Expression and Characterization of Hepatitis B Virus Precore-Core Antigen in E. coli

Lanford, Robert E.; Notvall, Lena; Dreesman, Gordon R.; Harrison, Christopher; Lockwood, Don E.; Burk, Kenneth H.

doi:10.1089/vim.1987.1.97

Cited by 8 publications

(5 citation statements)

References 36 publications

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“…Similar steady-state levels of HBV core proteins were observed between wild-type and mutant viruses. Briefly, cell lysates harvested 3 days posttransfection were analyzed by Western blotting using a rabbit anticore antibody (21). The same filter was also probed with a monoclonal antibody to the HBVspecific pre-S1 protein as an internal control for equal loading and transfection efficiency (17; data not shown).…”

Section: Resultsmentioning

confidence: 99%

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Low-Level Secretion of Human Hepatitis B Virus Virions Caused by Two Independent, Naturally Occurring Mutations (P5T and L60V) in the Capsid Protein

Pogam

Yuan

Sahu

et al. 2000

J Virol

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The functional significance of naturally occurring variants of human hepatitis B virus (HBV) remains largely unknown. Previously, we reported an immature secretion phenotype caused by a highly frequent mutation at amino acid 97 of the HBV core (capsid) protein (HBcAg). This phenotype is characterized by a nonselective and excessive secretion of virions containing an immature genome of single-stranded viral DNA. To extend our study of virion secretion to other naturally occurring variants, we have characterized mutations at HBcAg codons 5, 38, and 60 via site-directed mutagenesis. Although the phenotype of the mutation at codon 38 is nearly identical to that for the wild-type virus, our study reveals that a single mutation at codon 5 or 60 exhibits a new extracellular phenotype with significantly reduced virion secretion yet maintains normal intracellular viral DNA replication. A complementation study indicates that the mutant core protein alone is sufficient for the "lowsecretion" phenotype. Furthermore, the low-secretion phenotype of the codon 5 mutant appears to be induced by the loss of a parental proline residue, rather than by the gain of a new amino acid. Our study underscores the core protein as another crucial determinant in virion secretion, in addition to the known envelope proteins. Our present results suggest that a very precise structure of both ␣-helical and nonhelical loop regions of the entire HBcAg molecule is important for virion secretion. The low-secretion variants may contribute to the phenomenon of gradually decreasing viremia in chronic carriers during the late phase of persistent infection.Hepatitis B virus (HBV) is an important human infectious agent. Worldwide, at least 300 million individuals are chronic HBV carriers. Chronic infection with HBV leads to the development of cirrhosis and liver cancer (31, 32). Naturally occurring variants have been found frequently in chronic HBV carriers. The potential role of these variants in chronicity and pathogenesis remains unclear, since no universal assay is available to evaluate these diverse HBV variants. For example, naturally occurring variants of HBV core antigen (HBcAg) have been frequently reported. This 183-amino-acid protein has multiple functions, including interaction with the pregenomic RNA and polymerase during encapsidation, polymerization with itself to form the nucleocapsid, import of relaxed circular DNA to the nucleus, and targeting to the endoplasmic reticulum for envelope formation (3,7,16,25,26,37). In addition, HBcAg is structurally related to the secreted e antigen (27), and thus certain mutations in HBcAg should also be present in the e antigen. Because of the highly versatile nature of the HBV core protein, it is difficult to predict which functional assays should be used for the study of HBcAg variants in order to yield more informative results. Therefore, the biological significance of these mutations remains unclear.Previously, we described several missense mutations in HBcAg which coincide with major histocompatibili...

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Section: Resultsmentioning

confidence: 99%

“…Core protein expression was detected by Western blot analysis using a rabbit anticore antibody (21). Detection of HBsAg and HBeAg was performed using the Abbott HBsAg kit and HBe (rDNA) kit, respectively (42).…”

Section: Plasmid Constructs (I) Y38h L60v Y38h/l60vmentioning

confidence: 99%

Low-Level Secretion of Human Hepatitis B Virus Virions Caused by Two Independent, Naturally Occurring Mutations (P5T and L60V) in the Capsid Protein

Pogam

Yuan

Sahu

et al. 2000

J Virol

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“…Serological response against HBc is considered a marker of exposure to HBV infection (Kao, 2008). The recombinant HBV core protein has been successfully expressed using a variety of cell systems including bacteria (Lanford et al, 1987;Pasek et al, 1979), yeast (Miyanohara et al, 1986), mammalian cells (Roossinck et al, 1986), and insect cells (Lanford & Notvall, 1990). HBV core protein possesses the property to self-assemble in VLPs eliciting a strong immune response when inoculated in mice (Lanford & Notvall, 1990).…”

Section: Discussionmentioning

confidence: 99%

Detection of antibodies against domestic cat hepadnavirus using baculovirus‐expressed core protein

Fruci

Profio

Palombieri

et al. 2022

Transbounding Emerging Dis

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A novel orthohepadnavirus (domestic cat hepadnavirus [DCH]) similar to human hepatitis B virus has been recently detected in serum and liver samples from domestic cats with chronic hepatitis and hepatocellular carcinoma. Molecular investigations by independent research groups around the world have revealed positivity rates ranging from 6.5% to 12.5% in blood samples and up to 14.0% in liver tissue. In this study, we screened an age-stratified collection of feline sera (n = 256) by using an antibody detection enzyme-linked immunosorbent assay based on the recombinant core antigen of DCH (DCHc). Specific antibodies (DCHc Abs) were detected with a prevalence of 25.0%. The DNA of DCH was detected in 35.9% (23/64) of seropositive cats and only in 1.0% (2/192) of seronegative animals. Based on the serological (IgG and IgM anti-DCHc) and virological status, the possible stages of DCH infection were predicted.

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“…Total cellular RNA extracted from liver tissue (24) was subjected to agarose gel electrophoresis in 2.2 M formaldehyde (25). Nucleic acids were transferred to GeneScreenPlus membranes according to manufacturer's protocols, and hybridizations (23) were performed in 1% SDS, 1 M NaCl at 65°C using nick-translated plasmid pN-1 as a probe; an intact HBV genome cloned at the EcoRI site of pUCl8 (18). Autoradiography was as described above.…”

Section: Immunoprecipitation Of F"s]methionine-labeledmentioning

confidence: 99%

“…Immunofluorescence. Hepatocytes grown on collagentreated glass coverslips were washed three times in phosphatebuffered saline, air dried, fixed in acetone and processed for indirect immunofluorescence (17) with rabbit antisera directed against either HBsAg (Calbiochem, San Diego, CA) or HBcAg (18).…”

mentioning

confidence: 99%

In vitro replication and expression of hepatitis B virus from chronically infected primary chimpanzee hepatocytes

Jacob¹,

Eichberg²,

Lanford³

1989

Hepatology

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Primary chimpanzee hepatocytes were maintained in vitro utilizing a serum-free medium. Hepatocyte functions were sustained throughout the culture period as demonstrated by the synthesis and secretion of liver-specific plasma proteins characteristic for differentiated hepatocytes. Hepatocyte cultures established from a chimpanzee chronically infected with human hepatitis B virus exhibited the synthesis and secretion of hepatitis B virus proteins into the medium. In addition, the de novo replication of hepatitis B virus was documented by the recovery of virus, exhibiting an endogenous DNA polymerase activity, from the tissue culture medium. Therefore, both the long-term maintenance of differentiated hepatocytes and the expression of hepatitis B virus from these primary cultures were sustained in the serum-free medium.

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Expression and Characterization of Hepatitis B Virus Precore-Core Antigen in E. coli

Cited by 8 publications

References 36 publications

Low-Level Secretion of Human Hepatitis B Virus Virions Caused by Two Independent, Naturally Occurring Mutations (P5T and L60V) in the Capsid Protein

Low-Level Secretion of Human Hepatitis B Virus Virions Caused by Two Independent, Naturally Occurring Mutations (P5T and L60V) in the Capsid Protein

Detection of antibodies against domestic cat hepadnavirus using baculovirus‐expressed core protein

In vitro replication and expression of hepatitis B virus from chronically infected primary chimpanzee hepatocytes

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