Hans Will

Regulation of p53 activity in nuclear bodies by a specific PML isoform

Covalent modification of the promyelocytic leukaemia protein (PML) by SUMO‐1 is a prerequisite for the assembly of nuclear bodies (NBs), subnuclear structures disrupted in various human diseases and linked to transcriptional and growth control. Here we demonstrate that p53 is recruited into NBs by a specific PML isoform (PML3) or by coexpression of SUMO‐1 and hUbc9. NB targeting depends on the direct association of p53, through its core domain, with a C‐terminal region of PML3. The relocalization of p53 into NBs enhances p53 transactivation in a promoter‐specific manner and affects cell survival. Our results indicate the existence of a cross‐talk between PML‐ and p53‐dependent growth suppression pathways, implying an important role for NBs and their resident proteins as modulators of p53 functions.

A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [gamma-32P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.

Naturally occurring variants of hepatitis B virus.

Publisher Summary Although most living biological systems have developed intricate mechanisms to keep the genetic information stable, many viruses, including hepatitis B virus (HBV) can undergo rapid and drastic sequence changes. This discusses the data from extensive research on the genetic variability of HBV done during the past decade and the implications for the life cycle of the virus, immune recognition, and pathogenesis. It also discusses currently available data on the epidemiology of mutants and their functional significance. It is still largely unknown whether mutants, particularly those with combinations of different mutations, have an impact on liver disease, virus persistence, and course and outcome, especially of chronic infection. Therefore, more long-term follow-up studies, analyses of complete genomes, and functional studies of variants in appropriate animal models are needed. Important questions to be answered in the future concern the driving forces for the selection of HBV variants and the influence of mutations on immune recognition. Answering these questions will deepen the understanding of the virus–host interplay and eventually help to develop new strategies for the successful therapy and eradication of HBV infection.

A new hepatitis B virus strain in patients with severe anti-HBe positive chronic hepatitis B

In hepatitis B virus carriers who are anti-HBe positive despite ongoing viral replication (HBcAg in liver and HBV-DNA in serum) the natural course of hepatitis is severe and the response to interferon is low. We investigated whether a new hepatitis B virus (HBV) strain could be involved. A translational termination codon at the carboxyterminal end of the pre-C region responsible for the lack of HBeAg secretion was found in 18 of 19 HBV clones isolated from seven pedigreed patients with this clinical syndrome. The same findings were confirmed by direct sequencing. One of these patients underwent a liver transplant and HBV infection of the new liver resulted in high titered viremia and intrahepatic expression of HBcAg, without detectable HBeAg in serum. Another patient was superinfected by hepatitis delta virus (HDV) and developed high titres of total and IgM anti-HD. In spite of this, chronic hepatitis remained unchanged during 7 years of follow-up. These data strongly suggest that a viable precore minus mutant of hepatitis B virus is responsible for the lack of HBeAg in the serum of these patients. The HBV variant may explain the peculiar geographic distribution of anti-HBe positive hepatitis. The variations in the virus genome sequence may cause the more severe form of liver disease and modify the pathogenicity in the case of HDV superinfection.

Hepatitis B virus antigen-specific T-cell activation in patients with acute and chronic hepatitis B

Since the hepatitis B virus is noncytopathic, it is generally believed that the individual specific immune response determines the course of infection. The lack of data about hepatitis B virus-specific T-cell reactions in acute infection led us to investigate the specific cellular immune response of infected individuals in terms of proliferation, and gamma-interferon and lymphotoxin production. Our results demonstrate that peripheral blood mononuclear cells (PBMNC) from patients with acute and chronic hepatitis B respond weakly to HBsAg. In contrast, patients with acute hepatitis show a vigorous response to the nucleocapsid antigen (HBcAg) in terms of proliferation and lymphokine production, while only few chronic virus carriers gave a proliferative response. Either of the antigens could activate lymphocytes to produce gamma-interferon and lymphotoxin, cytokines which may modulate antiviral immune response.

Full-length and truncated versions of the hepatitis B virus (HBV) X protein (pX) transactivate the cMYC protooncogene at the transcriptional level

The products of the human hepatitis B virus (HBV) and woodchuck hepatitis B virus X genes (pXs) transactivate homologous and heterologous genes including the HBV-X and core promoters, the human immunodeficiency viruses 1 (HIV-1) and 2 (HIV-2) long terminal repeats and the beta interferon regulatory sequences. We report here that pX is also able to influence the expression of both extrachromosomal transfected c-myc regulatory sequences and endogenous c-myc gene. pX acts by increasing transcription of the c-myc gene and do not affect c-myc mRNAs stability. The presence of the first AUG of the X-ORFs is indeed necessary for the production of an active pX. The very carboxyterminus of the pX protein is dispensable for this transactivating activity and at least one domain important for its action is located between aminoacids 103 and 117.

Monoclonal antibodies against chromosomal proteins of Drosophila melanogaster

Total nuclear protein from the embryonic D. melanogaster cell line Kc and crude hydroxyapatite fractions thereof were used for immunization of mice. From the spleen cells of these mice we established 755 permanent lymphoid cell lines using the hybridoma technique originally developed by Köhler and Milstein (1975). Radioimmunoassay showed 455 of these cell lines secreted antibodies which bound to component(s) contained in the antigen mixtures used for immunization. Screening of 311 cell lines using indirect immunofluorescence revealed 58 lines whose antibodies showed a highly selective staining pattern on polytene chromosomes from the salivary glands of D. melanogaster third instar larvae. Eight of these cell lines were cloned and further characterized. We were able to order the staining patterns into three distinct classes based on the staining behaviour of the monoclonal antibodies: staining of active regions, staining of phase dark bands or staining of most interbands. The molecular weight of those antigens against which the monoclonal antibodies were directed was determined in SDS polyacrylamide gels.

Prevalence and type of Pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area

The sequence variability in the pre-C region of the hepatitis B virus (HBV) genome in the serum of 42 anti-HBe antibody positive carriers with chronic hepatitis B was studied by PCR and direct sequencing to determine prevalence and type of HBV pre-C mutants in a highly endemic area. Except for one, all patients were infected with viruses containing mutations in the pre-C region which prevent precore and e-antigen (HBeAg) expression: 33 were infected predominantly or exclusively with variants containing a stop codon; two had a mixture of wild-type and a pre-C stop codon mutant virus; three had precore variants with mutations of the pre-C initiation codon and two of them an additional stop codon; four had a frameshift mutation; and one had two stop codons. One patient was infected with viruses which contained a mutation creating an amino acid exchange which should not prevent precore and HBeAg expression. These data demonstrate that in an endemic area a higher prevalence and even broader spectrum of pre-C HBV mutants are found than has been recognized previously in anti-HBe positive patients with chronic hepatitis B.

A new hepatitis B virus variant in a chronic carrier with multiple episodes of viral reactivation and acute hepatitis

To examine whether there are HBV variants not yet described which cannot express HBe due to a mutation in the pre-C region, and, if they exist, whether they cause a particular course of infection and disease, we analyzed the HBV genomes of a HBs/anti-HBe positive chronic carrier who had several episodes of acute reexacerbations of chronic hepatitis with at least two viremic phases. Direct sequencing of the precore/core and the pre-S regions of the HBV sequences of both viremic phases amplified by the polymerase chain reaction revealed that they were very similar to each other but substantially divergent from published HBV genomes. Both virus populations contained a mutation in the first nucleotide of the pre-C translation initiation codon (AUG/CUG) which prevents HBeAg expression. These data demonstrate the existence of a new HBV variant which can enter a latent phase from which it can be reactivated with acute reexacerbation of liver inflammation.

Hepatitis B virus transcription in the infected liver.

Hepatitis B virus (HBV) transcription was studied in the liver of an infected chimpanzee and compared with HBV transcription in heterologous systems. Besides the well characterized 2.3‐kb surface antigen mRNA produced in most systems, a second major transcript was identified in the liver. This 3.8‐kb transcript (+/‐ 300 bases) is slightly larger than the HBV genome and is probably involved both in core/e antigen synthesis and in HBV replication via reverse transcription. In addition, minor variants of the 2.3‐kb surface antigen mRNA were characterized as probably being involved in the expression of HBsAg‐related minor proteins. Finally, several potential transcription signals, identified on the HBV genome using heterologous expression systems, were found to be poorly active if at all in the infected liver, thereby stressing the importance of HBV transcription studies performed with liver material.