Functional analysis of HBV genomes from patients with fulminant hepatitis

Sterneck, Martina; Kalinina, Tatyana; Günther, Stephan; Fischer, Lutz; Santantonio, T.; Greten, H.; Will, Hans

doi:10.1002/hep.510280530

Cited by 56 publications

(54 citation statements)

References 45 publications

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“…For Western blot analysis the beads were boiled for 5 minutes in Laemmli sample buffer and subjected to separation through a 15% polyacrylamide gel. For Southern blot analysis, HBV DNA was purified, 22 including DNAse pretreatment for digestion of inputtransfected DNA. Fig.…”

Section: Methodsmentioning

confidence: 99%

“…HBV DNA and plasmids used. Wt virus and M7 represent full-length HBV genomes described previously 22 used in linear form with sticky ends for transfection and for plasmid constructs. These genomes circulate in cells and all viral proteins are expressed from authentic promoters.…”

Section: Methodsmentioning

confidence: 99%

“…22 To eliminate any trace of transfected plasmid the viral particles resuspended in phosphate-buffered saline were treated with DnaseI. Similar amounts of mutant and Wt viral particles were exposed to different concentration of NP-40 for 40 minutes at 30°C.…”

Section: Np-40 Treatment Of Concentrated Viral Particlesmentioning

confidence: 99%

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Deficiency in virion secretion and decreased stability of the hepatitis B virus immune escape mutant G145R

et al. 2003

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Hepatitis B virus with a G145R mutation in the small surface protein is considered the quintessential immune escape mutant because it frequently is found in vaccinated individuals with breakthrough infections and liver transplant recipients under anti-hepatitis B surface antigen (HBsAg) immunoglobulin prophylaxis. Nowadays the prevalence of the variant progressively increases. However, because spread of a virus depends not only on immune pressure but also on the viral phenotype, we investigated the biologic properties of the G145R variant. The G145R mutation was introduced into wild-type (Wt) virus genome by in vitro mutagenesis. After transfection into human hepatoma cells, the DNA, RNA, and protein synthesis and viral secretion ability of the mutant were studied. Furthermore, cotransfection studies were performed with the G145R variant and a Wt virus S-protein expressing construct and vice versa. Production and stability of viral messenger RNAs (mRNAs), DNA, and proteins were not affected by the G145R mutation. In contrast, secretion of mutant virions was reduced significantly. Only 20% of virions were found in the medium of G145R variant-transfected cells compared with Wt virus. Furthermore, mutant virions were more sensitive to detergent treatment suggesting a diminished stability. In cotransfection studies, Wt virus S-protein rescued secretion of mutant virions, whereas mutant S-protein had a transdominant negative effect on secretion of Wt virus. Both mechanisms may support persistence of the defective mutant in a mixed population with Wt virus. In conclusion, the significant defect of the G145R mutant for secretion of infectious virions and the diminished stability of mutant virions may limit global spread of the mutant. H epatitis B virus (HBV) exhibits a mutation rate more than 4 orders of magnitude higher than that of other DNA viruses. 1 Naturally occurring variants may accumulate over time if they have a survival benefit over wild-type (Wt) virus, such as a higher ability to replicate DNA, an enhanced secretion ability, an increased infectivity or stability of virions, and so forth. In addition, certain variants may be selected under immune pressure or therapy with antiviral drugs.Nowadays, immune-escape variants that emerge under active and/or passive vaccination and are responsible for vaccination-breakthrough infections are of particular clinical relevance. Several such mutants carrying single or multiple amino acid substitutions in the major antigenic region of the virus, the a-determinant of the small surface protein, have been described. [2][3][4] The mutant by far most frequently detected all over the world in vaccinated individuals and liver transplant recipients treated with monoclonal or polyclonal anti-hepatitis B surface antigen (HBsAg) hyperimmunoglobulin carries a glycine to arginine substitution at amino acid position 145 (G145R). 3,[5][6][7][8][9][10][11] There is no doubt that despite selection of immuneescape variants, vaccination programs have so far been highly effective in reduci...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Deficiency in virion secretion and decreased stability of the hepatitis B virus immune escape mutant G145R

et al. 2003

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“…Thus, the unique genetic framework of the predominant HBV quasispecies with multiple mutations within a gene or genes associated with FHB have been studied. 8,20,25,33 These more recent studies, including the report by Kalinina et al, suggest that multiple mutations throughout the genome may contribute to (1) an increase in viral replication fitness, (2) a change in viral gene expression, or (3) an alteration of cytotoxic T lymphocyte epitopes. These virologic markers either alone or in various combinations and the interplay between the virus and the host are clearly important in the development of FHB.…”

Section: Hbv Mutants Associated With Fulminant Hepatitismentioning

confidence: 99%

“…Earlier studies from this group had shown that the infectious clones from the same samples exhibited enhanced replication levels. 8 The enhanced replication and the severe defect in viral particle secretion most likely contributed to the fulminant clinical course of recurrent HBV infection posttransplantation. The patient was also hepatitis B e antigen (HBeAg) negative.…”

mentioning

confidence: 99%

Hepatitis B virus mutants and fulminant hepatitis B: Fitness plus phenotype

Bartholomeusz

2001

Hepatology

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The human hepatitis B virus (HBV) is the smallest known double-stranded DNA virus of man that replicates its genome through a novel mechanism of reverse transcription using the RNA-replicative intermediate and the viral pregenomic RNA. 1 Viral pregenomic RNA is synthesized from molecules of covalently closed circular (ccc) DNA, the major transcriptional template of the virus, functioning as a viral minichromosome within the nucleus of infected cells. 2 The reverse transcriptase of HBV lacks a conventional proofreading function, which is found in higher-order polymerases and, not surprisingly then, HBV exhibits a mutation rate more than 10-fold higher than other DNA viruses. The rates for nucleotide substitutions vary depending on the stage of disease. The natural evolutionary rate for the HBV genome in chronic hepatitis B is approximately 1.4 to 3.2 ϫ 10 Ϫ5 substitutions/site/year, 3 whereas in the liver transplantation setting, it is almost 100-fold higher. 4 Longitudinal studies have shown that these HBV mutations are not distributed evenly over the entire genome but rather tend to cluster into mutational patterns in particular parts of the viral DNA; in particular, the basal core promoter (BCP), the precore region and the "a" determinant of the viral envelope. 5 Importantly, the type and number of mutations that accumulate in the HBV genome over time have been shown to be a marker of the duration and/or severity of the liver disease, or the type and intensity of the immune response. 5 The outcome of HBV infection depends on the interplay between the virus, the hepatocyte, and the host's immune response. 6 Under normal circumstances, HBV is not cytopathic and liver damage is the result of the host's immune response targeting infected hepatocytes. However, during special or unusual circumstances, HBV appears to be directly cytopathic for hepatocytes often causing unique histopathologic conditions such as fibrosing cholestatic hepatitis, which, before the advent of specific antiviral therapy, 7 was invariably fatal. The molecular virologic and cellular basis for fulminant hepatitis B (FHB) has been the focus of intensive investigation, but to date the results have been controversial. In the report by Kalinina et al. in this issue of HEPATOLOGY, the investigators, have provided the first evidence, using in vitro functional studies that mutations in the S gene of the HBV isolated at the time of FHB had caused a severe defect in viral particle secretion. Earlier studies from this group had shown that the infectious clones from the same samples exhibited enhanced replication levels. 8 The enhanced replication and the severe defect in viral particle secretion most likely contributed to the fulminant clinical course of recurrent HBV infection posttransplantation. The patient was also hepatitis B e antigen (HBeAg) negative. REGULATION OF HEPADNAVIRAL cccDNA: ROLE OF THE VIRAL ENVELOPEVirus persistence in infected cells during chronic infection depends on the maintenance of the HBV cccDNA pool. 9 This pool of transcript...

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Detection of mutations in hepatitis B virus enhancer 2/core promoter and X protein regions in patients with fatal hepatitis B virus infection

et al. 2000

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The enhancer 2/core promoter and the X protein regions located upstream of the precore and core regions in hepatitis B virus regulate expression of core/e antigen peptides. Mutations in the precore and core regions have been reported to be associated closely with the severity of type B hepatitis, and regions regulating expression of these peptides may also be involved in severe liver damage. Mutations were examined in regions that may be related to fatal liver diseases. Nucleotide sequences and deduced amino acid sequences from 20 patients with fatal type B hepatitis (12 with fulminant hepatitis and 8 with severe exacerbation) and 10 patients with self-limited acute hepatitis were analyzed. There were 50 nucleotide alterations in the enhancer 2/core promoter region of virus from 12 patients with fulminant hepatitis (average 4.1/case), 37 alterations in 8 patients with severe exacerbation (4.6/case), and 10 mutations in 10 cases of acute hepatitis (1.0/case). The numbers of amino acid mutations in X protein were 53 in 12 cases of fulminant hepatitis (4.4/case), 27 in 8 cases of severe exacerbation (3.3/case), and 9 in 10 cases of acute hepatitis (0.9/case). In fatal cases, approximately 50% of the nucleotide mutations were located within the region spanning nucleotides 1741-1777 (14.2% of the enhancer 2/core promoter region) and 30% of the amino acid mutations in X protein were located within the region containing codons 122-132 (7.1% of X protein). In addition to mutations in the precore and core regions, mutations in the enhancer 2/core promoter and the X protein regions may be associated with the pathogenesis of fatal B hepatitis infection.

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Functional analysis of HBV genomes from patients with fulminant hepatitis

Cited by 56 publications

References 45 publications

Deficiency in virion secretion and decreased stability of the hepatitis B virus immune escape mutant G145R

Deficiency in virion secretion and decreased stability of the hepatitis B virus immune escape mutant G145R

Hepatitis B virus mutants and fulminant hepatitis B: Fitness plus phenotype

Detection of mutations in hepatitis B virus enhancer 2/core promoter and X protein regions in patients with fatal hepatitis B virus infection

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