Hepatitis B virus DNA forms in nuclear and cytoplasmic fractions of infected human liver.
Human liver tissues obtained at autopsy from two patients chronically infected with hepatitis B virus (HBV) were found to contain several distinct species of HBV DNA. Southern blot analysis using a nick-translated HBV [32P]DNA probe identified specific DNA bands migrating at the positions expected for linear double-stranded DNA of 3.6 and 2.0 kb. These DNA bands were shown to represent relaxed circular and closed circular (supercoiled) HBV DNA, respectively. In addition to these distinct bands several minor bands as well as a heterogeneous population of HBV DNA molecules were present. When infected cell nuclei were isolated, and the nuclear and cytoplasmic nucleic acid separately analyzed, the nuclear fraction contained the 2.0-kb DNA species. This species was shown to be supercoiled 3.2-kb HBV DNA by electron microscopy, restriction endonuclease digestion, and thermal denaturation. The cytoplasmic fraction contained DNA forms similar to those found in virions isolated from plasma (i.e., migration in the position of linear double-stranded molecules of 3.6 and 3.2 kb) and no supercoiled DNA was detected. Particles isolated from the cytoplasmic fraction were able to incorporate dNTPs into viral DNA sequences. Southern blot analysis of the nucleic acid isolated from the particles revealed the presence of HBV DNA forms migrating in positions expected for 3.6- and 3.2-kb linear double-stranded molecules as well as a heterogeneous population of HBV molecules. The 3.6- and 3.2-kb species were identified as relaxed circular and double-stranded linear genome-length HBV DNA. Digestion of the viral nucleic acid with pancreatic ribonuclease increased the electrophoretic mobility of a portion of the heterogeneous HBV molecules and resulted in the appearance of a distinct 1.9-kb DNA band suggesting the same viral DNA was complexed with RNA. Experiments to be reported elsewhere showed this DNA species to be genome-length minus-strand HBV DNA which was released from DNA-RNA hybrid molecules by RNase digestion. Thus, supercoiled HBV DNA exists free in the nucleus of infected liver cells and cytoplasmic particles contain relaxed circular and linear HBV DNA as well as a heterogeneous population of HBV DNA and DNA-RNA hybrid molecules, and a DNA polymerase reaction in the particles results in incorporation of dNTP into DNA strands of these molecules.
D002467:Cell Nucleus / Q000032:analysis*; D003593:Cytoplasm / Q000032:analysis; D004278:DNA, Superhelical / Q000302:isolation & purification; D004279:DNA, Viral / Q000302:isolation & purification*; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification; D006509:Hepatitis B / Q000382:microbiology*; D006514:Hepatitis B Surface Antigens / Q000032:analysis; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000201:enzymology / Q000382:microbiology*; D008854:Microscopy, Electron; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization
D004278:DNA, Superhelical; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D004259:DNA-Directed DNA Polymerase