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Biomedical subjects

H Schaller

Publications and source records attributed to H Schaller.

At least 55 records · Page 3Linked to original sources
PubMed1996

Hepatitis B virus replication--an update.

Hepatitis B virus (HBV), the causative agent of type B hepatitis in humans, is the prototypic member of the hepadnaviridae, a family of small enveloped DNA-containing viruses with pronounced host and tissue specificity. This property has greatly hampered progress in understanding the initial events of infection, i.e. attachment, penetration and uncoating. After the discovery, originally made with the duck hepatitis B virus (DHBV), that hepadnaviruses replicate by reverse transcription, DNA transfection of cloned wild-type and mutant HBV genomes into cell lines supporting virion formation has revealed the molecular mechanisms of the late steps of the infectious cycle in some detail. During the last few years, such studies have emphasized the differences between hepadnaviral and retroviral replication. Very recent research, however, indicates that the border separating the two viral families may not be as strict as previously thought. In this article, we will briefly summarize the pertinent differences, and will then focus on the new data, with particular emphasis on the initiation of reverse transcription.

Gene Products, pol↗
PubMed1995

Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage.

Apoptosis occurs in the normal liver and in various forms of liver disease. The CD95 (APO-1/Fas) (CD95) receptor mediates apoptosis, and liver cells in animal models are acutely sensitive to apoptosis initiated by this receptor. We have used primary human hepatocytes as a model system to investigate CD95-mediated apoptotic liver damage. Treatment of fresh human hepatocytes with low concentrations of agonistic antibodies against CD95 resulted in apoptosis of > 95% of the cultured liver cells within 4 and 7.5 h. Immunohistology of a panel of explanted liver tissues revealed that hepatocytes in normal livers (n = 5) and in alcoholic cirrhosis (n = 13) expressed low constitutive levels of CD95. CD95 receptor expression was highly elevated in hepatocytes in hepatitis B virus-related cirrhosis (n = 9) and in acute liver failure (n = 8). By in situ hybridization CD95 ligand messenger RNA expression was absent in normal liver but detected at high levels in livers with ongoing liver damage. In cases of hepatitis B virus-related cirrhosis and acute hepatic failure, ligand expression was found primarily in areas with lymphocytic infiltration. In contrast, in patients with alcoholic liver damage, high CD95 ligand messenger RNA expression was found in hepatocytes. These findings suggest that liver destruction in hepatitis B may primarily involve killing of hepatocytes by T lymphocytes using the CD95 receptor-ligand system. In alcoholic liver damage, death of hepatocytes might occur by fratricide and paracrine or autocrine mechanisms mediated by the hepatocytes themselves.

Antibodies, Monoclonal↗
PubMed1995

HBx protein of hepatitis B virus interacts with the C-terminal portion of a novel human proteasome alpha-subunit.

Two-hybrid protein interaction screening in yeast was used to identify proteins that interact with the HBx nonstructural protein of hepatitis B virus (HBV). A new human member of the proteasome alpha-subunit family was obtained. Its protein sequence closely resembles the 28 kD subunits from other organisms. The interaction with HBx was abolished by a two amino-acid insertion behind position 128 in HBx, in a region previously found to be essential for its transcriptional transactivation function. These data support a model of HBx acting indirectly on transcriptional processes. By binding to a specific proteasome alpha-subunit, HBx might interfere with degradative processes, thereby enhancing the half-life of different transcription factors and other nuclear regulatory proteins. Interaction with the Hu 28k proteasome subunit could thus provide a unifying explanation for the markedly pleiotropic effects of HBx.

Amino Acid Sequence↗
PubMed1995

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.

Animals↗
PubMed1994

A preliminary chemotaxonomic review of Kopsia (Apocynaceae).

The present paper is the result of a botanical and chemical study of the Indo-Pacific genus Kopsia (Plumerioideae-Rauvolfieae) Apocynaceae. Some of the species of this genus possess medicinal uses. A survey of the accepted taxa and their synonymy, based on the study of types, is presented. Studies on alkaloids provides chemotaxonomic information. Indeed, in some cases, chemical relationships occur between species belonging to the same botanical group.

Alkaloids↗
PubMed1994

Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime reverse transcription.

Hepadnavirus DNA minus strands are covalently linked at their 5' terminus to the viral P gene product, which has been taken to indicate that the hepadnaviral polymerase polypeptide itself also functions as a protein primer for initiating reverse transcription of the RNA pregenome. The present study confirms this indication by identifying the nucleotide-linked amino acid in the P protein sequence of the duck hepatitis B virus (DHBV). In a first set of experiments, mutational analysis of three phylogenetically conserved tyrosine residues in the DNA terminal (TP) domain indicated that of these, only tyrosine 96 was essential for both viral DNA synthesis in transfected cells and priming of DNA synthesis in a cell-free system. This assignment was confirmed by direct biochemical analysis: tryptic peptides from the DHBV P protein, 32P labelled at the priming amino acid by the initiating dGTP and additionally labelled internally by [35S]methionine, were isolated and analyzed in parallel to reference peptides synthesized chemically and 33P labelled by a tyrosine kinase. Mobility in high-performance liquid chromatography, as well as the release in stepwise amino acid sequencing of phospholabel and of [35S]methionine, identified the priming amino acid unequivocally as the tyrosine in the sequence 91KLSGLYQMK99, which is located in the center of the TP domain. Conserved sequence motifs surrounding Tyr-96 allow the prediction of the priming tyrosine in other hepadnaviruses. Weak sequence similarity to picornavirus genome-linked polypeptides (VPgs) and similar gene organization suggest a common origin for the mechanisms that use protein priming to initiate synthesis of viral DNA genomes or RNA genomes from an RNA template.

Amino Acid Sequence↗
PubMed1993

Two-codon insertion mutations of the HBx define two separate regions necessary for its trans-activation function.

A panel of mutants of the hepatitis B virus X gene (HBx) was constructed by oligonucleotide-directed insertion of two codons (Arg Pro) at 10- or 20-amino-acid intervals along the entire gene. These mutants were tested in transiently transfected HepG2 cells for their effects on HBx trans-activation of an AP1-CAT reporter plasmid. The effects of HBx mutations on the mRNA and protein stability were also determined. Our results reveal two separate internal domains of the HBx, one around amino acid residue 68 and the other between residues 110 and 139, that are necessary for its activity. Mutations in the amino terminus (to position 45), between the two necessary domains and in the extreme carboxyl terminal portion of HBx, have little or no effect on its activity.

Amino Acid Sequence↗
PubMed1993

Hepatitis B virus replication.

Hepatitis B virus (HBV) is the causative agent of B-type hepatitis in humans and the prototypic member of the hepadnaviruses. It is a small enveloped DNA virus that replicates via reverse transcription. Although hepadnaviruses are similar to retroviruses in basic life cycle and genome organization, recent studies have revealed several unique hepadnaviral strategies that optimize exploitation of their extremely small circular DNA genomes.

Animals↗
PubMed1993

Mechanisms governing hepadnaviral nucleocapsid assembly.

Reverse transcription of an RNA pregenome is the central step in the replication cycle of the hepatitis B viruses. This reaction takes place within the viral nucleocapsid composed of the core protein, product(s) of the P (pol) gene and the RNA pregenome. As the enzymatic activities required reside in the P-protein it plays a major role in the hepadnaviral life cycle. This article summarizes recent data on structure and function of the hepadnaviral P-protein and discusses its important role in the early steps of nucleocapsid assembly.

Capsid↗
PubMed1993

Hepadnavirus infection requires interaction between the viral pre-S domain and a specific hepatocellular receptor.

To better define the molecules involved in the initial interaction between hepadnaviruses and hepatocytes, we performed binding and infectivity studies with the duck hepatitis B virus (DHBV) and cultured primary duck hepatocytes. In competition experiments with naturally occurring subviral particles containing DHBV surface proteins, these DNA-free particles were found to interfere with viral infectivity if used at sufficiently high concentrations. In direct binding saturation experiments with radiolabelled subviral particles, a biphasic titration curve containing a saturable component was obtained. Quantitative evaluation of both the binding and the infectivity data indicates that the duck hepatocyte presents about 10(4) high-affinity binding sites for viral and subviral particles. Binding to these productive sites may be preceded by reversible virus attachment to a large number of less specific, nonsaturable primary binding sites. To identify which of the viral envelope proteins is responsible for hepatocyte-specific attachment, subviral particles containing only one of the two DHBV surface proteins were produced in Saccharomyces cerevisiae. In infectivity competition experiments, only particles containing the large pre-S/S protein were found to markedly reduce the efficiency of DHBV infection, while particles containing the small S protein had only a minor effect. Similarly, physical binding of radiolabelled serum-derived subviral particles to primary duck hepatocytes was inhibited well only by the yeast-derived pre-S/S particles. Together, these results strongly support the notion that hepadnaviral infection is initiated by specific attachment of the pre-S domain of the large DHBV envelope protein to a limited number of hepatocellular binding sites.

Animals↗
PubMed1992

Expression of the P-protein of the human hepatitis B virus in a vaccinia virus system and detection of the nucleocapsid-associated P-gene product by radiolabelling at newly introduced phosphorylation sites.

Hepatitis B virus (HBV) contains a particle-associated DNA polymerase/reverse transcriptase activity encoded by the P (pol) open reading frame. Due to its low abundance, the corresponding protein has so far escaped direct detection and structural analysis. As a first step to overcome these difficulties, a series of recombinant vaccinia viruses was constructed and used for the synthesis in human hepatoma cells of both the authentic full length protein and of its functional domains. Pulse chase experiments demonstrated that the P-proteins had very short half lives in striking contrast to the viral core protein expressed in parallel with the same system. No evidence was obtained for a specific proteolytic processing of the P-protein as occurring with retroviral pol gene products. Overexpression of P-protein by recombinant vaccinia viruses was then employed to develop a highly sensitive detection method based on the in vitro phosphorylation of newly introduced target sites for protein kinase A. The usefulness of this method was demonstrated in the analysis of encapsidated P-gene products that were transiently expressed from an appropriately modified HBV genome. The results obtained indicate that the P-protein acts unprocessed, at least during the initial steps of nucleocapsid assembly and reverse transcription, and that a fraction of the P-protein molecules is linked as such to the viral DNA. Direct detection of the hepadnaviral P-protein by in vitro phosphorylation should greatly facilitate future analyses on P-protein structure and function.

Base Sequence↗
PubMed1992

Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome.

Hepadnaviruses, as well as other pararetroviruses, express their pol (P) gene product unfused to the preceding core gene implying that these retroelements have developed a mechanism for initiating assembly and replication that is principally different from the one used by retroviruses and retrotransposons. We have analysed this mechanism for the human hepatitis B virus by using a newly developed, highly sensitive detection method based upon radiolabelling of the P protein at newly introduced target sites for protein kinase A. The results obtained demonstrate that polymerase encapsidation depends on the concomittant encapsidation of the HBV RNA pregenome and that packaging of the viral RNA, in turn, depends on the presence of P protein. Loss of P protein encapsidation by mutations inactivating the HBV RNA encapsidation signal epsilon could be compensated by trans-complementation with recombinant RNA molecules carrying the epsilon sequence. Thus, in contrast to retroviral replication, the interaction of the hepadnaviral P protein and the RNA genome at its packaging signal appears to be crucial for initiating the formation of replication-competent nucleocapsids. Furthermore, RNA control of P protein packaging stringently limits the number of polymerase molecules that can be encapsidated.

Capsid↗
PubMed1992

Duck hepatitis B virus infection of hepatocytes is not dependent on low pH.

The pH dependency for initiation of infection by the hepadnavirus duck hepatitis B virus (DHBV) was investigated in primary duck hepatocytes. First, an infection assay was developed using a radioimmunoblot to measure DHBV e antigen secreted into tissue culture fluid from infected hepatocytes. The quantity of this viral marker was proportional to the duration of inoculation and the amount of DHBV used as inoculum. The role of pH in initiation of DHBV infection was investigated by using this assay, but no dependence on low pH was found. DHBV was able to infect hepatocytes in the presence of NH4Cl and monensin, agents that raise the pH in intracellular vesicles and prevent penetration of viruses dependent on low pH in endosomes. In control experiments, infection by Semliki Forest virus, which is low pH dependent, was inhibited, whereas herpes simplex virus type 1 infection, which is pH independent, occurred. Attempts to trigger DHBV-cell fusion by exposure of DHBV prebound to hepatocytes to mildly acidic pH were unsuccessful. In these experiments, it was also observed that internalization of DHBV occurred only between pH 6.8 and 8.0. Additionally, in the absence of cells, infectivity of DHBV was stable at pH 4.6 to 4.8, which is lower than the pH encountered in endosomes (pH 5 to 6.6). Thus, no evidence for a role for mildly acidic pH in the initiation of DHBV infection was found. Therefore, we propose that the infection route followed by DHBV resembles that of the group of enveloped viruses, including herpesviruses, that fuse with their host cells at neutral pH.

Ammonium Chloride↗