Structure Of Hepatitis Research Articles

Hepatitis B viral core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped

The structure of hepatitis B virus (HBV) nucleocapsids has been revealed in great detail by cryoelectron microscopy. How nucleocapsids interact with surface antigens to form enveloped virions remains unknown. In this study, core mutants with N-terminal additions were created to address two questions: (1) can these mutant core proteins still form nucleocapsids and (2) if so, can the mutant nucleocapsids interact with surface antigens to form virion-like particles. One plasmid encoding an extra stretch of 23 aa, including six histidine residues, fused to the N terminus of the core protein (designated HisC183) was expressed in Escherichia coli and detected by Western blot. CsCl gradient and electron microscopy analyses indicated that HisC183 could self-assemble into nucleocapsids. When HisC183 or another similar N-terminal fusion core protein (designated FlagC183) was co-expressed with a core-negative plasmid in human hepatoma cells, both mutant core proteins self-assembled into nucleocapsids. These particles also retained kinase activity. Using an endogenous polymerase assay, a fill-in HBV DNA labelled with isotope was obtained from intracellular nucleocapsids formed by mutant cores. In contrast, no such signal was detected from the transfection medium, which was consistent with PCR and Southern blot analyses. Results indicate that core mutants with N-terminal extensions can form nucleocapsids, but are blocked during the envelopment process and cannot form secreted virions. The mutant nucleocapsids generated from this work should facilitate further study on how nucleocapsids interact with surface antigens.

The structure of hepatitis B envelope and molecular variants of hepatitis B virus.

Accumulated evidence in recent years has shown that the variation of hepatitis B virus (HBV) genomes may have profound implications for our understanding of hepatitis B pathogenesis and prevention. Attention has focused on areas of the outer envelope coded by the S gene which are involved in the induction of a protective neutralising antibody response, and mutations which directly affect the production of C gene products, one of which is considered as a target for immune T cells involved in virus clearance. This review highlights recent experimental data which emphasizes the role of such mutations in the establishment and maintenance of chronic HBV infections and focuses attention on the significance of HBV variants with respect to the expanding use of HBV vaccines for mass immunization.

Site-directed mutagenesis of cysteine residues of hepatitis B surface antigen. Analysis of two single mutants and the double mutant.

The structure of hepatitis B surface antigen (HBsAg) is mainly maintained by an intricate disulfide network responsible for most of its structural and antigenic properties. Characterization of three cysteine-replacement mutants of HBsAg has been performed by both structural and immunological methods. Replacement of Cys121 or Cys124 with serine results in mutant proteins that show diminished binding titres to both monoclonal antibodies and to a polyclonal serum, indicating that a structural change has taken place. Circular dichroism analysis shows that the substitution of either of these two residues also diminishes the helical content of the protein. However, the double mutant, in which both cysteine residues have been simultaneously changed, reverts the properties of the single mutations, and shows similar behaviour to the wild-type protein. Both the single and double cysteine mutants are efficiently glycosylated and secreted from Chinese hamster ovary cells and, in all cases, the mutant proteins assemble into spherical particles of similar buoyant density to both the wild-type and serum derived HBsAg.

Proteolytic activity of hepatitis A virus 3C protein

Although the genome organization and overall structure of hepatitis A virus are similar to those of other picornaviruses, nothing is known about the protein-processing pathways used by this virus to generate its capsid and nonstructural proteins from the polyprotein precursor. RNA transcripts of cloned hepatitis A virus cDNAs representing parts of the P2 and P3 regions of the genome were translated in rabbit reticulocyte lysates in vitro, and the translation products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after immunoprecipitation with specific antisera. Pulse-chase experiments demonstrated rapid cleavage at the P2-P3 junction, followed by further but incomplete processing at the 3C-3D junction. Mutation of the 3C coding sequence eliminated all cleavages. Efforts to demonstrate intermolecular cutting of the P2-P3 cleavage site by active 3C or 3CD sequences were unsuccessful; thus, it is likely that this cleavage occurs by intramolecular reaction, in cis.

The structure of hepatitis B surface antigen and its antigenic sites

AbstractHepatitis B virus infects about 200 million people worldwide yearly. The consequences of the infection range from mild, self‐limiting hepatitis with full recovery and immunity to chronic infection with liver disease of varying severity, including fulminant hepatitis and death. Alternatively, individuals may become healthy carriers of the virus and thus serve as a reservoir of infection. In addition, all chronic carriers of the virus are also at risk for development of primary hepatocellular carcinoma later in life. Efforts to combat this disease on a global scale require an inexpensive vaccine. Studies of the hepatitis B viral coat protein (HBsAg) have permitted the development of two generations of vaccine to date, and detailed structural studies of its antigenic sites may eventually allow the development of a completely synthetic (and therefore inexpensive) vaccine in the future.

Anti-HBs antibody of normal human subjects predominantly binds 54 K and 60 K dalton HBs polypeptides.

The structure of hepatitis B virus surface antigen (HBs) recognized by anti-HBs antibody was analyzed by western blotting using anti-HBs sera obtained from normal subjects, from rabbits immunized with purified HBs and commercially available goat serum. The HBs used had 7 components of 24 K, 27 K, 33 K, 36 K, 39 K, 43 K and 67-72 K daltons. Goat anti-HBs serum bound all of these components, while human and rabbit anti-HBs sera bound only two components (60 K and 54 K daltons), which were hardly visible in the gel even by silver staining. Mixing the 24 K and 27 K components, and the 24 K and 43 K components without reducing reagent produced several polymerized forms of HBs components including 60 K and 54 K polypeptides, which were recognized by anti-HBs rabbit serum. Other combinations of HBs components did not yield any new polymeric forms. Thus, it was concluded that the formation of anti-HBs antibody in normal subjects might predominantly require an antigenic structure of polymeric forms of specific combinations of HBs polypeptides, other than previously known antigenic determinants.

Electron microscopic and solution X-ray scattering observations on the structure of hepatitis B surface antigen.

The structure of the small, spherical hepatitis B surface antigen was studied by negative staining, freeze-fracture and freeze-etching electron microscopy and solution X-ray scattering techniques. The protein appears to be organized at the surface into a small number of morphological subunits which display two- and threefold axes of symmetry. The mean particle size was 18.3 nm by negative staining and 19.6 nm by freeze-fracture electron microscopy. The diameter of the individual subunits was about 7.5 nm with an intersubunit distance of about 10.0 nm. The lipid is distributed more homogeneously. Some heterogeneity of the particle structure is apparent which may be due to a slightly variable lipid-protein composition or incomplete or defective particle formation.

Structure of hepatitis B surface antigen. Correlation of subtype with amino acid sequence and location of the carbohydrate moiety.

Hepatitis B surface antigens (HBsAg) of both the adw and ayw subtypes have been purified from four different sources. These antigens have been compared by comparison of the products of tryptic hydrolysis performed under conditions which do not disrupt the overall particle morphology of HBsAg. The resultant peptides were compared by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and high performance liquid chromatography followed by amino acid analysis and Edman degradation of the isolated peptides. The same techniques were also applied to HBsAg which had been labeled with tritium in the carbohydrate moiety of the glycoprotein gp-30. These studies demonstrate that residues 122-150 of the protein p-25 and glycoprotein gp-30 occupy an exposed region of the HBsAg lipoprotein particle and contain the major attachment site for carbohydrate in the case of gp-30. The two subtypes were found to differ at two specific positions in this region, suggesting that this is an antigenically important area of the protein.

Structure of hepatitis B surface antigen. Characterization of the lipid components and their association with the viral proteins.

The lipid composition of hepatitis B surface antigen (HBsAg) (subtype adw) obtained from different carriers has been determined and proven to be truly characteristic of HBsAg and not subject to individual variation. Phosphatidylcholine (approximately 60%), cholesteryl ester (approximately 14%), cholesterol (approximately 15%), and triglycerides (approximately 3%) are the main HBsAg lipid constituents. The fatty acid composition of the different HBsAg lipid components is similar to that of other normal human serum lipoprotein. A photoactivatable hydrophobic probe, pyrenesulfonyl azide, has been used to determine what portions of the protein components of HBsAg are exposed to the HBsAg lipid matrix. Both major HBsAg protein components became randomly pyrenesulfonyl azide labeled in both the NH2-terminal and COOH-terminal tryptic fragments, therefore suggesting they are buried within the HBsAg lipids. A model for the arrangement of proteins in HBsAg is proposed whereby regions within the NH2-terminal and COOH-terminal parts of the two major HBsAg protein components are buried within the lipid matrix of HBsAg particles, while the antigenically important residue 122-150 region is exposed to the aqueous environment.

Structure of hepatitis B Dane particle DNA before and after the Dane particle DNA polymerase reaction.

DNA isolated from the hepatitis B antigen form known as the Dane particle was examined by electron microscopy before and after the endogenous Dane particle DNA polymerase reaction. The most frequently occurring form was an untwisted circular double-stranded DNA molecule approximately 1 mum in length. Less frequently occurring forms included circular DNA of approximately unit length and having one or more small single-stranded regions, similar circular molecules with one or more tails either shorter or longer than 1 mum in length, and very small circular molecules with tails. There was no increase in frequency or length of tails after a DNA polymerase reaction, suggesting that tails were not formed during this reaction. The mean length of circular molecules increased by 23% when DNA was spread in formamide compared with aqueous spreading, suggesting that single-stranded regions are present in most of the molecules. The mean length of circular molecules obtained from aqueous spreading increased by 27% after a Dane particle DNA polymerase reaction. This indicates that single-stranded regions were converted to double-stranded DNA during the reaction.