Evolutionary conservation in the hepatitis B virus core structure: comparison of human and duck cores.

BACKGROUND: Hepatitis B virus is a major human pathogen which has been extensively studied, yet its structure is unknown. Cryo-electron microscopy of the viral cores expressed in Escherichia coli or isolated from infected liver provides a means for determining the structure of the hepatitis B nucleocapsid. RESULTS: Using cryo-electron microscopy and three-dimensional image reconstruction, we have determined the structures of duck and human hepatitis B virus cores and find that they have similar dimer-clustered T = 3 and T = 4 icosahedral organizations. The duck virus core protein sequence differs from the human in both length and amino acid content; however, the only significant structural differences observed are the lobes of density on the lateral edges of the projecting (distal) domain of the core protein dimer. The different cores contain varying amounts of nucleic acid, but exhibit similar contacts between the core protein and the nucleic acid. Immunoelectron microscopy of intact cores has localized two epitopes on the core surface corresponding to residues 76-84 and 129-132. CONCLUSIONS: The bacterial expression system faithfully reproduces the native hepatitis B virus core structure even in the absence of the complete viral genome. This confirms that proper assembly of the core is independent of genome packaging. Difference imaging and antibody binding map three sequence positions in the structure: the C terminus and the regions near amino acids 80 and 130. Finally, we suggest that the genome-core interactions and the base (proximal) domain of the core dimer are evolutionarily conserved whereas the projecting domain, which interacts with the envelope proteins, is more variable.

Ultrastructural study of hepatitis B virus in biliary epithelial cells of duck liver.

Liver specimens from the Shanghai Ma ducks congenitally infected with duck hepatitis B virus (DHBV) were examined by immunohistochemical technique and electron microscopy. All the 12 serum DHBV positive ducks showed varying degrees of positive straining in hepatocytes. In 8 of the 12 ducks, DHBV antigen was discovered in the cytoplasm of biliary epithelial cells. Conventional electron microscopy revealed two kinds of virus particles in the biliary epithelial cells: 1. the incomplete virus, 40-50 nm in diameter and spherical in shape with an outer membrane, located mainly in the dilated cisternae of rough endoplasmic reticulum of the cells in large amounts; 2. the complete virions, 55-60 nm in diameter, were spherical with an outer membrane and located mainly in the cytoplasmic vesicles in small amount. We believe the particles found in the biliary epithelial cells in this study were DHBV particles. It is most likely that the infection and replication of DHBV not only take place in the liver cells but also in the biliary epithelial cells.

[Ultrastructural study and localization of duck hepatitis B virus in the livers of experimentally infected ducks].

An electron microscopic study was performed to investigate the occurrence and localization of duck hepatitis B virus (DHBV) in the livers of infected Beijing ducks. DHBV-DNA was found in trace amounts in liver extract on 9th hours after infection by Southern blot hybridization and was found in serum on the 4th day of infection. Virus particles were recognized in the hepatocytes as early as the 3rd day of infection. Many incomplete virus particles, (50 to 80 nm in diameter), and a few complete virus particles, (45-65 nm in diameter), were seen in cisternae of rough and smooth endoplasmic reticula of hepatocytes, clusters or scattered core particles, (35-38 nm in diameter), were seen in the cytoplasm, some core particles were found in nuclei of liver cells where core assembly might be occur. The similarities and differences of the localizations of DHBV and HBV in duck liver cells are discussed.

[Ultrastructural study of duck hepatitis B virus in biliary epithelial cells of duck liver].

Liver specimens from Shanghai Ma ducks infected with duck hepatitis B virus (DHBV) were examined by immunohistochemical technique and electron microscopy. The results showed that DHBV and DHBV antigen were found not only in the hepatocytes but also in the biliary epithelial cells of infected ducks. Electron microscopy also revealed that incomplete spherical particles, 40-50 nm in diameter, were present in the dilated cisternae of rough endoplasmic reticulum (RER), and complete spherical virions, 55-65 nm in diameter, existed in the cytoplasmic vesicles and cytoplasm in small amounts. The results of the present study seem to confirm the possibility of infection and replication of DHBV not only in the liver cells but also in the biliary epithelial cells of ducks.

Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay.

Replication of hepadnaviruses involves reverse transcription of an intermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymerase which has DNA-dependent DNA polymerase, reverse transcriptase, and RNase H activities. Biochemical studies of the polymerase protein(s) have been limited by the inability to purify useful quantities of functional enzyme from virus particles and, until recently, to express enzymatically active polymerase proteins in heterologous systems. An activity gel assay which detects in situ catalytic activities of DNA polymerases after electrophoresis in partially denaturing polyacrylamide gels was used by M.R. Bavand and O. Laub (J. Virol. 62:626-628, 1988) to show the presence of DNA- and RNA-dependent DNA polymerase activities associated with hepatitis B virus particles produced in vitro. This assay has provided the only means by which hepadnavirus polymerase proteins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantities of enzymatically active polymerase protein(s), we have devised a protocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles contain DNA- and RNA-dependent DNA polymerase activities, as shown in the endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins associated with these purified DHBV core particles. These enzymatically active proteins appear larger than, approximately the same size as, and smaller than an unmodified DHBV polymerase protein predicted from the polymerase open reading frame. This is the first report of the detection of active hepadnavirus core-associated DNA polymerase proteins derived from a natural host.

Local average intensity-based method for identifying spherical particles in electron micrographs.

A method is presented that reliably detects spherical viruses from a wide variety of noisy low-contrast electron micrographs. Such detection is one of the first image analysis steps in the computer-aided reconstruction of three-dimensional density distribution models of viruses. Particle detection is based on the comparison of intensity in a circular area and in the surrounding ring followed by a number of tests to validate the potential particles. The only required input from the user in addition to the micrograph is an approximate radius of the particle. The method has been implemented as program ETHAN that has been tested for several different data sets. ETHAN has also successfully been used to detect DNA-less virus particles for an actual reconstruction.