Naturally occurring mutations of hepatitis B virus and outcome of chronic infection: is there an association?
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Carrier State↗
Biomedical subjects
S Günther
Publications and source records attributed to S Günther.
Nucleotide binding of an ADP analog to cooperating sites of chloroplast F1-ATPase (CF1).
Pre-steady state nucleotide binding to the chloroplast F1-ATPase (CF1) was measured in a stopped-flow apparatus by monitoring the change of fluorescence intensity of TNP-ADP upon binding. The analysis of the time courses led to the proposal of a mechanism of nucleotide binding with the following characteristics. (a) It involves three sites binding nucleotides in a concerted manner. (b) Each binding site is able to undergo a conformational change from a loose binding state into a state refraining from any direct release of the bound nucleotide into the medium. Only the reverse reaction via the loose binding state enables release out of the tight binding state. (c) Due to very strong negative cooperativity, a maximum of two of the three sites can be found in the state of tight binding. (d) Cooperativity between the three sites leads to a slower nucleotide binding of the second nucleotide compared to the first nucleotide. Furthermore, the conformational change from the loose binding state to the tight binding state is slowed down if one of the other sites already is in the tight binding state. Three-sites mechanisms in which rotation leads to an exchange of the properties of the binding sites failed to simulate the observed time courses of nucleotide binding. However, as the experimental set up was designed to prevent catalysis taking place, our results entirely agree with the current finding that rotation requires catalytic turnover of the enzyme.
Adenosine Diphosphate↗
Naturally occurring variants of hepatitis B virus.
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Animals↗
Absence of mutations in the YMDD motif/B region of the hepatitis B virus polymerase in famciclovir therapy failure.
BACKGROUND/AIMS: Nucleoside analogues such as lamivudine and famciclovir are potent drugs for treatment of chronic hepatitis B virus infection. Breakthrough infections during lamivudine therapy are associated with mutations in the YMDD motif and putative B region of the HBV polymerase. This study investigated whether failure of famciclovir therapy is also associated with presence or emergence of particular mutations in the HBV polymerase. METHODS: We analyzed longitudinally the sequence of the priming and polymerase domain in seven patients with primary non-response to therapy and two patients with a breakthrough during therapy. Two patients who responded to therapy served as a control. RESULTS: The YMDD motif and the B region were conserved in all isolates. V-->I changes at position 555 just downstream of the YMDD motif were observed before and during therapy in a virus subpopulation of two patients with a primary non-response. In patients with a breakthrough, 378-V-->I and 424-N-->D mutations emerged in the N terminal part of the polymerase domain during follow-up. Lamivudine rescue therapy initiated in four patients, including a patient infected with YMDD(555-V-->I) variants, efficiently reduced viremia. CONCLUSIONS: These data indicate that failure of famciclovir therapy can occur independently of mutations in the YMDD motif or B region of the HBV polymerase and provide a rationale for rescue therapy with lamivudine.
2-Aminopurine↗
Effect of interferon alpha on hepatitis B virus replication and gene expression in transiently transfected human hepatoma cells.
BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFNalpha), which results in efficient reduction of the viral load only in 10-20% of treated patients. The mechanisms induced by IFNalpha resulting in reduction of viremia in responding patients are unknown. The aim of this study was to characterize HBV-specific IFNalpha-induced intracellular inhibitory mechanisms and IFNalpha-sensitive HBV targets. METHODS: To determine the antiviral activity, cells transiently transfected with HBV DNA were treated with IFNalpha and thereafter, viral products were quantified at different time points. RESULTS: Time-dependent reduction of RNA, replicative DNA-intermediates, core protein and secreted HBsAg/HBeAg levels was observed in IFNalpha-treated cells. Viral RNA levels were reduced most effectively early post-treatment whereas those of core protein and replicative intermediates decreased later. By expression of subgenomic HBV sequences, an RNA target region mediating IFNalpha-induced RNA degradation was mapped. CONCLUSIONS: These data indicate that HuH7 cells transiently transfected with HBV-DNA represent a system well suited for detailed analysis of IFNa-induced antiviral mechanisms and HBV targets. At least two IFNalpha-induced HBV-specific antiviral activities are active in this system: one reduces the levels of core protein and replicative intermediates, the other leads to posttranscriptional degradation of HBV-RNA. Based on the established in vitro system a detailed characterization of the IFNalpha-sensitive RNA-region and of factors mediating this intracellular antiviral effect is feasible. This may lead to the development of novel strategies for therapy of chronic hepatitis.
Carcinoma, Hepatocellular↗
Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1).
The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.
Adenosine Diphosphate↗
Apoptosis induced by modified ribonucleosides in human cell culture systems.
The in vitro modulation of apoptosis and cell proliferation by modified in comparison with non-modified ribonucleosides was investigated for the first time using peripheral blood lymphocytes, HL-60 cells and Caco-2 cells as human cell culture models. Modulating effects of several ribonucleosides were found in the range of 10(-7)-10(-3) mol/l. The following ribonucleosides induced significant apoptosis of HL-60 cells: adenosine, N6-dimethyladenosine, N6-(2-isopentenyl)-adenosine, N2-dimethylguanosine. A significant apoptotic effect on PBL was found with N6-dimethyladenosine and N6-(2-isopentenyl)-adenosine. N6-Dimethyladenosine, N6-(2-isopentenyl)-adenosine and guanosine had a pronounced inhibitory effect on Caco-2 cell apoptosis. Regarding the known function of ribonucleosides as pathobiochemical marker molecules for cancer, the possibility of a selective apoptotic effect against malignant cells is discussed.
Adenosine↗
Di- and oligosaccharide substrate specificities and subsite binding energies of pig intestinal glucoamylase-maltase.
The substrate specificity of pig intestinal glucoamylase-maltase was investigated. The alpha-1, beta-2-glycosidic bond of the disaccharide sucrose was not hydrolyzed. Various substrates with alpha-1,4-glycosidic bonds (maltose, maltooligosaccharides) were hydrolyzed with high maximal reaction velocities. Reduction lowered the rate of hydrolysis drastically: k'0 decreases from 75 s-1 for maltose to 3 s-1 for maltitol while the K(m) value increases from 4.2 to 50 mM. Leucrose with alpha-1,5-glycosidic bond was hydrolyzed with a k'0 value of 8 s-1 and a K(m) value of 74 mM. Disaccharides with alpha-1,6-glycosidic bonds were hydrolyzed with extremely low rates: for isomaltose and isomaltulose k'0 values of 5 and 3 s-1, respectively, and K(m) values of 90 and 42 mM, respectively, were observed. Again reduction lowers the k'0 values: The corresponding disaccharide alcohols alpha-D-glucopyranosyl-1,6-sorbitol and alpha-D-glucopyranosyl-1,6-mannitol, like isomaltooligosaccharides, were not hydrolyzed. Regarding the conformation of sucrose, leucrose, and maltose previously determined by molecular dynamics simulations, a reasonable explanation for the different rates of hydrolysis could be postulated. Based on the enzyme kinetic parameters for the series of maltooligosaccharides, subsite affinities (A1) according to the subsite theory were calculated as 7.5 (A1), 17 (A2), 3.4 (A3), and 1.3 kJ/mol (A4) for subsites 1, 2, 3, and 4, respectively. The intrinsic rate constant k'int was estimated at 76 s-1.
Animals↗
Analysis of hepatitis B virus populations in an interferon-alpha-treated patient reveals predominant mutations in the C-gene and changing e-antigenicity.
It is largely unknown whether hepatitis B virus (HBV) sequence variation during chronic infection hampers HBV immune recognition or the antiviral effect of cytokines on HBV production. Here we have analyzed which region of the HBV genome changes most drastically during an interferon-alpha (IFNalpha)-stimulated immune response. In addition, we have investigated whether the mutations affect viral replication, gene expression, and immune recognition of the mutant viral proteins. The study was performed with full-length HBV genomes taken longitudinally from a patient who transiently cleared HBV and seroconverted to anti-HBe during a long-term IFNalpha treatment. We found a replacement of the predominant virus population during IFNalpha therapy The virus populations differed mainly by a cluster of nucleotide changes in the C-gene and a pre-S2 deletion. Most of the newly emerging mutations localized within core/HBe B-cell epitopes, changed HBe antigenicity toward mono- and polyclonal antibodies, and also influenced the reactivity of the anti-HBc/e antibodies of the patient. All genomes tested expressed less HBeAg than wild-type HBV, while replication and IFNalpha susceptibility were similar. These data indicate that IFNalpha therapy can lead to the emergence of HBV variants with mutations mainly affecting recognition of the core/HBe proteins by antibodies. Taken together, the type of core/HBe-specific B-cell immune response, the sequence of the corresponding epitopes, and the HBe expression level appear to contribute to the decision on viral clearance or persistence.
Amino Acid Sequence↗
Food proteins as precursors of peptides modulating human cell activity (short communication).
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Apoptosis↗
Functional analysis of HBV genomes from patients with fulminant hepatitis.
Two previous case reports suggest that hepatitis B virus (HBV) core promoter variants with a high replication competence contribute to the pathogenesis of fulminant hepatitis B (FHB). We recently found in HBV genomes from patients with FHB an accumulation of mutations within the core promoter region. Therefore, the aim of this study was to investigate the phenotype of these HBV variants. Replication competence and expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) of viral genomes from seven patients with FHB and one patient with fulminant recurrent hepatitis after liver transplantation were analyzed by transfection experiments in human hepatoma cells. Compared with wild-type virus, the HBV variants from the seven patients with FHB produced similar or slightly lower levels of intracellular replicative intermediates and extracellular viral particles. In contrast, the HBV genomes from the patient with fulminant recurrent hepatitis synthesized and secreted significantly more HBV DNA. All genomes tested expressed similar or even higher levels of HBeAg compared with wild-type virus, except for those from four patients with a precore stop codon mutation in the respective dominant viral populations. The level of HBsAg produced by all variant genomes was similar or reduced compared with wild-type virus. These data indicate that in some cases HBV variants with enhanced replication competence and/or a defect in HBeAg expression may contribute to the development of FHB. However, neither phenotype is an essential prerequisite; thus, an additional role of other viral or host factors in the pathogenesis of FHB is suggested.
Base Sequence↗
Neonatal fulminant hepatitis B: structural and functional analysis of complete hepatitis B virus genomes from mother and infant.
Transmission of hepatitis B virus (HBV) from anti-hepatitis B e (anti-HBe)-positive carrier mothers to their infants may result in neonatal fulminant hepatitis B (FHB). We investigated whether HBV variants with a particular DNA sequence and functional phenotype, responsible for FHB, are selected during transmission. Full-length HBV genomes from a mother-infant pair were completely sequenced and transfected into human hepatoma cells. The dominant neonatal and maternal HBV populations were nearly identical (homology 99.8%) and showed a precore stop codon mutation, T-1762 and A-1764 substitutions in the core promoter region, and pre-S2 start codon mutations. Cells transfected with variants from mother and child, compared with wild-type virus, synthesized and released a similar number or fewer HBV DNA-containing particles. In conclusion, no particular HBV strain emerged during neonatal FHB. In this case, a de novo infection with variants showing a defect in HBe antigen and pre-S2 protein synthesis but not a high replication competence probably contributed to the fulminant disease course.
Adult↗
Wild-type levels of pregenomic RNA and replication but reduced pre-C RNA and e-antigen synthesis of hepatitis B virus with C(1653) --> T, A(1762) --> T and G(1764) --> A mutations in the core promoter.
Hepatitis B virus (HBV) isolates with A-1762 to T and G-1764 to A mutations in the core promoter have been associated with active hepatitis, severe liver disease following liver transplantation, hepatocellular carcinoma and acute fulminant courses--in the latter case combined with a C-1653 to T mutation. In this study, a mutant core promoter region containing the T-1653, T-1762 and A-1764 mutations was placed into the context of a wild-type HBV genome and analysed by transfection. The mutations reduced the level of pre-C mRNA (by 55%) and e-antigen secretion. In contrast, no significant effects on the levels of pregenome/C and pre-S/S mRNAs, intracellular core, polymerase, and pre-S /S2 proteins and secreted S-antigen were observed. The amount of progeny virus DNA in the cells and in the culture medium was increased marginally, if at all.
Base Sequence↗
Amplification of full-length hepatitis B virus genomes from samples from patients with low levels of viremia: frequency and functional consequences of PCR-introduced mutations.
To facilitate the investigation of hepatitis B virus (HBV) sequence variation, we recently established a method for functional analysis of PCR-amplified full-length HBV genomes. This study aimed at estimating the number of mutations introduced during amplification of genomes from samples from patients with low levels of viremia and their influence on replication and antigen expression. Wild-type HBV DNA template molecules in concentrations like those present in samples from patients with very low levels of viremia were amplified, sequenced (30 kb total), and functionally tested. We found that Taq polymerase and a Taq-Pwo polymerase mixture introduced an average of 5.7 and 3.1 mutations per genome, respectively, corresponding to polymerase error rates of 12.1 x 10(-5) and 6.0 x 1(0-5). One of 8 genomes (12%) amplified with Taq polymerase, but 7 of 17 genomes amplified with Taq-Pwo polymerases (41%), remained replication competent. All replication-competent genomes expressed HBs and HBe antigens and had an average of only 0.9 mutations per genome. In contrast, replication-defective genomes had an average of 5.4 mutations, which frequently also disturbed viral antigen expression. From these data we conclude that many of the replication-competent HBV genomes from a clinical specimen will retain their replication and antigen expression phenotypes even after extensive amplification with Taq-Pwo polymerases. Because replication competence is highly sensitive to random mutations, it is the best marker for the identification of HBV genomes with few or no PCR-introduced mutations.
Cloning, Molecular↗
A new class of defective hepatitis B virus genomes with an internal poly(dA) sequence.
Sequence heterogeneity of hepatitis B virus (HBV) is increasingly recognized to play a role in virus-host interaction. We have used a recently established method for HBV full-length genome amplification to search for novel types of HBV variants and to investigate further the sequence heterogeneity of HBV genome populations. Using this method, a substantial fraction of HBV genomes much shorter than wildtype size was found in some sera and liver biopsies from infected patients. Cloning and sequencing of a number of these HBV genomes as well as hybridization studies revealed a new minor class of HBV genomes with an internal poly(dA) sequence approximately 60 to more than 100 nucleotides long in 4 of 10 patients. The 5'-ends of the internal poly(dA) sequences are located at positions corresponding to the authentic processing/polyadenylation sites of the RNA pregenome, whereas the positions of the 3'-ends are variable due to different sizes of adjacent deletions. These data suggest that the poly(A) tail of the pregenomic RNA is occasionally reverse transcribed by the HBV P-protein and during this process a deletion seems to be introduced into the DNA minus strand. We propose a mechanism by which this could be accomplished during DNA minus strand synthesis.
Base Sequence↗
Heterogeneity and common features of defective hepatitis B virus genomes derived from spliced pregenomic RNA.
Defective hepatitis B virus (HBV) genomes derived from packaging and reverse transcription of spliced RNA pregenomes were reported to be associated with progression to chronic infection. Since only two types with similarly spliced regions were characterized so far we reasoned that additional "spliced" genome variants may exist. Therefore, we isolated a large number of defective HBV genomes from sera of seven chronic carriers by full-length PCR. Forty-eight were found to contain deletions caused by splicing as identified by cloning, subgenomic PCR, and sequencing. In total, 11 types of spliced genomes derived from excision of 10 different introns were present in various combinations in each serum. This diversity resulted from alternative usage of five splice donor and four acceptor sites present in most but not all HBV genotypes. All spliced genomes shared sequence elements essential for replication as well as for transcription of the pre-C and pregenome/C mRNAs and the X mRNA. Moreover, all contained the coding regions for the X protein and for precore/core or precore/ core fusion proteins but lacked the pre-S/S gene promoters. These data demonstrate substantial and HBV genotype-dependent diversity of spliced genomes from which a variety of aberrant precore/core fusion proteins and normal X protein but no functional envelope and P proteins could be expressed. These genomes and the encoded proteins may play a role in the viral life cycle, persistence, and pathogenesis.
Cloning, Molecular↗
Acarbose and 1-deoxynojirimycin inhibit maltose and maltooligosaccharide hydrolysis of human small intestinal glucoamylase-maltase in two different substrate-induced modes.
The inhibition of the glucoamylase-maltase-catalyzed maltose and maltooligosaccharide hydrolysis by acarbose and 1-deoxynojirimycin has been demonstrated. Acarbose and 1-deoxynojirimycin act as potent competitive inhibitors with Ki = 0.8 microM for the hydrolysis of maltose and with Ki values of 0.4 and 0.3 microM, respectively, for the hydrolysis of maltooligosaccharides. In a previous work (Günther et al., Arch. Biochem. Biophys. 327, 295-302, 1996) using maltitol and maltobionate as inhibitors we were able to discriminate two different binding modes for glucoamylase-maltase: a maltose and an oligosaccharide binding mode. Here we found that structurally quite different substances, namely, the pseudotetrasaccharide acarbose and the monomeric glucose analog 1-deoxynojirimycin, act as competitive inhibitors for maltose and maltooligosaccharide hydrolysis. The Ki values for all used maltooligosaccharides are nearly equal, but for maltose hydrolysis the Ki values are significantly higher by a magnitude factor of two. The differences concerning Ki values can be explained by means of the two-binding-mode model.
1-Deoxynojirimycin↗
Naturally occurring hepatitis B virus genomes bearing the hallmarks of retroviral G-->A hypermutation.
Two hypermutated genomes of hepatitis B virus (HBV) were cloned from sera of chronic virus carriers. Twelve percent and 26% of guanosine residues were replaced by adenosine, with the transitions being erratically distributed along the genome. G-->A substitutions showed a strong dinucleotide preference, decreasing in the order GpA > GpG > > GpC > or = GpT. Such traits are typical of retroviral G-->A hypermutation which results from cDNA synthesis coinciding with fluctuations in the intracellular [dTTP]/[dCTP] ratio. The observations offer an explanation for the high prevalence of HBV variants bearing a tryptophan 28-->stop codon in the pre-core region of carriers with chronic active or fulminant hepatitis. The HBV hypermutants indicate that a small proportion of hepatocytes have distorted dNTP pools, which might have implications for the fidelity of hepatocyte DNA replication or repair.
Adenine↗