Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus

Lenhoff, Raymond J.; Summers, Jesse

doi:10.1128/jvi.68.7.4565-4571.1994

Cited by 140 publications

(80 citation statements)

References 18 publications

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“…12 Sitedirected mutagenesis was used to produce a single nucleotide change, which resulted in the single amino acid change from glycine to glutamic acid at residue 133 (G133E) in the large envelope protein. 13 The coding of the DHBV polymerase open reading frame was not affected by this single nucleotide change.…”

Section: Methodsmentioning

confidence: 91%

“…Virus was concentrated by precipitation with polyethylene glycol (10% final concentration). 13 The titer of virus was determined by Southern blot hybridization following selective DNA extraction of enveloped virus particles, as previously described. 13,16,17 Animals.…”

Section: Methodsmentioning

confidence: 99%

“…13 The titer of virus was determined by Southern blot hybridization following selective DNA extraction of enveloped virus particles, as previously described. 13,16,17 Animals. Day-old ducklings obtained from Metzer Farms (Redland, CA) were used for two independent experiments, experiment 1 and experiment 2.…”

Section: Methodsmentioning

confidence: 99%

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Acute liver injury following infection with a cytopathic strain of duck hepatitis B virus

1999

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A variant avian hepadnavirus that has been shown to destroy hepatocytes in vitro was found to be cytopathic in vivo. A single amino acid change of glycine to glutamic acid at position 133 (G133E) in the preS protein of duck hepatitis B virus (DHBV) caused an increase in the intranuclear pool of viral covalently closed circular DNA (cccDNA), resulting in a transient elevation of viral replication and eventual hepatocyte destruction. In vivo viral infection with the G133E virus was compared with infection with wild-type virus over a 72-day period. Birds were inoculated with virus at day 2 post-hatch to ensure a high percentage of infected hepatocytes and potential persistence of virus. Birds infected with the G133E virus had increased periportal cellular proliferation and numerous lysed apoptotic hepatocytes following 100% infection of hepatocytes. The liver damage within G133E virus-infected birds subsided over time, resulting in mild chronic hepatitis that was similar to that observed within wild-type virusinfected birds. The subsidence of liver damage in G133E virus-infected birds coincided with a reduction of viral cccDNA to wild-type virus levels in the liver. Our study indicates that maintenance of wild-type levels of viral cccDNA promotes persistence of virus infection by establishing a noncytopathic infection. (HEPATOLOGY 1999;29:563-571.)Hepadnaviruses cause persistent productive infections of each infected hepatocyte. During the initiation of infection, the relaxed circular DNA (rcDNA) genome in the virion is converted to covalently closed circular DNA (cccDNA) in the nucleus. Viral cccDNA is used as the template for viral RNA transcription, resulting in the production of a viral RNA molecule (pregenome) that is greater than one genome in length. The RNA pregenome is encapsidated within a viral core particle, where it is reverse-transcribed by the viralencoded DNA polymerase to produce viral minus-strand DNA. 1 Plus-strand DNA synthesis is then carried out by the same viral DNA polymerase using the viral DNA minusstrand as a template. The final DNA product contained with the capsid is a double-stranded molecule that is held in a circular conformation by a short region of base-pairing between the 5Ј end of the two strands. This form of viral DNA is referred to as relaxed circular DNA, or rcDNA. The viral capsids that contain rcDNA associate with the viral envelope proteins at the endoplasmic reticulum and are secreted as virions. Alternatively, rcDNA molecules may enter into the nucleus of the infected hepatocyte, where they are converted to additional molecules of cccDNA and cause increased levels of viral replication. The production of new viral cccDNA is regulated by the viral large envelope protein (preS) by a mechanism that has not been biochemically defined. 2 Presumably, an interaction between the preS protein and rcDNAcontaining capsids is necessary for enveloped virus formation and promotes secretion of viral rcDNA from the hepatocyte as virions. Because defects in preS that specifically inhibit ...

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Section: Methodsmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

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Acute liver injury following infection with a cytopathic strain of duck hepatitis B virus

1999

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“…For immunoblot analysis of the medium of transfected cells viral particles were precipitated with polyethylene glycol using the protocol of Lenhoff and Summers. 23 Then viral proteins were detected as given above.…”

Section: Methodsmentioning

confidence: 99%

A dominant hepatitis B virus population defective in virus secretion because of several S-gene mutations from a patient with fulminant hepatitis

et al. 2001

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There is increasing evidence that certain pathogenic hepatitis B virus (HBV) variants may play a role in the pathogenesis of fulminant hepatitis (FHB). Recently, we isolated from a patient with fulminant recurrent hepatitis B after liver transplantation variants with enhanced replication competence and a possible defect in viral particle secretion. Both viral features may have contributed to the severity of the disease. The aim of this study was to prove the secretion defect of these variants, to analyze the consequences, and to identify the responsible viral mutations. The variant genomes and appropriate wild-type/variant hybrid genomes were functionally characterized after transfection in human hepatoma cells. Two cloned genomes and the polymerase chain reaction (PCR)-amplified mixture of full-length genomes showed a block in viral particle secretion. This was caused by a combination of amino acid changes in the Sprotein including the mutation G145R frequently emerging after hyperimmunoglobulin treatment. The mutations induced retention of the surface proteins in an endoplasmic reticulum (ER)-like compartment, but no intracellular accumulation. These data provide evidence for the in vivo existence of a dominant HBV population with a severe defect in The occurrence of hepatitis B virus (HBV) variants as minor or major viral populations in infected patients with fulminant, acute, and chronic hepatitis B was amply documented in the past. 1-5 HBV variants can differ from wild-type virus (WT) both in the DNA and viral protein sequences. Sequence variability and mutations were found in regulatory regions as well as in structural and nonstructural proteins. There is increasing evidence for a role of specific hepadnaviral variants in establishing chronic infection, virus/host cell interaction, and hepatopathogenesis. 4,5 In addition, several epidemic outbreaks of fulminant hepatitis B infections, which could be traced back to one chronically infected carrier, argue for an impact of viral variants in the pathogenesis of fulminant hepatitis (FHB). 6-9 Furthermore, it was shown that viral strains isolated from patients with FHB are particularly pathogenic for chimpanzees. 10 Three animals that were infected with serum of a child who transmitted FHB to 2 pediatricians developed severe hepatitis with unusually high transaminases for these animals. Based on the hypothesis that certain pathogenic HBV strains may cause or contribute to FHB several studies tried to identify structural characteristics of viral variants of these patients by determining the DNA and amino acid sequences of certain regions or the complete HBV genomes. 11-14 These experiments revealed that certain mutations, such as the precore stop codon mutation A-1896 and the core promoter mutations T-1762 and A-1764 occur more frequently in patients with FHB compared with chronic carriers, but no mutation specific for this disease course could be identified. However, this does not exclude a role of HBV variants in the pathogenesis of FHB because several differen...

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“…In the experimental setting used here (i.e., transfection of hepatoma cells), it is impossible to determine selectively the amount of virion-associated HBV DNA in the culture medium by Southern blotting because substantial amounts of nonenveloped core particles often are released from transfected cells. Therefore, for specific analysis of virion-associated HBV DNA, virions were separated from core particles by native agarose gel electrophoresis 23 and subsequent detection of viral proteins and DNA by Western and Southern blot analysis, respectively ( Fig. 3).…”

Section: G145r Substitution Does Not Affect Dna Virus Replication or mentioning

confidence: 99%

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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Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus

Cited by 140 publications

References 18 publications

Acute liver injury following infection with a cytopathic strain of duck hepatitis B virus

Acute liver injury following infection with a cytopathic strain of duck hepatitis B virus

A dominant hepatitis B virus population defective in virus secretion because of several S-gene mutations from a patient with fulminant hepatitis

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

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