Endogenous DNA Polymerase-Positive Core Particles from Hepatitis-Infected Hepatocytes

Abstract

The purification is described of liver-derived hepatitis B core antigen (HBe Ag) from human hepatocytes demonstrating only intranuclear particles by electron microscopy. The purified preparation contained 5 × 10u particles per ml. The particles were mono-dispersed and relatively free of background material. DNA-dependent polymerase activity was present and had greater activity than an equal number of cores derived from Dane particles isolated from plasma. Specificity of the polymerase reaction was confirmed by precipitation of the activity with specific anti-HBc antiserum. A proportion of the liver-derived core particles was nonreactive for DNA polymerase activity. The polymerase-positive population of particles had a larger size than the polymerase-negative population of liver-derived cores as evidenced by gel filtration in Sepharose 4B.

Biochemical and Biophysical Properties of Hepatitis B Core Particles Derived from Dane Particles and Infected Hepatocytes

“…Two preparations of cores obtained from infected hepatocytes were purified from two different livers by the method previously described [10] and outlined briefly in 'Materials and Methods'. Three criteria were used to judge the purity of these preparations.…”

“…Unlabeled, purified Dane-and liver-derived core particles were diluted in PBS-BSA to the same particle per ml concentration (1.5 x 1010) based on electron microscopic particle counts [10]. A time course of incorporation experiment was performed over a 3-hour incubation period at 37° for detection of the endogenous DNA-dependent DNA polymerase activity.…”

“…A human liver containing intranuclear core particles was obtained from an HBsAg/Wiv-positive individual who died of chronic glomerulonephritis. Clinical description of the patient, as well as electron micrographs of hepatocytes, have been presented elsewhere [10].…”

“…An immunoradiometric assay was performed essentially as described previously [10]. The wells of polyvinyl micro titer plates (No.…”

“…Purified HBcAg was prepared in our laboratory as previously described [10]. Briefly, 20 g of liver tissue which had been frozen and thawed once was homogenized in a Sorvall omnimixer with 80 ml of extraction buffer containing 0.05 m Tris-HCl, 0.04 M KC1,0.005 M sodium acetate, and 0.5% (w/v) Triton X-100, pH 8.0.…”