Claus H. Schröder

Hepatitis B Virus Genome Is Organized into Nucleosomes in the Nucleus of the Infected Cell

Hepatitis B virus (HBV) nucleoprotein complexes were isolated from nuclei of the human hepatoblastoma cell line HepG2.2.15. Under conditions of physiological ionic strength, the complexes sedimented at a rate corresponding to about 82 S. They contained viral DNA, histone, and nonhistone proteins. For DNA a circular, covalently closed structure was shown both by CsCl gradient centrifugation and electron microscopy. Spread preparations revealed the typical “beads-on-a-string” appearance of nucleosomally organized DNA. The average number of nucleosomes was 18, resulting in a biochemical repeat unit of HBV chromatin of approximately 180 base pairs of DNA. This value was confirmed by experiments analyzing the structure of the HBV chromatin by the use of micrococcal nuclease. Electron microscopy demonstrated that exposure to high ionic strength conditions resulted in removal of nucleosomes from the complexes, but also revealed proteinaceous structures remaining bound to viral DNA molecules. The nature of these residual proteins is discussed. Since native nucleoprotein complexes could be precipitated with HBV-core antibodies, core protein appeared to be one of the nonhistone proteins.

Replacement of Glycoprotein B Gene Sequences in Herpes Simplex Virus Type 1 Strain ANG by Corresponding Sequences of the Strain KOS Causes Changes of Plaque Morphology and Neuropathogenicity

DNA sequences encoding glycoprotein B (gB) derived from herpes simplex virus type 1 (HSV-1) strain KOS321 were transferred to HSV-1 ANG. In cotransfection experiments the cloned HSV-1 KOS BamHI G fragment served as donor, and genomic DNA of two ANG variants as recipients. One of these variants, HSV-1 ANG path, expresses gC and the other, C18, was a spontaneous gC-negative mutant. Both ANG strains are of the syncytial (syn) phenotype whereas HSV-1 KOS321 is non-syncytial (syn+). Recombinants were identified by means of a monoclonal antibody which selectively recognizes gBKOS. Among the HSV-1 ANG path/gBKOS recombinants, the majority displayed an altered plaque morphology, i.e. they were of the syn+ phenotype. In contrast all of the C18/gBKOS recombinants were of the syn phenotype. The possibility that the mutant C18 carries a syn mutation not present in the parental strain could be excluded. Marker transfer experiments involving subfragments of the gB gene mapped the syn mutation of HSV-1 ANG path to a locus within the gene that has been previously termed syn 3. Subclones of HSV-1 ANG path were established either directly or after intermittent transfection or cotransfection with the KOS BamHI G fragment. The pathogenicity in mice of these clones was compared. The data obtained indicated that at high frequency, the BamHI G fragment confers apathogenicity.

Experimental infection of inbred mice with herpes simplex virus. II. Interferon production and activation of natural killer cells in the peritoneal exudate

The peritoneal exudate cells (PEC) represent the first line of defence against the virus in the mouse model of intraperitoneal (i.p.) infection with herpes simplex virus (HSV). We have therefore studied interferon production and activation of natural killer (NK) cells in vitro in PEC of HSV-injected mice. Injection of HSV caused a marked increase in NK cell activity, as reported by others. PEC from HSV-injected mice also produced high titres of interferon. This observation may be important since induction of interferon appears to be the primary event whereas activation of NK cells - as generally accepted - represents a secondary effect of the interferon produced. The HSV-induced NK cells shared the properties of NK cells in that they were sensitive to a monoclonal anti-theta antibody and to a monoclonal anti-Qa 5 antibody. In contrast, the cells producing interferon were not sensitive to either antibody. Irradiation (200 R) of the mice 24 h before injection of the virus decreased interferon production by more than 90%. The identity of the interferon-producing cells is unknown, but they may represent B cells.

Mutations on Free and Integrated Hepatitis B Virus DNA in a Hepatocellular Carcinoma: Footprints of Homologous Recombination

Hepatitis B virus nucleotide sequences derived from a hepatocellular carcinoma with free and multiply integrated viral DNAs were determined. Based on a comparison within the X-gene region, cloned free viral DNA previously had been attributed to two distinct groups of preC minus genomes. The comparison of the complete sequence identified one of the genome equivalents as a recombinant between members of these groups. Four different integrated viral DNA elements were cloned and analysed. Similarity to either one of two DNAs representing the two groups of free viral DNA on one hand and the presence of certain mutations only on integrated DNA on the other hand, allowed to recognize distinct segments within the integrants. The data suggest a contribution of different but related genotypes to contiguous stretches of integrated viral DNA via homologous recombination. On this basis an evolutionary relationship between free and integrated DNAs of the preC and the preC minus genotype could be recognized when short sequence segments were compared. The observed coexistence on a given integrated DNA of segments homologous to free viral DNA and of segments homologous to another integrated DNA is consistent with (1) a long lasting association of individual genotypes with dividing cells and (2) multiple integration events being the result of a series of steps not separated by a long time span.

Herpes simplex virus type 1-induced interferon production and activation of natural killer cells in mice.

Cultures of peritoneal exudate cells (PEC) of non-immune C57BL/6 mice reacted to infectious herpes simplex virus type 1 (HSV), but not to non-infectious virions by producing interferon. Similarly, when the virus preparations were injected intraperitoneally (i.p.) and interferon was determined in the wash-out fluid, interferon could only be detected after injection of infectious HSV. All interferons were mixtures of interferon-alpha and -beta. The cell that produced interferon was not sensitive to anti-theta serum or anti-asialo-GM1 serum, it was retained on nylon wool columns and removed by plastic adherence. Silica treatment of PEC abolished HSV-induced interferon production whereas irradiation by 2000 R was without effect. Pure cultures of mouse T cells did not produce interferon when treated with infectious HSV whereas pure cultures of mouse bone marrow macrophages did produce interferon. Thus, we suggest that in PEC the cells which produced interferon were of the macrophage lineage. In further experiments we have analysed in vivo activation of natural killer (NK) cells in the peritoneal cavity of mice. Non-infectious virions were as effective as infectious HSV and doses of HSV too low to induce measurable titres of interferon caused a marked activation of NK cells.

Protection of Mice by an Apathogenic Strain of HSV-1 Against Lethal Infection by a Pathogenic Strain of HSV-1

DBA/2 and C57BL/6 mice were infected intraperitoneally with two different strains of herpes simplex virus type 1, HSV-1 ANG and HSV-1 WAL. Unlike HSV-1 WAL, HSV-1 ANG was apathogenic by this mode of infection. Furthermore, infection with HSV-1 ANG protected mice of both inbred strains against infections with lethal doses of pathogenic HSV-1 WAL. This protection was observed when the apathogenic virus was given with the pathogenic virus or 4 to 24 h before it.

Overexpression of p53 protein is not directly related to hepatitis B x protein expression and is associated with neoplastic progression in hepatocellular carcinomas rather than hepatic preneoplasia.

p53 mutations and binding of p53 to hepatitis B virus (HBV) x protein (HBx) have been suggested as alternative mechanisms of development of hepatocellular carcinomas (HCCs) in man, both processes resulting in intracellular accumulation of the protein which is detectable by immunohistochemical approaches. We have examined p53 expression in 149 explanted human livers, including 39 cases infected with HBV and 35 bearing HCC. p53 was demonstrated immunohistochemically in 51% of HCC samples (18/35), localized mainly in fast growing poorly differentiated areas. Accumulation of mutant p53 was verified by immunoprecipitation in most of the positive HCC samples (14/15), implying occurrence of p53 mutations. No cells positive for p53 were found in 354 preneoplastic hepatocellular lesions examined. This indicates that p53 mutation is associated with progression, rather than early development, of HCC in the low-aflatoxin B(1)-exposed region. The intracellular distribution patterns of p53 and HBx were different, with the former within nuclei and the latter confined to cytoplasmic compartment. HBx did not coimmunoprecipitate with p53. These data indicate that p53-HBx binding is infrequent, if it really occurs, in HBV-infected human liver, and that it cannot be a common mechanism of HBV-associated hepatocarcinogenesis. In addition, p53 accumulation was also observed in some parenchymal and ductular (oval) cells in cirrhotic livers and, more frequently, in fulminant hepatitis, being independent of HBx expression, and seemingly associated with the damage and/or regeneration of liver parenchyma, perhaps merely reflecting a cellular stress response.

Mouse Monoclonal Antibody Directed against Hepatitis B Virus X Protein Synthesized in Escherichia coli: Detection of Reactive Antigen in Liver Cell Carcinoma and Chronic Hepatitis

A mouse monoclonal antibody directed against the protein product of the hepatitis B virus X open reading frame was prepared. This antibody was used to screen liver tissue sections from patients with chronic hepatitis (CH) and patients with liver cell carcinoma (LCC). Reactive antigen was detected by immunohistochemistry in about 30% auf the samples from CH patients and in about 80% of the samples from LCC patients regardless of whether tumor or surrounding nontumor tissue was analyzed. A predominant localization of the antigen in the cytoplasm was observed. In liver sections of CH patients the presence of HBx or HBx-related protein appeared to correlate with the presence of the classical viral antigens HBs- and/or HBcAg. A similar correlation was not found in liver or tumor tissue samples from LCC patients. The occurrence of X-monoclonal-antibody-reactive protein (Xarp) at a low frequency in liver tissue from patients without hepatitis B virus related disease suggests that Xarp in some cases may not be identical with the putative viral X antigen.

Replication of hepatitis B virus in a hepatocellular carcinoma.

Hepatitis B virus transcripts and DNA from paired samples of neoplastic and nonneoplastic liver tissue of HBsAg seropositive patients were analyzed. The data obtained support the view that transcription of integrated DNA is frequent, both in neoplastic as well as in nonneoplastic liver tissue. In the case of one patient, integrated and free forms of hepatitis B virus DNA were detected in the tumor. Complete cycles of viral replication in this tumor were suggested by the following markers: (i) DNA and RNA intermediates expected to occur during replication of the viral genome, (ii) HBcAg and HBsAg, (iii) core and Dane particles. Viral DNA cloned from tumor tissue was proven to be replication competent in a transient replication assay. Five independent clones of viral DNA were established and found to be closely related at the nucleotide level. A preX open reading frame and a stop codon within preC were common features. In tissue surrounding the tumor, a nonreplicative state of virus infection prevailed, characterized by free viral DNA exclusively of the covalently closed, circular form. The replication of the viral DNA appeared to be blocked at the level of transcription.

Characterization of a Tree Shrew Herpesvirus Isolated from a Lymphosarcoma

Summary A herpesvirus was isolated from a lymphosarcoma culture of tree shrews (Tupaia belangeri) and termed Tupaia herpesvirus 2 (THV-2). Electron microscopy of THV-2 revealed the presence of virus particles with nucleocapsids of about 100 nm surrounded by large envelopes compatible with virions of the herpesvirus group. An extensive host range study revealed the Tupaia embryonic fibroblasts are the cells of choice for the efficient propagation of THV-2. This cell line was used for the continued propagation and plaque assay of THV-2. The mol. wt. of the virus DNA was found to be 100 × 106. The buoyant density of THV-2 was 1.724 g/ml. The DNA of THV-2 was compared to the DNA of herpes simplex virus (HSV) and to another previously isolated herpesvirus from apparently healthy tree shrews (THV-1). The analysis was carried out using the restriction endonuclease EcoRI and the cleavage pattern of THV-2 DNA resulted in DNA fragments which were different from those of HSV-1 DNA and distinguishable from the DNA of THV-1.