Josef Köck

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. (HEPATOLOGY 2005;42:301–309.)

Efficient Infection of Primary Tupaia Hepatocytes with Purified Human and Woolly Monkey Hepatitis B Virus

ABSTRACT The Asian tree shrew, Tupaia belangeri, has been proposed as a novel animal model for studying hepatitis B virus (HBV) infection. Here, we describe a protocol for efficient and reproducible infection of primary tupaia hepatocytes with HBV. We report that human serum interferes with HBV binding to the hepatocytes, thus limiting the maximum multiplicity of infection. Purification of HBV virions by gradient sedimentation greatly enhances virus binding and infectivity. Covalently closed circular DNA was clearly detectable by Southern blot analysis and newly synthesized single-stranded HBV DNA was visible 2 weeks postinoculation. Primary tupaia hepatocytes are also susceptible to infection with the recently discovered woolly monkey hepatitis B virus (WMHBV) but not to woodchuck hepatitis virus infection. Compared to HBV, WMHBV replicated at a higher rate with single-stranded DNA detectable within the first week postinoculation. Primary tupaia hepatocytes should represent a useful system for studying early steps of HBV and WMHBV infection.

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.

Hepatitis B Virus Nucleocapsids Formed by Carboxy-Terminally Mutated Core Proteins Contain Spliced Viral Genomes but Lack Full-Size DNA

ABSTRACT 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.

Hypermutation of hepatitis B virus genomes by APOBEC3G, APOBEC3C and APOBEC3H.

Hepatitis B virus (HBV) is a DNA virus that causes liver disease and replicates by reverse transcription of an RNA template. Previous studies have reported that HBV genomes bearing G-->A hypermutation are present at low frequency in human serum. These mutations are most likely due to the activity of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) cytosine deaminases, cellular proteins known to confer innate immunity against retroviruses by generating lethal hypermutations in viral genomes. This study assessed APOBEC3G, APOBEC3C and APOBEC3H, three members of this protein family present in human liver, for their ability to edit HBV genomes. Transfection of human HepG2 hepatoma cells with a plasmid encoding the APOBEC3C protein resulted in abundant G-->A mutations in the majority of newly formed HBV genomes. By contrast, transfection of APOBEC3G- and APOBEC3H-encoding plasmids only marginally increased hypermutation rates above the level caused by the cytosine deaminases naturally present in HepG2 cells. APOBEC3G- and APOBEC3H-mediated hypermutation, however, was clearly revealed by transfection of chicken LMH hepatoma cells, which lack endogenous cytosine deaminases. These results indicate that APOBEC3G, APOBEC3C and APOBEC3H have the ability to edit HBV DNA and that each protein is likely to contribute to various degrees to the generation of modified genomes in human liver cells.

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. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005.)41:247–256

Mutations of the serine protease inhibitor, Kazal type 1 gene, in patients with idiopathic chronic pancreatitis

OBJECTIVE:The pathogenesis of chronic pancreatitis (CP) is poorly understood. Genetic studies revealed mutations in the cationic trypsinogen gene and an increased frequency of cystic fibrosis gene mutations in patients with CP. Recently, a point mutation (N34S) in the gene encoding the serine protease inhibitor, Kazal type 1 (SPINK1), was found in approximately 20% of patients with CP. The aim of our study was to determine the frequency of the N34S SPINK1 gene mutation in a well-defined patient cohort with idiopathic CP (ICP) and to compare the incidence with healthy controls. In addition, we investigated the impact of this mutation on the long-term course of CP.METHODS:Fourteen patients with early-onset and four patients with late-onset CP of our well-defined pancreatitis cohort were enrolled in the present study, and 397 healthy individuals served as a control population. Coding exonic and the flanking intronic sequences of SPINK1 were investigated by direct DNA sequencing. The mutations found were confirmed by melting curve analysis. In addition, the N34S mutation was detected by analyzing the DNA fragments generated by digestion with restriction enzyme TspR I. Clinical data of patients with the N34S mutation were compared with those without mutations.RESULTS:The N34S mutation was detected in six of 14 (43%) patients with early-onset ICP. One patient was homozygous, and five patients were heterozygous for this mutation. The N34S mutation in a heterozygous state was found in four of 397 healthy controls (1.0%). The different allele frequency observed (seven of 28 vs four of 794) was significant (odds ratio = 66, 95% CI = 18–242, p < 0.0001). The clinical course was similar in patients with a mutation compared with those without a mutation. No other SPINK1 mutations were detected. The N34S mutation was not found in patients with late-onset ICP.CONCLUSIONS:Our results indicate that the N34S mutation in the SPINK1 gene is strongly associated with ICP, especially with the early-onset type. The natural course is similar in patients with mutations compared with SPINK1 mutation-negative patients. The N34S mutation may easily be screened for by restriction digestion with TspR I.

Trypsinogen gene mutations in patients with chronic or recurrent acute pancreatitis.

Three-point mutations (R117H, N21I, A16V) within the cationic trypsinogen gene have been identified in patients with hereditary pancreatitis (HP). A genetic background has also been discussed for idiopathic juvenile chronic pancreatitis (IJCP), which closely mimicks the clinical pattern of HP, and alcoholic chronic pancreatitis because only a small number of heavy drinkers develop pancreatitis. This prompted us to screen 104 patients in our well-defined pancreatitis cohort for the currently known cationic trypsinogen gene mutations. The R117H mutation was detected in seven patients (six patients of two clinically classified HP families, one patient with clinically classified IJCP) and the A16V mutation in one IJCP patient. No cationic trypsinogen gene mutations were found in the remaining 96 patients with chronic and recurrent acute pancreatitis of various etiologies. Our results demonstrate the need for genetic testing to exclude HP, particularly in the presence of an atypical or unknown family history. In addition, cationic trypsinogen gene mutations are no predisposing factor in patients with chronic and recurrent acute pancreatitis of different etiologies.

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.

Human hepatitis B virus production in avian cells is characterized by enhanced RNA splicing and the presence of capsids containing shortened genomes.

Experimental studies on hepatitis B virus (HBV) replication are commonly done with human hepatoma cells to reflect the natural species and tissue tropism of the virus. However, HBV can also replicate, upon transfection of virus coding plasmids, in cells of other species. In such cross-species transfection experiments with chicken LMH hepatoma cells, we previously observed the formation of HBV genomes with aberrant electrophoretic mobility, in addition to the those DNA species commonly seen in human HepG2 hepatoma cells. Here, we report that these aberrant DNA forms are mainly due to excessive splicing of HBV pregenomic RNA and the abundant synthesis of spliced DNA products, equivalent to those also made in human cells, yet at much lower level. Mutation of the common splice acceptor site abolished splicing and in turn enhanced production of DNA from full-length pgRNA in transfected LMH cells. The absence of splicing made other DNA molecules visible, that were shortened due to the lack of sequences in the core protein coding region. Furthermore, there was nearly full-length DNA in the cytoplasm of LMH cells that was not protected in viral capsids. Remarkably, we have previously observed similar shortened genomes and non-protected viral DNA in human HepG2 cells, yet exclusively in the nucleus where uncoating and final release of viral genomes occurs. Hence, two effects reflecting capsid disassembly in the nucleus in human HepG2 cells are seen in the cytoplasm of chicken LMH cells.