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Biomedical subjects

R E Streeck

Publications and source records attributed to R E Streeck.

At least 37 records · Page 2Linked to original sources
PubMed1992

Deletion and insertion mutants of HBsAg particles.

We have found previously that hybrid 22-nm HBsAg particles can be created by insertion of short antigenic sequences into the HBV major envelope protein. We have now performed a detailed deletion mutagenesis of the S gene of HBV encoding HBsAg. Deletion of the 51 C-terminal amino acids including most of the third and all of the fourth hydrophobic domain of the S protein did not affect particle assembly and secretion. However, secretion of 22-nm particles was abolished by minor deletions in the N-terminal region. Insertion and deletion/substitution mutants carrying a poliovirus epitope at the N-terminus and the preS1 region at the C-terminus have been characterized.

DNA Mutational Analysis↗
PubMed1992

Human papillomavirus type 42: new sequences, conserved genome organization.

We report the nucleotide sequence and genome organization of the human papillomavirus type 42. HPV42 DNA was isolated from vulvar papillomas. It has been detected in benign forms of proliferative lesions only. The genome of HPV42 is 7917 bp long and shows the open reading frame pattern conserved in all HPVs sequenced so far. HPV42 has no high degree of sequence homology to any of the known HPVs. It shows characteristics previously found either exclusively in HPVs associated with invasive carcinomas or exclusively in nongenital HPVs. Therefore it cannot be readily ascribed to any of the established subgroups of human papillomaviruses.

Base Sequence↗
PubMed1992

Deletions in the hepatitis B virus small envelope protein: effect on assembly and secretion of surface antigen particles.

The small envelope S protein of hepatitis B virus carrying the surface antigen has the unique property of mobilizing cellular lipids into empty envelope particles which are secreted from mammalian cells. We studied the biogenesis of such particles using site-directed mutagenesis. In this study, we describe the effect of deletions in the N-terminal hydrophobic and hydrophilic domains of the S protein. Whereas short overlapping deletions of hydrophilic sequences flanking the first hydrophobic domain were tolerated, larger deletions of the same sequences were not. Conversely, the hydrophilic region preceding the second hydrophobic domain was not permissive for even short deletions. Deletion of part or all of the first hydrophobic domain also completely blocked secretion, confirming that the entire apolar region serves an essential function. Most of the secretion-defective deletion mutants still entered the secretory pathway and translocated at least the second hydrophilic domain across the membrane of the endoplasmic reticulum. These mutants appeared to remain arrested in a membrane-associated configuration in the endoplasmic reticulum or the cis-Golgi compartment but preserved their capacity for oligomerization with the wild-type S protein. While secretion of wild-type S protein was specifically blocked by the formation of intracellularly retained mixed envelope aggregates, secretion of an unrelated protein (interleukin 9) was completely unaffected.

Base Sequence↗
PubMed1991

Genome organization and nucleotide sequence of human papillomavirus type 39.

The 7833-bp nucleotide sequence of human papillomavirus type 39 (HPV39), which is associated with genital intraepithelial neoplasias and invasive carcinomas, has been determined. The genome organization deduced from the sequence shares characteristic features with other genital papillomaviruses. According to sequence comparisons, HPV39 most closely resembles HPV18 and may be a member of a subgroup of genital papillomaviruses distinct from the HPV16/31/33 group. As a novel feature, we report a 1.3-kb open reading frame on the DNA strand which lacks major open reading frames in the other sequenced HPV genomes.

Base Sequence↗
PubMed1991

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.

Capsid↗
PubMed1990

Enhancement of gene expression by somatic hybridization with primary cells: high-level synthesis of the hepatitis B surface antigen in monkey Vero cells by fusion with primary hepatocytes.

Vero cells transfected with the S gene encoding the surface antigen (HBsAg) of the hepatitis B virus (HBV) synthesize HBsAg at low levels. We have obtained a large increase in S gene expression by somatic hybridization of Vero cells with primary hepatocytes, which are the natural target cells for HBV infection. Fusion with cells other than hepatocytes did not enhance expression of the S gene. The Vero/hepatocyte hybrid clones analyzed are stable and have maintained a high level of HBsAg synthesis over prolonged periods. Hybrid cell lines may be of general interest for the high-level synthesis of proteins using cloned genes.

Animals↗
PubMed1989

An expression vector for high-level protein synthesis in Vero cells.

We have constructed two new multi-purpose cloning vectors, pNI1 and pNI2, that carry the Escherichia coli gene Ecogpt encoding the enzyme xanthine-guanine phosphoribosyl transferase as a dominant selective marker. The Ecogpt gene is under the control of either the long-terminal-repeat promoter of mouse mammary tumor virus, pNI1, or the simian virus 40 early promoter, pNI2. Another feature of the vectors is a polylinker preceded by the human metallothionein IIA promoter. We have used pNI2 for the synthesis of the hepatitis B surface antigen (HBsAg) at a high level in monkey Vero cells. We show that gene amplification and a concomitant stable increase of HBsAg synthesis can be achieved in these cells using modified selective medium containing hypoxanthine, aminopterin and thymidine, i.e., increasing the aminopterin and decreasing the hypoxanthine concentrations.

Animals↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1988

Presentation and immunogenicity of the hepatitis B surface antigen and a poliovirus neutralization antigen on mixed empty envelope particles.

Insertion of a synthetic DNA fragment encoding a poliovirus neutralization epitope into the S gene encoding the major envelope protein of hepatitis B virus has yielded hybrid (HBsPolioAg) particles closely resembling authentic 22-nm antigen (HBsAg) particles by expression of the modified gene in mammalian cells. In mice, these hybrid particles induce neutralizing antibodies against poliovirus but only weak immune response to HBsAg (F. Delpeyroux, N. Chenciner, A. Lim, Y. Malpièce, B. Blondel, R. Crainic, S. Van der Werf, and R. E. Streeck, Science 233:472-474, 1986). By cotransfection with different plasmids carrying either modified or unmodified S genes, we have now obtained mixed particles presenting both HBsAg and HBsPolioAg. When such particles were inoculated into rabbits, antibodies to both poliovirus and to HBsAg were induced. Moreover, the titers of neutralizing antibodies to poliovirus induced by HBsPolioAg were much higher than those previously obtained in mice. The design of multivalent particles carrying various peptide sequences or presenting several heterologous epitopes may therefore be possible.

Animals↗
PubMed1987

Identification of vaccinia promoters by heterologous expression of hepatitis B surface antigen in mouse cells infected by recombinant vaccinia viruses.

DNA fragments preceding open reading frames in a conserved segment of the vaccinia virus genome (Plucienniczak A., et al. (1985) Nucleic Acids Res. 13, 985-998) were cloned into plasmids upstream of the S gene of the hepatitis B virus encoding the surface antigen (HBsAg). Recombinant vaccinia virus obtained after insertion of these constructs into the thymidine kinase gene were used to infect mouse 1D cells. HBsAg was assayed in cellular supernatants. A strong promoter was thus identified in a 295 bp fragment preceding the coding region of the 147 kDa subunit of the vaccinia RNA polymerase.

Animals↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

Amplifiable expression vectors for mammalian cell lines.

We have constructed a prototype of a general expression vector for mammalian cells, which carriers a strong promoter active in a variety of tissues and animal species for expression of the gene of interest and a truncated gene for amplification in selective medium. Expression of the S gene of HBV encoding the surface antigen was used to evaluate various versions of this vector. The human metallothionein IIA promoter was found to be particularly efficient for expression of this gene, both in mouse L and monkey Vero cells. Including a promoterless tk gene in the vector led to gene amplification in Ltk- cells by selection of TK+ variants in selective medium containing hypoxanthine, aminopterine and thymidine (HAT medium). Concomitant increases of the S gene expression levels were initially 50-100 fold. Although many clones were unstable, even in selective medium, some maintained the high expression levels for over one year.

Animals↗
PubMed1986

A poliovirus neutralization epitope expressed on hybrid hepatitis B surface antigen particles.

The hepatitis B virus (HBV) envelope protein carrying the surface antigen (HBsAg) is assembled with cellular lipids in mammalian cells into empty viral envelopes. In a study to evaluate the capacity of such particles to present foreign peptide sequences in a biologically active form, in-phase insertions were created in the S gene encoding the major envelope protein. One of the sequences inserted was a synthetic DNA fragment encoding a poliovirus neutralization epitope. Mammalian cells expressing the modified gene secreted hybrid particles closely resembling authentic 22-nanometer HBsAg particles. These particles reacted with a poliovirus-specific monoclonal antibody and induced neutralizing antibodies against poliovirus. The results indicate that empty viral envelopes of HBV may provide a means for the presentation of peptide sequences and for their export from mammalian cells.

Animals↗
PubMed1986

Expression of immunogenically reactive diphtheria toxin fusion proteins under the control of the pR promoter of bacteriophage lambda.

The tox228 gene encoding the non-toxic, immunologically cross-reactive CRM228 mutant diphtheria toxin (DT) has been cloned downstream of the PR promoter and the cro translational initiation region of bacteriophage lambda carried by plasmid pCQV2 (Queen, 1983). Efficient transcription but no appreciable amount of a translational product corresponding to complete DT could be detected in Escherichia coli hosts. Deletion of 320 bp from the C-terminal region of the B-fragment of DT, and fusion of the truncated tox228 gene to lacZ yielded several hybrid beta-galactosidases (beta Gal) in an E. coli lon- strain in addition to beta Gal. The various DT fragments fused to beta Gal were immunologically reactive and were identified with antibodies specifically directed against the A- or the B-fragment of DT. Antibodies raised against the DT-beta Gal fusion proteins in guinea pigs cross-reacted with wild-type DT and its B-fragment and protected Vero cells in tissue culture against the lethal action of DT. Immunized guinea pigs survived upon injection of a five-fold lethal dose of wild-type DT.

Amino Acid Sequence↗
PubMed1986

Expression of the hepatitis B virus large envelope protein in Saccharomyces cerevisiae.

The yeast vector pPV2 has been constructed for inducible expression of non-fused proteins from the PHO5 promoter. Signals of the URA3 gene are used for transcription termination. The 226-amino-acid 'major' and the 389-amino-acid 'large' envelope protein of hepatitis B virus (HBV) have been produced in Saccharomyces cerevisiae following insertion of the S gene or of the entire pre-S region and the S gene, respectively, of HBV into pPV2. Although normally only a minor constituent of the viral envelope, the 'large' protein forms particles with cellular lipids similar to those composed of the 'major' envelope protein. Such particles carry pre-S1, pre-S2, and S-encoded epitopes and, in addition, a receptor for polymerized human serum albumin.

Gene Expression Regulation↗
PubMed1986

Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer.

The 7,909-nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer, has been determined and used to deduce the corresponding genome arrangement. Extensive sequence homologies and other genetic features are shared with the related oncogenic virus, human papillomavirus type 16, especially in the major reading frames. A surprising difference was found in the noncoding region of human papillomavirus type 33 as, unlike all other sequenced papillomaviruses, it contains a perfect 78-base pair tandem repeat.

Base Sequence↗
PubMed1986

The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens.

The proteins encoded by the open reading frames of papillomavirus genomes and the minor polypeptides detected in purified virions are still poorly defined. We show here by its expression in Escherichia coli that the open reading frame L2 of human papillomavirus type 1a codes for a minor structural protein of Mr 76,000. Antisera raised against a truncated L2-beta-galactosidase fusion protein in which the conserved N-terminal region of L2 is missing are type specific for human papillomavirus type 1 virions and are reactive at high dilutions. Expression of the L2-encoded type-specific antigens thus provides a powerful new tool for the identification of papillomaviruses.

Antigens, Viral↗