2 and preS1 antigenic domains.

Yeast cells have been engineered to express mixed HBsAg particles containing the S and a modified large (L*) protein. Their construction resulted in reduced protease sensitivity, reduced glycosylation and complete inactivation of the polymerized human albumin binding site. The particles exposed the S, preS1 and preS2 antigenic determinants and induced an immune response against the three domains. Highly purified preparations have been obtained and are presently being tested in human volunteers.

The large surface protein of hepatitis B virus is retained in the yeast endoplasmic reticulum and provokes its unique enlargement.

The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology.

Sequence analysis of the putative structural genes of hepatitis C virus from Japanese and European origin.

cDNA fragments encoding the putative structural genes of the hepatitis C genome were isolated from a plasma pool of Japanese non-A, non-B hepatitis patients and from sera of individual Spanish patients. From the Japanese plasma pool a series of E1 clones was obtained that showed 88-98% homology among each other, both at the nucleotide and amino acid level. Compared to the sequences published by the Chiron Corporation and Takeuchi et al., the amino acid homology was 75-79% and 91-94%, respectively. Analysis of the core and E2/NS1 genes showed a high conservation of the core sequence and a high sequence variation in the 5' end of the E2/NS1 gene. The E1 gene of one Spanish isolate showed greater homology to the Chiron than to the Japanese sequence. Another Spanish isolate was more homologous to the Japanese sequence indicating that both hepatitis C genotypes are present in Europe. Analysis of the E1 gene of an isolate derived from a single patient with a 5-year interval revealed nine nucleotide and five amino acid changes.

Simultaneous synthesis and assembly of various hepatitis B surface proteins in Saccharomyces cerevisiae.

Yeast transposon of class-1-based vectors, allowing integration at a series of chromosomal loci by homologous recombination with resident transposons, were constructed. Using such vectors, we have introduced several copies of an expression cassette encoding the major hepatitis B surface protein as well as expression cassettes encoding the middle (M) or/and the large (L) surface protein into Saccharomyces cerevisiae. In extracts of such strains, coassembly of the different proteins into a single lipoprotein structure is observed. This was demonstrated by immunoprecipitation of the major protein using monoclonal antibodies directed specifically against epitopes that are present only on the M or the L protein. These results show that hepatitis B surface antigen envelope proteins synthesized in yeast are able to assemble into structures composed of different polypeptides. This opens the possibility of producing in yeast a variety of particles carrying well-defined amounts of preS epitopes on their surface. Also, one can envisage the production of mixed particles containing different foreign epitopes on their surface, in defined relative abundance, which could be useful for vaccine applications.

Construction and characterization of a Saccharomyces cerevisiae strain (RIT4376) expressing hepatitis B surface antigen.

A host/vector system suitable for large-scale production of HBsAg has been constructed and optimized in terms of the expression plasmid and yeast host strain in order to permit fermentation to very high cell densities. The final expression plasmid contains the coding sequence of the major HBsAg protein (P24) flanked by the promoter sequences from a glycolytic gene and by the transcription-termination region of the ARG3 gene. The host/vector system was found to be genetically stable under large-scale fermentation conditions as demonstrated by nucleotide sequencing and restriction mapping experiments. The P24 protein is recovered from yeast as particles whose physiochemical properties are very similar to those of plasma-derived HBsAg.

Development of a hepatitis B vaccine from transformed yeast cells.

The production in yeast cells of the recombinant DNA hepatitis B vaccine of SmithKline Biologicals involves an optimized fermentation process followed by cell disruption and extraction, together with other soluble yeast components of the surface antigen of the hepatitis B virus. The subsequent purification process includes precipitation steps, ion exchange and gel permeation chromatography, and caesium chloride ultracentrifugation. The yeast-derived antigen occurs as spherical particles containing the non-glycosylated HBsAg polypeptide, lipid, and Tween 20. The purity of the polypeptide is above 95% and confirmed by the absence of an immune response to yeast-derived contaminants in vaccinees. Yeast DNA levels were less than 10 pg/vaccine dose. Various biochemical analyses showed that the recombinant polypeptide was faithfully expressed and did not undergo unwanted processing or degradation during fermentation or purification. These results indicate that the recombinant HBsAg can be effectively produced in yeast and processed to a high degree of purity to yield HBsAg particles displaying most of the characteristic properties of plasma-derived HBsAg.

Production in yeast of hepatitis B surface antigen by R-DNA technology.

Yeast synthesizes the surface antigen protein of Hepatitis B virus when the structural gene is fused to the promoter from the ARG3 gene. Analysis of extracts and total cells shows that the primary translation product can be identified as a poorly antigenic monomer with an estimated molecular weight of 22K. In cell extracts Y-HBsAg is in the form of 20 nm particles which, like serum derived particles, are highly immunogenic in mice and monkeys. Yeast derived surface antigen is thus a viable alternative to the present serum derived HBV vaccines.

Characterization of the coat protein mRNA of southern bean mosaic virus and its relationship to the genomic RNA.

RNA isolated from southern bean mosaic virions contains, in small amount, a subgenomic RNA (molecular weight, 0.38 x 10(6)) that serves in vitro as an mRNA for southern bean mosaic virus coat protein. The RNA has a 5'-linked protein indistinguishable from the protein linked to the 5' end of full-length genomic RNA. Its base sequence, determined to 91 bases from the 3' end, is identical to the 3'-terminal sequence of the genomic RNA. The results suggest that the coat protein messenger sequence exists as a "silent" cistron near the 3' end of the genomic RNA.

Messenger RNA for the coat protein of southern bean mosaic virus.

When RNA from southern bean mosaic virus is fractionated on a sucrose gradient, the resulting absorbance profile shows a major peak corresponding to 1.4 x 10(6) MW together with a considerable amount of slower sedimenting material. The RNA from these gradient fractions was translated in wheat embryo extracts and reticulocyte lysates. Only RNA in the molecular weight range 0.3 - 0.4 x 10(6) was found to induce synthesis of coat protein (designated P3). RNA of molecular weight 1.4 x 10(6) induced synthesis of three proteins, P1, P2, and P4.

Translation of southern bean mosaic virus RNA in wheat embryo and rabbit reticulocyte extracts.

Southern bean mosaic virus RNA was translated in wheat embryo extracts and in nuclease-treated rabbit reticulocyte lysates. Four principal products were synthesized: two related products with molecular weights of 105,000 and 75,000, a product with a molecular weight of 29,000 that closely resembled coat protein, and a product with a molecular weight of 14,000. Their proportion depended on ionic conditions and the translational system used.

Southern bean mosaic viral RNA has a 5'-linked protein but lacks 3' terminal poly(A).

Nuclease digestion of SBMV RNA releases a protein of molecular weight approximately 12,000 in addition to the four mononucleotides. The lack of reactivity of SBMV RNA to polynucleotide kinase and the absence of a capping group suggest that the protein is covalently attached to the 5' end of the RNA. RNA sequencing shows that the 3' terminus of SBMV RNA is not polyadenylated.

The influence of the PO1A1 mutation upon recombination in Escherichia coli K12.

Genetic recombination in Escherichia coli is a highly regulated process involving multiple gene products. We have investigated the role of DNA polymerase I in this process by studying the effect of the po1A1 mutation upon DNA transfer and conjugation in otherwise isogenic suppressor-free strains of E. coli K-12. It was found that the po1A1 mutation greatly reduces recombination in Hfr crosses (a factor of 20 in Pol+ x Po1A1 crosses and more than a factor of 100 in Po1A1 X Po1A1 crosses). However, since the po1A1 mutation reduces the strains capacity to act as a recipient for an F-prime and the analysis of recombination transfer gradients revealed no differences between Po1+ and Po1- strains, it is concluded that DNA polymerase I probably affects the transfer and/or stability of donor DNA rather than the recombinational process itself.