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Biomedical subjects

Josef Köck

Publications and source records attributed to Josef Köck.

10 recordsLinked to original sources
PubMed2005

Hepatitis B virus mutations associated with fulminant hepatitis induce apoptosis in primary Tupaia hepatocytes.

Hepatitis B virus (HBV) core promoter mutations have been implicated in the pathogenesis of fulminant hepatitis B. Due to the limited availability of primary human hepatocytes, the functional characterization of HBV mutants has been performed predominantly in transformed cells, which may not represent ideal model systems for studying virus-cell interactions. We and others have shown that primary hepatocytes of the tree shrew Tupaia belangeri support HBV infection and replication. In this study, we used primary Tupaia hepatocytes to analyze the phenotype of two HBV core promoter mutations that have been associated with a clinical outbreak of fatal fulminant hepatitis. Similar to previous findings in human hepatoma cells, the HBV core promoter mutations resulted in enhanced viral replication and core expression. Surprisingly, however, the presence of the mutations had a marked effect on hepatocyte viability not previously observed in hepatoma cells. Reduced cell viability was found to be due to the induction of apoptosis, as evidenced by caspase-3 activation and nuclear fragmentation. In conclusion, HBV mutants exhibit a novel phenotype in primary hepatocytes distinctly different from previous findings in hepatoma cell lines. This phenotype may have important implications for the understanding of the fulminant clinical course associated with HBV mutations.

Animals↗
PubMed2005

APOBEC-mediated interference with hepadnavirus production.

APOBEC3G is a cellular cytidine deaminase displaying broad antiretroviral activity. Recently, it was shown that APOBEC3G can also suppress hepatitis B virus (HBV) production in human hepatoma cells. In the present study, we characterized the mechanisms of APOBEC-mediated antiviral activity against HBV and related hepadnaviruses. We show that human APOBEC3G blocks HBV production in mammalian and nonmammalian cells and is active against duck HBV as well. Early steps of viral morphogenesis, including RNA and protein synthesis, binding of pregenomic RNA to core protein, and self-assembly of viral core protein, were unaffected. However, APOBEC3G rendered HBV core protein-associated full-length pregenomic RNA nuclease-sensitive. Ongoing reverse-transcription in capsids that had escaped the block in morphogenesis was not significantly inhibited. The antiviral effect was not modulated by abrogating or enhancing expression of the accessory HBV X protein, suggesting that HBV X protein does not represent a functional homologue to the HIV vif protein. Furthermore, human APOBEC3F but not rat APOBEC1 inhibited HBV DNA production. Viral RNA and low-level DNA produced in the presence of APOBEC3F or rat APOBEC1 occasionally displayed mutations, but the majority of clones were wild-type. In conclusion, APOBEC3G and APOBEC3F but not rat APOBEC1 can downregulate the production of replication-competent hepadnaviral nucleocapsids. In contrast to HIV and other retroviruses, however, APOBEC3G/3F-mediated editing of nucleic acids does not seem to represent an effective innate defense mechanism for hepadnaviruses.

APOBEC-1 Deaminase↗
PubMed2005

Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes.

BACKGROUND/AIMS: Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection. METHODS: Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera. RESULTS: Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil. CONCLUSIONS: uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.

Adenine↗
PubMed2004

Hepatitis B virus nucleocapsids formed by carboxy-terminally mutated core proteins contain spliced viral genomes but lack full-size DNA.

The carboxy-terminal sequence of the hepatitis B virus (HBV) core protein constitutes a nucleic acid binding domain that is rich in arginine residues and contains three serine phosphorylation sites. While dispensable for capsid assembly, this domain is involved in viral replication, as demonstrated by the effects of mutations on RNA packaging and/or reverse transcription; however, the underlying mechanisms are poorly understood. Here we tested a series of core protein mutants in which the three serine phosphorylation sites were replaced by glutamic acid, in parallel with a previously described deletion variant lacking the 19 C-terminal amino acid residues, for their ability to support viral replication in transfected hepatoma cells. Replacement of all serines and the deletion gave rise to nucleocapsids containing a smaller than wild-type DNA genome. Rather than a single-stranded DNA intermediate, as previously thought, this was a 2.0-kbp double-stranded DNA molecule derived from spliced pregenomic RNA (pgRNA). Interestingly, full-length pgRNA was associated with nucleocapsids but was found to be sensitive to nuclease digestion, while encapsidated spliced RNA and 3' truncated RNA species were nuclease resistant. These findings suggest that HBV pgRNA encapsidation is directional and that a packaging limit is determined by the C-terminal portion of the core protein.

Amino Acid Sequence↗
PubMed2003

Inhibitory effect of adefovir and lamivudine on the initiation of hepatitis B virus infection in primary tupaia hepatocytes.

Adefovir dipivoxil and lamivudine are two safe and efficacious drugs licensed for the treatment of chronic hepatitis B virus (HBV) infection. Both drugs inhibit the viral polymerase, resulting in a profound suppression of virus production. Blocking the viral polymerase may also affect the initiation of HBV infection, because HBV virions harbor a partially double-stranded genome and productive infection requires completion of viral plus-strand DNA synthesis with subsequent formation of covalently closed circular DNA (cccDNA). To address this issue, we used primary hepatocytes from the tree shrew Tupaia belangeri that were recently shown to be susceptible to HBV infection. Treatment of cells with either drug partially inhibited initial HBV cccDNA formation. Adefovir was more effective than lamivudine, resulting in a 3-fold reduction of RNA synthesis and viral surface antigen production. However, prevention of initial cccDNA formation was incomplete even after combined treatment, whereas de novo synthesis of viral replicative intermediates was completely suppressed. A possible explanation for this observation is the genomic plus-strand gap of less than 200 bases in some virions, limiting the window for antiviral action. In conclusion, nucleos(t)ide analogues can target initial plus-strand DNA repair and reduce but not completely block HBV infection.

Adenine↗
PubMed2002

Cis-preferential recruitment of duck hepatitis B virus core protein to the RNA/polymerase preassembly complex.

Hepadnaviral replication requires the concerted action of the polymerase and core proteins to ensure selective packaging of the RNA pregenome into nucleocapsids. Virus assembly is initiated by cis-preferential binding of polymerase to the encapsidation signal straightepsilon, present on pregenomic RNA. Using the duck hepatitis B virus (DHBV) model, we analyzed how core protein is recruited to the RNA/polymerase preassembly complex. Two sets of trans-complementation assays were performed in cotransfected hepatoma cells. First, a replication-competent DHBV construct was tested for its ability to rescue replication of genomes bearing mutations within the core region. Self-packaging of wild-type pregenomes was more efficient than cross-packaging of core-deficient pregenomes, and this bias was strongly enhanced if mutant pregenomes coded for self-assembly-competent, but packaging-deficient, core proteins. Second, the site of wild-type core protein translation, i.e., pregenomic RNA (cis) or separate messenger RNA (trans), was analyzed for its effect on the phenotype of a previously described dominant-negative (DN) DHBV core protein mutant. This mutant forms chimeric nucleocapsids with wild-type core proteins and blocks reverse transcription within most, but not all, mixed particles. Strikingly, suppression of viral DNA synthesis by the mutant increased 100-fold when wild-type core protein was provided in trans. Our results suggest that recruitment of core protein to the DHBV preassembly complex occurs in a cis-preferential manner. This mechanism may account for the leakiness of DN DHBV core protein mutants targeting reverse transcription.

Binding Sites↗
PubMed2002

Virus replication and virion export in X-deficient hepatitis B virus transgenic mice.

The function of the X protein (pX) in the replication cycle of mammalian hepadnaviruses is enigmatic. Using tissue culture experiments it has been shown that the X gene product is not central to hepatitis B virus (HBV) replication and virion export. However, at present it is still unclear whether this also applies to the in vivo situation. Using a terminally redundant X-deficient HBV DNA construct, transgenic mice were established that exhibited high-level expression of the viral core protein in liver and kidneys. Importantly, replicative DNA intermediates and mature viral genomes could be detected in the liver and serum of these mice, respectively. These findings indicate that, in the in vivo model of transgenic mice, the HBV X (HBx) gene product is not required for HBV replication and virion secretion.

Animals↗