Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019163 (hepatitis B)
 38,309 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our studies on the assembly of hepatitis B virus capsids or core particles in Xenopus oocytes have demonstrated that unassembled p21.5 core proteins ("free p21.5") provide a pool of low-molecular-mass precursors for core-particle assembly. Here we have characterized this material. Free p21.5 sedimented through gradients of 3-25% sucrose (wt/vol) as a single protein species of approximately 40 kDa, corresponding to a p21.5 dimer. On nonreducing SDS/polyacrylamide gels, free p21.5 migrated as disulfide-linked p21.5 dimeric species of 35 and 37 kDa. Truncated core proteins lacking most or all of the 36-amino acid protamine region at the p21.5 carboxyl terminus were also found to behave as disulfide-linked dimers with appropriately reduced molecular masses. Our experiments failed to reveal monomeric core proteins or stable intermediates between dimers and capsids along the assembly pathway. We conclude that hepatitis B virus core particles are most likely assembled by aggregating 90 (or possibly 180) disulfide-linked p21.5 dimers. We discuss similarities between the assembly of hepatitis B virus capsids and simple T = 3 plant virus and bacteriophage structures.
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PMID:Hepatitis B virus capsid particles are assembled from core-protein dimer precursors. 143 93

The hepatitis B virus capsid or core protein (p21.5) binds nucleic acid through a carboxy-terminal protamine region that contains nucleic acid-binding motifs organized into four repeats (I to IV). Using carboxy-terminally truncated proteins expressed in Escherichia coli, we detected both RNA- and DNA-binding activities within the repeats. RNA-binding and packaging activity, assessed by resolving purified E. coli capsids on agarose gels and disclosing their RNA content with ethidium bromide, required only the proximal repeat I (RRRDRGRS). Strikingly, a mutant in which four Arg residues replaced repeat I was competent to package RNA, demonstrating that Arg residues drive RNA binding. In contrast, probing immobilized core proteins with 32P-nucleic acid revealed an activity which (i) required more of the protamine region (repeats I and II), (ii) appeared to bind DNA better than RNA, and (iii) was apparently modulated by phosphorylation in p21.5 derived from Xenopus oocytes. Deletion analysis suggested that this activity may depend on an SPXX-type DNA-binding motif in repeat II. Similar motifs found in repeats III and IV may also function to bind DNA. On the basis of these observations, together with a reinterpretation of recent studies showing that capsid protein mutants cause defects in viral genome replication, we propose a model suggesting that hepadnavirus capsid proteins participate directly in the intracapsid reverse transcription of RNA into DNA. In this model, repeat I binds RNA whereas the distal repeats are progressively recruited to bind elongating DNA strands. The latter motifs may be required for replication to be energetically feasible.
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PMID:RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. 150 Dec 73

In the spherical capsid of hepatitis B virus (HBV), intermolecular disulfide bonds cross-link the approximately 180 p21.5 capsid protein subunits into a stable lattice. In this study, we used mutant capsid proteins to investigate the role that disulfide bonds and the four p21.5 Cys residues (positions 48, 61, 107, and 185) play in capsid assembly and/or stabilization. p21.5 Cys residues were either replaced by Ala or removed (Cys-185) by carboxyl-terminal truncation, creating Cys-minus mutants which were expressed in Xenopus oocytes via microinjected synthetic mRNAs. Fractionation of radiolabeled oocyte extracts on 10 to 60% sucrose gradients revealed that Cys-minus core proteins resolved into the nonparticulate and capsid forms seen for wild-type p21.5. On 5 to 30% sucrose gradients, nonparticulate Cys-minus core proteins sedimented as dimers of approximately 40 kDa. We conclude that Cys residues and disulfides are not required for the assembly of either HBV capsids or the dimers that provide the precursors for capsid assembly. Since assembly presumably demands an appropriate p21.5 tertiary structure, it is unlikely that Cys residues are required for proper p21.5 folding. However, Cys residues stabilize isolated p21.5 structures, as evidenced by the marked reduction in stability of Cys-minus dimers and capsids (i) in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and (ii) upon protease digestion. We discuss these results in the context of the HBV life cycle and the role of Cys residues in other proteins.
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PMID:Cys residues of the hepatitis B virus capsid protein are not essential for the assembly of viral core particles but can influence their stability. 150 Dec 80

Little is known about the assembly of the 28-nm nucleocapsid or core particle of hepatitis B virus. Here we show that this assembly process can be reconstituted in Xenopus oocytes injected with a synthetic mRNA encoding the hepatitis B virus capsid protein (p21.5). Injected oocytes produce both a nonparticulate p21.5 species (free p21.5) and capsid particles. We describe rapid and simple methods for fractionating these species on a small scale either with step gradients of 10 to 60% (wt/vol) sucrose or by centrifugation to pellet the particles, and we characterize the oocyte core particles. Free p21.5 exhibits chemical and physical properties distinctly different from those of particles. Free p21.5 is partially cleaved by proteinase K, whereas core particles are almost completely resistant to cleavage. This suggests that the carboxyl-terminal protamine region, the main target for proteases within p21.5, is exposed in free p21.5 but faces the interior of the p21.5 core particle. Finally, pulse-chase experiments demonstrated that free p21.5 can be chased almost quantitatively into core particles, establishing that free p21.5 is fully competent to form particles and represents an assembly intermediate on the pathway for core particle formation. However, core particle assembly appears very dependent on p21.5 concentration and is rapidly compromised if the p21.5 concentration is lowered. The advantages of oocytes for studying assembly are discussed.
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PMID:Characterization of hepatitis B virus capsid particle assembly in Xenopus oocytes. 156 May 38

We have analyzed the translocation of hepatitis B virus (HBV) precore (PC) proteins by using Xenopus oocytes injected with a synthetic PC mRNA. The PC region is a 29-amino-acid sequence that precedes the 21.5-kDa HBV capsid or core (C) protein (p21.5) and directs the secretion of core-related proteins. The first 19 PC amino acids provide a signal peptide that is cleaved with the resultant translocation of a 22.5-kDa species (p22.5), in which the last 10 PC residues precede the complete p21.5 C polypeptide. Most p22.5 is matured to 16-20 kDa species by carboxyl-terminal proteolytic cleavage prior to secretion. Here we show that some four unexpected PC proteins of 24 to 25 kDa are produced in addition to the secretion products described above. Protease protection and membrane cosedimentation experiments reveal that all PC proteins behave as expected for proteins that are translocated into the lumen of the endoplasmic reticulum except for the single largest PC protein (p25), which is not translocated. Like p21.5, p25 is a phosphoprotein that localizes to the oocyte cytosol and nucleus, and protease digestion studies suggest that the two molecules have similar two-domain structures. Radiosequencing of immobilized p25 demonstrates that it contains the intact PC signal peptide and represents the unprocessed translation product of the entire PC/C locus. Thus, while many HBV PC protein molecules are correctly targeted to intracellular membranes and translocated, a significant fraction of these molecules can evade translocation and processing.
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PMID:Hepatitis B virus p25 precore protein accumulates in Xenopus oocytes as an untranslocated phosphoprotein with an uncleaved signal peptide. 172 93

The location of hepatitis B virus (HBV) nucleocapsid (core particle) assembly in infected cells remains controversial. Some lines of evidence implicate the nucleus; others favor the cytoplasm. Via injection of a synthetic mRNA encoding the HBV nucleocapsid protein (p21.5), we have expressed both unassembled p21.5 and nucleocapsidlike core particles in Xenopus oocytes. Subcellular fractionation reveals that approximately 91% of the unassembled p21.5 and 95% of the core particles are cytoplasmic, with only 9 and 5%, respectively, in the nucleus. We present evidence showing that unassembled p21.5 equilibrates between nucleus and cytoplasm by passive diffusion and that intact core particles do not enter the nucleus. To examine the role of the nucleus in core particle formation, we expressed p21.5 in surgically anucleate oocytes. We show that anucleate oocytes support efficient core particle formation, indicating that (i) the nucleus is not essential for assembly and (ii) the cytoplasm can assemble most core particles found in oocytes. On the basis of our data, we propose that in oocytes, most core particle assembly (up to 95%) occurs in the cytoplasm, but that at least approximately 5% of the cellular core particles are assembled in the nucleus and remain there. We discuss the implications of these findings for the formation of replication-competent core particles in infected cells.
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PMID:Production of hepatitis B virus nucleocapsidlike core particles in Xenopus oocytes: assembly occurs mainly in the cytoplasm and does not require the nucleus. 189 94

The nucleocapsid (hepatitis B core Ag (HBcAg] of the hepatitis B virus is a particulate Ag composed of a single polypeptide (p21). Although a non-particulate form of HBcAg designated hepatitis B e Ag (HBeAg) shares significant amino acid identity, the immune responses to these Ag appear to be regulated independently. This report describes the use of recombinant HBcAg and HBeAg to examine and compare murine T cell and B cell recognition of these related Ag. The HBcAg preparation was stable at pH 7.2 and 9.6 and expressed HBc antigenicity. However, the antigenicity of the HBeAg preparation was pH dependent. At pH 9.6 the HBeAg preparation was non-particulate and expressed HBe antigenicity exclusively; however, at pH 7.2 it was particulate and expressed both HBc and HBe antigenicities. Although this "hybrid" particle most likely does not exist naturally, it is a unique research reagent to investigate the interrelationship between HBcAg and HBeAg. HBcAg was significantly more immunogenic in terms of in vivo antibody production as compared to either the non-particulate or particulate forms of HBeAg. Nevertheless, in most murine strains HBcAg and HBeAg were equivalently immunogenic and crossreactive at the level of T cell activation. The disparity between anti-HBc and anti-HBe antibody production is best explained by the observation that HBcAg can function as a T cell-independent Ag whereas HBeAg is T cell dependent even when present within the same particulate structure as HBcAg. Furthermore, HBcAg was shown to function efficiently as an immunologic carrier moiety for the DNP hapten in athymic as well as euthymic mice in contrast to conventional carrier proteins. These results have implications relevant to the human immune responses to HBcAg and HBeAg during infection, and to vaccine development.
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PMID:Comparative immunogenicity of hepatitis B virus core and E antigens. 246 May 43

Hepatitis B virus (HBV)-related antigens produced by the human hepatoma cell line (HB611 cell), which had been transfected with a cloned HBV DNA and established as a stable producer of HBV (T. Tsurimoto, A. Fujiyama, and K. Matsubara, 1987, Proc. Natl. Acad. Sci. USA 84, 444-448), were investigated immunochemically and morphologically. All HBV-related antigens, HBV surface (HBsAg), e (HBeAg), and core (HBcAg), were semiquantitatively examined by the respective reversed passive hemagglutination assay (RPHA). RPHAs for HBcAg and for HBeAg were characterized as reacting only to the core particles and to the free form of nucleocapsid proteins, respectively. The amounts of HBsAg and nucleocapsid protein in culture medium were roughly related to the number of viable cells. The amount of core particles was, instead, proportional to the number of dead cells. Relative amounts of HBsAg, core particles, and nucleocapsid proteins in culture medium, cell surface, and cell lysate were determined and it was found that HBsAg and nucleocapsid proteins were effectively secreted into culture medium but core particles were not. Molecular species of nucleocapsid proteins were identified to be p17 and p18 (HBeAg polypeptides) in the culture medium and HBeAg polypeptides and p21.5 (HBcAg polypeptide) in the cytosol fraction. The p21.5 was preferentially found in the nuclear fraction.
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PMID:Intra- and extracellular distribution and immunochemical characterization of hepatitis B virus nucleocapsid proteins produced by a human hepatoma cell line transfected with cloned viral DNA. 253 6

This study was performed to determine the relationship of the activation of ras and c-myc oncogenes in human hepatocellular carcinoma to the hepatitis B virus gene expression or the presence of hepatitis B virus DNA/RNA at the cellular level. This was done using immunocytochemical analysis with two different antibodies on serial sections. In addition, immunocytochemical assay for the detection of ras p21 or c-myc protein was performed in combination with in situ hybridization for hepatitis B virus DNA/RNA using 35S-labeled hepatitis B virus DNA as a probe. Investigation of a total of 14 paired human hepatocellular carcinoma and adjacent nontumorous hepatic tissues revealed enhanced expression of ras p21 in one human hepatocellular carcinoma whereas c-myc protein was found in one paired human hepatocellular carcinoma and nontumorous tissue of the same patient. Only a small proportion of human hepatocellular carcinoma cells or hepatocytes among a large number of cells on a given section showed enhanced expression, and the distribution of the oncogene product-expressing cells was focal. However, the cells overexpressing these oncogenes did not show hepatitis B surface antigen in the serial sections. Furthermore, the combined immunocytochemical and in situ hybridization assays revealed that human hepatocellular carcinoma cells overexpressing ras p21 did not show hepatitis B virus DNA/RNA, whereas some human hepatocellular carcinoma cells and nontumorous hepatocytes located away from the foci of oncogene-expressing cells gave positive signals. These findings suggest that continued expression of HBsAg or the presence of hepatitis B virus DNA/RNA in a given human hepatocellular carcinoma cell id not necessary for enhanced expression of ras or c-myc proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A lack of direct role of hepatitis B virus in the activation of ras and c-myc oncogenes in human hepatocellular carcinogenesis. 284 52

Expression of the ras oncogene p21 product by hepatocytes of cirrhotic liver with hepatocellular carcinoma (HCC) was examined immunohistochemically using mouse monoclonal antibody RAP-5. At the concentration of antibody used, histologically normal liver tissues were negative for p21 antigen, whereas hepatocytes of cirrhotic nodules from 80 of 92 HCC patients (87.4%), and 10 of 32 patients without HCC (59.4%) were positive. This difference was statistically significant (p less than 0.05). The incidence of p21 expression by hepatocytes was significantly higher in macronodular cirrhotic patients than in those with micronodular cirrhosis (p less than 0.05) and tended to be higher in those positive for hepatic hepatitis B virus markers than in those that were negative (p less than 0.1). All 16 patients with liver cell dysplasia, and 17 of 18 with adenomatous hyperplasias showed increased expression of p21 antigen. In HCC it was detected on tumor cells of 63 of 101 patients (62.4%). Characteristically, its expression in well-differentiated HCC was mild and uniformly diffuse, and in moderately differentiated tumors was markedly heterogeneous in both intensity and distribution, whereas no expression was observed in cells of poorly differentiated HCC. These observations suggest that elevated ras p21 antigen expression is important in the development of both cirrhotic nodules and HCC, but that after tumor development, its sustained elevation is no longer necessary for cell proliferation and progression through the grades of anaplasia.
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PMID:Immunohistochemical detection of ras oncogene p21 product in liver cirrhosis and hepatocellular carcinoma. 303 55


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