Peter Hans Hofschneider

Hepatitis B virus transactivator MHBst: activation of NF-kappa B, selective inhibition by antioxidants and integral membrane localization.

C‐terminal truncation of the middle surface antigen from hepatitis B virus (MHBs) gives rise to a novel transactivating protein, called MHBst. In this study we show that MHBst like the HBx protein of HBV, can cause nuclear appearance of NF‐kappa B DNA binding activity and induce various kappa B‐controlled reporter genes. While an inhibitor of protein kinase C could not block gene induction by MHBst, the antioxidants N‐acetyl‐L‐cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) could potently suppress transactivation at mM and microM concentrations, respectively. Also, kappa B‐dependent gene induction by the transactivator HBx was blocked. The effects were selective because PDTC did not interfere with MHBst and HBx‐induced activation of the c‐fos promoter/enhancer, nor with the basal activity of several other reporter genes lacking functional NF‐kappa B binding motifs. Our data suggest that induction of a prooxidant state is crucial for the activation of NF‐kappa B by MHBst and HBx and might be related to the hepatocarcinogenic potential of the viral proteins. MHBst had a subcellular localization unusual for a viral transactivator: it appeared to be an integral membrane protein of the endoplasmic reticulum.

The PreS2 activator MHBst of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice

The large hepatitis B virus (HBV) surface protein (LHBs) and C‐terminally truncated middle size surface proteins (MHBst) form the family of the PreS2 activator proteins of HBV. Their transcriptional activator function is based on the cytoplasmic orientation of the PreS2 domain. MHBst activators are paradigmatic for this class of activators. Here we report that MHBst is protein kinase C (PKC)‐dependently phosphorylated at Ser28. The integrity of the phosphorylation site is essential for the activator function. MHBst triggers PKC‐dependent activation of c‐Raf‐1/Erk2 signaling that is a prerequisite for MHBst‐dependent activation of AP‐1 and NF‐κB. To analyze the pathophysiological relevance of these data in vivo, transgenic mice were established that produce the PreS2 activator MHBst specifically in the liver. In these mice, a permanent PreS2‐dependent specific activation of c‐Raf‐1/Erk2 signaling was observed, resulting in an increased hepatocyte proliferation rate. In transgenics older than 15 months, an increased incidence of liver tumors occurs. These data suggest that PreS2 activators LHBs and MHBst exert a tumor promoter‐like function by activation of key enzymes of proliferation control.

Coxsackie B3 virus can replicate in cultured human foetal heart cells and is inhibited by interferon

Coxsackie B viruses (types 1 to 5) are the most frequent reported cause of acute viral myocarditis. To study the pathogenesis of the disease at the cellular level, we simulated an infectious situation by infecting cultured human foetal heart cells with Coxsackie B3 (CB3) virus. Successful replication of this virus could be demonstrated by the presence of virus particles inside cultivated foetal myocytes together with high titres of progeny virus of 10(8) plaque-forming units (PFU) per millilitre culture medium. Within 9 h of infection networks of myocytes lost their ability to contract spontaneously followed by disintegration and replacement by overgrowing fibroblasts which survived the infection. These cells produced CB3 virus continuously over several months, indicating carrier state infection of human myocardial fibroblasts. Human fibroblasts interferon (IFN-beta) was found to act as a potent inhibitor of the replication of this virus. Virus yields could be reduced from 1.2 x 1.8 x 10(5) PFU/ml culture medium when human heart cells were incubated with IFN-beta 20 h prior to challenge with a high input multiplicity of 50 PFU of CB3 virus per cell, demonstrating the major protective role of IFN-beta in CB3 viral infection. It thus appear that IFN-beta might become useful as an antiviral agent in the treatment of Coxsackie myocarditis.

Appearance of β-lactamase activity in animal cells upon liposome-mediated gene transfer

Abstract A restriction fragment, 875 bp, which encodes for a β-lactamase activity, was isolated from the Escherichia coli plasmid pBR322 DNA and entrapped in liposomes. The incubation of the DNA-liposomes with avian, murine, and human cultured cells results in the uptake of the DNA with the efficiency of around 2000 molecules per cell. Extracts of the recipient cells show a β-lactamase activity as demonstrated by spectroscopic and microbiological methods. These results indicate the expression of a prokaryotic gene in eukaryotic cells.

Integration of hepatitis B virus DNA: Evidence for integration in the single-stranded gap

The DNA of hepatitis B virus (HBV) can integrate into the genomes of infected human liver cells and may be related to the development of primary liver carcinoma (PLC). This report describes the analysis of three integrated HBV DNA sequences cloned from DNA of the human PLC cell line PLC/PRF/5. In all these sequences, integration of the viral DNA is at a site that is single-stranded (gap) in the unintegrated HBV DNA. Contrary to some speculations, there are no large terminal redundancies flanking the inserts as found in retroviral proviruses. The viral sequences show no obvious rearrangements in these clones, but integration can be associated with deletions in the viral DNA and also apparently in the host DNA.

Strand-specific detection of enteroviral RNA in myocardial tissue by in situ hybridization

In this report we describe the development and application of single-stranded RNA probes for strand-specific detection of enterovirus RNA in infected heart tissue by in situ hybridization. For synthesis of RNA probes a full-length reverse-transcribed, recombinant CVB3 cDNA was inserted into the transcription vector pSPT18. Run-off transcripts of plus-strand and minus-strand orientation were produced using either T7 or SP6 RNA polymerase. Binding specificity and sensitivity of the radioactively labelled RNA probes were determined by slot-blot hybridization. Due to the high degree of genetic identity among enteroviruses, the in vitro transcribed CVB3 RNA probes hybridized with various enterovirus serotypes, including group A and B coxsackieviruses and echoviruses, which are commonly implicated in human viral heart disease. Strand-specific in situ hybridization led to detection of viral plus-strand or minus-strand RNA in infected cell cultures and in myocardial tissue sections of infected mice. In consecutive sections either viral genomic plus-strand RNA or complementary minus-strand RNA were localized in the same infected myocardial cells. In situ hybridization with enterovirus-specific and highly sensitive single-stranded RNA probes is of particular interest for the diagnosis of myocardial infections and for studies concerning viral RNA replication.

Isolation and characterization of a ribonuclease III deficient mutant of Escherichia coli

SummaryA mutant of E. coli K 12 AB301 RNAase19-, selected for its inability to degrade double-stranded RNA, has been isolated and shown to have less than 1% of RNAase III-activity related to the parental strain.

Replication of the single-stranded DNA of the male-specific bacteriophage M13. Isolation of intracellular forms of phage-specific DNA.

The intracellular DNA has been isolated from Escherichia coli F+ cells infected with the small male-specific bacteriophage M13 and fractionated on a methylated albumin-kieselguhr column. Two infectious forms of phage-specific DNA were isolated and identified as a double-stranded replicative form and single-stranded DNA on the basis of their elution from the methylated albumin-kieselguhr column, infectivity to spheroplasts before and after heating, sedimentation at low ionic strength and buoyant density in CsCl. The replicative DNA was found to be resistant to thermal denaturation, suggesting that it may be a closed ring. It is estimated that there are at least 130 molecules of replicative DNA and 190 molecules of single-stranded DNA per infected bacterium.

Replication of the single-stranded DNA of the male-specific bacteriophage M13: Circular forms of the replicative DNA☆

Equilibrium centrifugation in caesium chloride, band sedimentation in alkaline solutions and electron microscopy have been used to study purified preparations of the M13 replicative DNA. The buoyant density of the M13 replicative DNA in a CsCl density gradient is 1.701, compared to a density of 1-710 for Escherichia coli DNA. When the replicative DNA is sedimented in alkaline solutions of CsCl (p = 1.35), two components are observed with uncorrected s-values of 48.6 +/- 0.2 and 15.2 +/- 0.5. Treatment of the replicative DNA with pancreatic DNase reduces the relative amount of the fast component in alkaline CsCl and similarly increases the relative amount of the slow component. Electron microscopic observation of the replicative DNA also shows two different forms of DNA, extended circles and condensed forms of DNA. The relative amount of condensed forms of DNA in a preparation of replicative DNA is equal to the relative amount of the fast component in alkaline velocity sedimentation. The average contour length of the extended circles is 2.19 +/- 0.07 micro.

β- and γ-Interferon in Chronic Active Hepatitis B: A Pilot Trial of Short-Term Combination Therapy

A controlled, randomized trial of a short-term, medium-dose combination therapy of β- and γ-interferon was performed in 20 patients with chronic active hepatitis B. According to clinical, biochemical, and histologic findings that were followed up for 16–24 mo, the combined treatment was successful in 5 of 10 patients. Two of the patients eliminated the virus completely, as confirmed by Southern blotting of hepatocellular deoxyribonucleic acid (DNA) against hepatitis B virus DNA. In the other 3 responders hepatitis B surface antigen persisted in the absence of hepatitis B e antigen, replicating hepatitis B virus DNA in the liver and inflammatory disease activity. Two of these responders with persistent hepatitis B surface antigen had hepatitis B virus DNA integrated into the hepatocyte genome and 1 responder had nonreplicating, episomal virus DNA. In the control group of 10 patients one spontaneous remission occurred. Antiviral treatment was significantly (p