Miller R H, Robinson W S
Virology. 1984 Sep;137(2):390-9. doi: 10.1016/0042-6822(84)90231-9.
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.
在对两名慢性感染乙肝病毒（HBV）患者进行尸检时获取的人肝组织中，发现含有几种不同类型的HBV DNA。使用缺口平移法标记的HBV [32P]DNA探针进行Southern印迹分析，鉴定出特定的DNA条带，其迁移位置与3.6 kb和2.0 kb线性双链DNA预期位置相符。这些DNA条带分别代表松弛环状和共价闭合环状（超螺旋）HBV DNA。除了这些明显的条带外，还存在几条较小的条带以及HBV DNA分子的异质群体。当分离感染细胞核并分别分析核和细胞质核酸时，核部分含有2.0 kb的DNA种类。通过电子显微镜、限制性内切酶消化和热变性分析表明，该种类为超螺旋3.2 kb HBV DNA。细胞质部分含有的DNA形式与从血浆中分离出的病毒颗粒中发现的类似（即迁移位置在3.6 kb和3.2 kb线性双链分子处），未检测到超螺旋DNA。从细胞质部分分离出的颗粒能够将脱氧核苷三磷酸掺入病毒DNA序列中。对从颗粒中分离出的核酸进行Southern印迹分析，显示存在迁移位置与3.6 kb和3.2 kb线性双链分子预期位置相符的HBV DNA形式以及HBV分子的异质群体。3.6 kb和3.2 kb的种类被鉴定为松弛环状和双链线性基因组长度的HBV DNA。用胰核糖核酸酶消化病毒核酸会增加部分异质HBV分子的电泳迁移率，并导致出现一条明显的1.9 kb DNA条带，表明相同的病毒DNA与RNA复合。在其他地方报告的实验表明，这种DNA种类是基因组长度的负链HBV DNA，通过核糖核酸酶消化从DNA - RNA杂交分子中释放出来。因此，超螺旋HBV DNA在感染的肝细胞细胞核中游离存在，细胞质颗粒含有松弛环状和线性HBV DNA以及HBV DNA和DNA - RNA杂交分子的异质群体，颗粒中的DNA聚合酶反应导致脱氧核苷三磷酸掺入这些分子的DNA链中。
