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H Schaller

Publications and source records attributed to H Schaller.

At least 73 records · Page 4Linked to original sources
PubMed1991

Genetic study and further biochemical characterization of a tobacco mutant that overproduces sterols.

A genetic and biochemical characterization is presented of a tobacco mutant that was previously shown to have an increased sterol content with an accumulation of biosynthetic intermediates. We first show that a precise regulation of the membrane sterol composition occurs in this mutant, via a selective esterification process. Indeed, sterols representing the usual end-products of the biosynthetic pathway are preferably integrated into the membranes as free sterols, whereas most of the intermediates pool is esterified and stored in cytoplasmic lipid droplets. It is further demonstrated that overproduction of sterols by the LAB1-4 mutant is due to a single nuclear and semi-dominant mutation. Finally, increase of biosynthesis and esterification of unusual sterols are shown to be responsible for the resistance of LAB1-4 calli to LAB170 250F, the triazole pesticide used to select this mutant. However, differentiated LAB1-4 tissues do not express the resistance trait, suggesting that sterol biosynthesis might not be the only site of action for the triazole at the plant level.

Crosses, Genetic↗
PubMed1991

Phosphorylation and rapid turnover of hepatitis B virus X-protein expressed in HepG2 cells from a recombinant vaccinia virus.

The human hepatitis B viral (HBV) genome contains a conserved open reading frame known as the X-gene which is capable of encoding a polypeptide of 16.565 kDa. The corresponding protein has so far not been identified directly in HBV-infected cells, but in transient transfection assays the X-gene encodes a product that functions as a transcriptional transactivator. To characterize the subcellular distribution, stability and post-translational modifications of X-protein in human hepatoma HepG2 cells, we have established a vaccinia virus expression system. As the major X-gene product, a protein with an apparent molecular weight of 16 kDa, and reacting with an X-protein-specific antiserum, was expressed from recombinant vaccinia virus. In indirect immunofluorescence assay, X-protein appeared to be distributed throughout the cells, with a tendency to localize at the nuclear periphery and to accumulate in granules as its levels increased. By subcellular fractionation, we found about one-third of X-protein associated with the fraction defined as the nuclear framework. In pulse-chase experiments, X-protein decayed with a bimodal half-life of 15 min and 3 h. X-protein having a half-life of about 15 min was found associated with the Triton X-100 detergent-soluble fraction of HepG2 cells, while that associated with the insoluble fraction turned over more slowly. By metabolic labeling with [32P] orthophosphate, we show that X-protein is capable of being phosphorylated. Modification by phosphorylation could play an important role in the regulation of X-protein function.

Amino Acid Sequence↗
PubMed1990

Translational inactivation of RNA function: discrimination against a subset of genomic transcripts during HBV nucleocapsid assembly.

Hepatitis B virus (HVB) is the prototype member of the hepadnaviridae, a family of small enveloped DNA viruses that replicate by reverse transcription. Assembly of replication-competent HBV nucleocapsids is based on specific interactions between the core protein, the product(s) of the P gene, and the RNA pregenome, which is marked for encapsidation by containing a sequence near its 5' end that acts in cis as an encapsidation signal. However, HBV produces several additional, almost identical, genomic transcripts that also bear the encapsidation sequence, but that are not encapsidated. The mechanism underlying this selection process has remained mysterious. Here we demonstrate that translating 80S ribosomes (but not scanning 40S ribosomal subunits) advancing into the encapsidation signal prevent its functioning. This finding reveals translational modulation of RNA function as a further regulatory mechanism employed by hepadnaviruses to utilize efficiently the restricted coding capacity of their extremely compact genome.

Amino Acid Sequence↗
PubMed1990

A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA.

The selective encapsidation of the hepadnaviral RNA pregenome from an excess of other viral and cellular mRNAs predicts specific protein-RNA interactions involving one or several sites on the pregenome. Using deletion analysis in a transient expression/packaging system in which all relevant viral proteins are provided in trans from a packaging-deficient helper genome, we identified near the 5'-end of the pregenome a 137 nucleotide sequence that is necessary and sufficient for RNA encapsidation; other parts of the 3 kb pregenome were found not to contribute to this process. The encapsidation sequence, which we call epsilon, possesses several interesting features with implications for the pregenome's function in RNA packaging, RNA translation and reverse transcription. (i) epsilon contains several indirect repeat sequences, suggesting a high degree of secondary structure, (ii) epsilon overlaps with the start signal for core gene translation, suggesting a mechanism to regulate the alternative use of the pregenome as core mRNA, (iii) epsilon does not contain the direct repeat sequences known to be involved in the initiation of viral DNA synthesis. Finally, our deletion analysis suggests that ribosomes translating the epsilon sequence from the precore start codon may interfere with genomic RNA packaging.

Base Sequence↗
PubMed1990

Complementation analysis of mutants defective in different steps of HBV reverse transcription.

The HBV P gene encodes a multifunctional polyprotein which contains the major enzymatic activities required for hepadnaviral reverse transcription (protein primer, DNA polymerase, and RNase H). Mutant HBV genomes unable to synthesize fully functional P gene products were analysed for their potential to be rescued by a second mutant HBV genome that either contained a wild type P gene (intergenic complementation) or a mutation in a functionally different P gene domain (intragenic complementation). This analysis was carried out by cotransfecting two mutants at a time into HepG2 cells and assaying for the production of core particles containing DNA polymerase activity. The results obtained demonstrate the existence of intergenic, but not of intragenic complementation. This indicates that the primary P gene product is not processed before core assembly, and furthermore that there is a rather free mixing of all HBV gene products in the HBV infected cell which can lead to the efficient propagation of defective viral genomes.

Defective Viruses↗
PubMed1990

Encapsidation of truncated human hepatitis B virus genomes through trans-complementation of the core protein and polymerase.

Mutational analyses and complementation tests were used to analyze the strategy of packaging and of replication of human hepatitis B virus (HBV). By creating new restriction enzyme sites and by varying the genome length of HBV mutants, we identified that the mutated genomes could be encapsidated through trans-complementation of the polymerase and/or core protein. This study demonstrates that the polymerase of HBV, similar to that of duck hepatitis B virus (DHBV), is synthesized de novo instead of through a core-polymerase fusion protein. The results also indicate that both the polymerase and the core protein can be supplied in trans during viral packaging, and that the complementation is not due to recombination between the cotransfected plasmids. Furthermore, HBV genome deleted down to 2.4 kb is still able to be encapsidated, as measured by the endogenous polymerase reaction. Taken together, these results provide a basis for using HBV as a vector to deliver foreign genes into hepatocytes and for defining the location of the packaging signal on the HBV genome.

Capsid↗
PubMed1990

In Vitro Selection of Calli Resistant to a Triazole Cytochrome-P-450-Obtusifoliol-14-Demethylase Inhibitor from Protoplasts of Nicotiana tabacum L. cv Xanthi.

The selection of biochemical mutants has been undertaken in order to elucidate regulatory and functional aspects of sterol biosynthesis in plants. 2-(4-Chlorophenyl)-3-phenyl-1-(1H-1,2,4- triazol-1-yl)-2,3-oxidopropane (LAB170250F), an experimental fungicide of the triazole family, was used as a selective agent. Indeed, this compound is a strong inhibitor of the cytochrome-P-450-obtusifoliol-14-demethylase in sterol biosynthesis. The selection strategy consisted of screening large populations of microcalli derived from ultraviolet-mutagenized protoplasts of Nicotiana tabacum L. cv Xanthi for resistance to a lethal concentration of LAB170250F. The best selective conditions were first determined, i.e. strength of the selection pressure as well as the time and duration of its application in the developmental process from protoplast to whole plant. Selection experiments resulted in the recovery of 40 resistant calli. These calli were divided into three classes according to the modification of their sterol content in response to LAB170250F. Some of these calli might be impaired in sterol biosynthesis, but most have a sterol profile identical to that of the control calli. This suggests that the toxic properties of LAB170250F are due to the parallel inhibition of sterol biosynthesis and of at least one additional unidentified target in the plant cell.

Journal Article↗
PubMed1990

The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation.

Encapsidation of the pregenomic RNA into nucleocapsids is a selective process which depends on specific RNA-protein interactions. The signal involved in the packaging of the hepatitis B virus (HBV) RNA pregenome was recently defined as a short sequence located near the 5' end of that molecule (Junker-Niepmann et al., EMBO J., in press), but it remained an open question which viral proteins are required. Using a genetic approach, we analyzed whether proteins derived from the HBV P gene play an important role in pregenome encapsidation. The results obtained with point mutations, deletions, and insertions scattered throughout the P gene clearly demonstrate that (i) a P gene product containing all functional domains is required both for the encapsidation of HBV pregenomic RNA and for packaging of nonviral RNAs fused to the HBV encapsidation signal, (ii) known enzymatic activities are not involved in the packaging reaction, suggesting that P protein is required as a structural component, and (iii) P protein acts primarily in cis, i.e., pregenomic RNAs from which P protein is synthesized are preferentially encapsidated.

Base Sequence↗
PubMed1990

Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity.

To correlate the hepatitis B virus P gene with the enzymatic activities predicted to participate in hepadnavirus reverse transcription, a series of P gene mutants containing missense mutations, in-phase insertions, and in-phase deletions was constructed by site-directed mutagenesis. These mutants were tested in the context of otherwise intact hepatitis B virus genomes for the ability to produce core particles containing the virus-associated polymerase activity. The results obtained suggest that the P protein consists of three functional domains and a nonessential spacer arranged in the following order: terminal protein, spacer, reverse transcriptase/DNA polymerase, and RNase H. The first two domains are separated by a spacer region which could be deleted to a large extent without significant loss of endogenous polymerase activity. In cotransfection experiments, all P gene mutants could be complemented in trans by constructs expressing the wild-type gene product but not by a second P gene mutant. This indicates that the multifunctional P gene is expressed as a single translational unit and independent of the core gene and furthermore that the gene product is freely diffusible and not processed before core assembly.

Amino Acid Sequence↗
PubMed1989

Biochemical characterization of a sterol mutant plant regenerated from a tobacco callus resistant to a triazole cytochrome-P-450-obtusifoliol-14-demethylase inhibitor.

We report here, for the first time, the biochemical characterization of a plant mutant impaired in sterol biosynthesis. A fertile plant was regenerated from a tobacco callus resistant to LAB170250F, a potent inhibitor of the cytochrome-P450-obtusifoliol-14-demthylase. The resistant callus and the leaves from the regenerated plant are characterized by profound qualitative and quantitative changes in their sterol content. Self-fertilization of this plant yielded seeds with the same biochemical features, indicating that the new phenotype is of mutational origin.

Chromatography, Gas↗
PubMed1989

Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins.

The expression strategy of the duck hepatitis B virus (DHBV) P gene, which is assumed to encode the viral reverse transcriptase, was investigated by mutational analysis. This study showed that P gene expression starts in the region where the P gene overlaps the viral core gene. However, in contrast to retroviral reverse transcriptases, which are expressed via gag-pol fusion protein intermediates, the DHBV P gene product was found to be synthesized starting at a P gene ATG codon. The resulting protein can complement polymerase-negative mutants in trans and can reverse transcribe viral pregenomic RNA that does not encode an active polymerase. These findings raise the question of how reverse transcription of cellular RNAs can be avoided in infected cells.

Animals↗
PubMed1989

Replication of duck hepatitis B virus in primary duck hepatocytes and its dependence on the state of differentiation of the host cell.

Primary duck hepatocytes obtained from Pekin ducks congenitally infected with duck hepatitis B virus were used to monitor expression of viral proteins and replication of viral DNA in cell culture. Duck hepatitis B virus core antigen, duck hepatitis B virus pre-surface antigen and duck hepatitis B virus DNA were detectable for at least 12 days after cell plating. Whereas expression of duck hepatitis B pre-surface antigen was constant during this time, expression of duck hepatitis B core antigen and of viral DNA rapidly declined. This diminished production of viral components in vitro was paralleled by a change of the hepatocytes toward a fibroblast-like morphology. Supplementation of cell culture medium with 2% dimethyl sulfoxide, a solvent known to maintain the differentiated state of cultured cells, retained competence of the cultured hepatocytes to express duck hepatitis B core antigen and duck hepatitis B virus DNA at high levels. In a second set of experiments, duck hepatitis B virus negative hepatocytes were infected with duck hepatitis B virus from serum of congenitally infected ducks. Dimethyl sulfoxide remarkably improved the competence of cultured duck hepatocytes to become productively infected. This function was maintained for at least 12 days postplating.

Animals↗
PubMed1989

A preliminary study on expression and significance of pre-S1 protein in liver tissue of patients with HBV infection.

Rabbit antiserum to Pre-S1 protein was used to establish peroxidase-antiperoxidase (PAP) and avidin-biotin-peroxidase complex (ABC) immunohistochemical techniques for detection of Pre-S1 protein in paraffin-embedded liver tissue. Pre-S1 protein could be expressed in hepatocyte cytoplasm and on membrane in some cases with chronic viral hepatitis, cirrhosis and hepatocellular carcinoma (HCC), and its expression was intimately associated with HBsAg, HBcAg in liver and HBV DNA in serum, indicating that pre-S1 protein may represent the essential component of hepatitis B virus (HBV) and also serve as one of the markers of HBV infection. The incidence of Pre-S1 protein was slightly lower in nontumorous liver of HCC than in other cases and Pre-S1 protein could not be detected in tumorous tissue of HCC suggesting that expression of pre-S1 protein may be suppressed in HCC cases.

Hepatitis B↗
PubMed1989

Detection of hepatitis B virus core gene products in sera and liver of HBV-infected individuals.

The C gene of hepatitis B virus (HBV) codes for at least two different proteins (p 21c and p 17e). To investigate the expression of C-gene-encoded proteins in vivo, serum and liver samples from HBsAg-positive patients as well as serial serum samples from an HBV-transfected chimpanzee were studied. Antibodies directed against bacterially synthesized C-fusion proteins were used in Western blots to test for the presence of p 21c and p 17e. In serial serum samples from the chimpanzee, p 21c and p 17e were detected concomitantly during the acute phase of the infection. When sera of patients with chronic HBV infection were studied, all sera containing p 17e were found to be positive also for p 21c. Sera positive for HBV DNA but negative for HBeAg were only positive for p 21c, indicating that HBeAg/p 17e is not an absolutely reliable marker for infectivity. In liver tissue specimens from 20 patients with HBV-related liver diseases, p 21c was detected in five cases, indicating viral replication. The p 17e antigen, however, was present only in low amounts in three of these five, suggesting that synthesis of p 21c and p 17e is not strictly coupled. C/Pol-gene-encoded fusion proteins were found in the liver tissue of only one patient with cirrhosis, supporting our previous finding that detectable levels of these proteins are expressed rarely.

Animals↗
PubMed1989

The secretory core protein of human hepatitis B virus is expressed on the cell surface.

Human hepatitis B virus (HBV) causes acute and chronic liver disease, which can result in tumor formation. An as yet unexplained phenomenon is that virus elimination usually correlates with the development of antibodies directed against the HBeAg, a secretory HBV core gene product which can be detected in the serum of infected patients. Expression of HBeAg in a human hepatoma cell line by using recombinant vaccinia viruses revealed that the HBeAg is not only secreted from HBeAg-producing cells but also incorporated into the outer cell membrane. No membrane-expressed core gene product could be detected when the cytoplasmic core protein (HBcAg) was expressed. Immune sera from patients who developed anti-HBe antibodies efficiently recognized the membrane-bound HBeAg, suggesting that surface-expressed HBeAg can serve as a target for an antibody-mediated elimination of HBV-infected cells.

Base Sequence↗
PubMed1989

Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus.

The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. The single conformational determinant responsible for HBc antigenicity in the assembled core (HBc) and a linear HBe-related determinant (HBe1) were both mapped to an overlapping hydrophilic sequence around amino acid 80; a second HBe determinant (HBe2) was assigned to a location in the vicinity of amino acid 138 but found to require for its antigenicity the intramolecular participation of the extended sequence between amino acids 10 and 140. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis B virus nucleocapsid.

Antibodies, Monoclonal↗
PubMed1989

Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease.

The hepatitis B virus (HBV) C gene directs the synthesis of two major gene products: HBV core antigen (HBcAg[p21c]), which forms the nucleocapsid, and HBV e antigen (HBeAg [p17e]), a secreted antigen that is produced by several processing events during its maturation. These proteins contain an amino acid sequence similar to the active-site residues of aspartic acid and retroviral proteases. On the basis of this sequence similarity, which is highly conserved among mammalian hepadnaviruses, a model has been put forward according to which processing to HBeAg is due to self-cleavage of p21c involving the proteaselike sequence. Using site-directed mutagenesis in conjunction with transient expression of HBV proteins in the human hepatoma cell line HepG2, we tested this hypothesis. Our results with HBV mutants in which one or two of the conserved amino acids have been replaced by others suggest strongly that processing to HBeAg does not depend on the presence of an intact proteaselike sequence in the core protein. Attempts to detect an influence of this sequence on the processing of HBV P gene products into enzymatically active viral polymerase also gave no conclusive evidence for the existence of an HBV protease. Mutations replacing the putatively essential aspartic acid showed little effect on polymerase activity. Additional substitution of the likewise conserved threonine residue by alanine, in contrast, almost abolished the activity of the polymerase. We conclude that an HBV protease, if it exists, is functionally different from aspartic acid and retroviral proteases.

Amino Acid Sequence↗