Hepatitis B viral DNA-RNA hybrid molecules in particles from infected liver are converted to viral DNA molecules during an endogenous DNA polymerase reaction.
Hepatitis B viral DNA-RNA hybrid molecules in particles from infected liver are converted to viral DNA molecules during an endogenous DNA polymerase reaction.
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DOI：
10.1016/0042-6822(84)90330-1
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发表时间：
1984-11
期刊：
影响因子：
3.7
通讯作者：
Roger H. Miller;Patricia L. Marion;William S. Robinson
中科院分区：
文献类型：
--
作者：
Roger H. Miller;Patricia L. Marion;William S. Robinson
从乙肝病毒感染患者的肝脏中提纯的颗粒以前曾被我们证明，当在DNA聚合酶反应混合物中孵育时，32P-脱氧核苷酸结合到病毒DNA和DNA-RNA杂化分子中。在这项研究中，来自感染了与乙肝病毒密切相关的病毒的鸭肝和地鼠肝的类似颗粒也被证明在DNA聚合酶混合物的体外培养中将32P-脱氧核苷酸结合到病毒特异的DNA和DNA-RNA杂化分子中。来自感染鸭乙型肝炎病毒的肝脏的颗粒含有不同种类的杂化分子，而来自感染黄鼠肝炎病毒的肝脏的颗粒含有密度与在乙肝颗粒中发现的相似的杂化分子，包括平均密度为1.57g/cm3的不同的分子种群。内源性DNA聚合酶与所有三种肝脏的颗粒发生短暂的聚合反应，导致32P-脱氧核苷酸掺入DNA-RNA杂交体的病毒DNA以及病毒DNA分子中。当反应继续进行时，当存在1000倍摩尔过量的未标记脱氧核苷酸时，观察到Cs2SO4密度梯度的DNA-RNA杂交区的[32P]DNA减少，而梯度DNA区的[32P]DNA成比例增加。这种影响在从新获得的感染了地鼠肝炎病毒的肝脏中分离出来的颗粒中表现得最为明显，其中杂交物种中90%的脉冲标记DNA在浮力密度为1.57g/cm3时似乎转化为具有纯DNA浮力密度(1.42g/cm3)的形式。病毒颗粒在4℃下保存，或预先冷冻感染的磨碎鼠肝脏，几乎完全取消了内源DNA聚合酶活性将32P-脱氧核苷酸结合到杂交分子中的能力，而结合到DNA分子中的能力显然没有受到影响。这些结果表明，在DNA-RNA杂交物和具有纯DNA浮力密度的分子中，不同的酶活性催化合成病毒DNA。因此，来自受感染肝脏的颗粒合成了DNA-RNA杂化分子的DNA，可以在颗粒中转化为具有纯DNA浮力密度的分子。这表明DNA-RNA杂交体可能是病毒DNA复制的中间体，并且乙肝病毒(以及与之密切相关的松鼠和鸭子病毒)的DNA复制机制不同于已知的其他DNA病毒。
Particles purified from the liver of hepatitis B virus-infected patients were previously shown by us to incorporate32P-deoxynucleotides into viral DNA and DNA-RNA hybrid molecules when incubated in a DNA polymerase reaction mixture. In this investigation, similar particles from duck and ground squirrel livers infected with viruses closely related to HBV were also shown to incorporate32P-deoxynucleotides into viral-specific DNA and DNA-RNA hybrid molecules when incubatedin vitroin a DNA polymerase reaction mixture. The particles from duck hepatitis B virus-infected liver contained a heterogeneous population of hybrid molecules, while those from ground squirrel hepatitis virus-infected liver contained hybrid molecules with densities similar to those found in HBV particles including a distinct population of molecules with an average density of 1.57 g/cm3. Brief endogenous DNA polymerase reactions with particles from all three livers, resulted in incorporation of32P-deoxynucleotides into viral DNA of DNA-RNA hybrid as well as viral DNA molecules. When the reaction was continued in the presence of a 1000-fold molar excess of unlabeled deoxynucleotides, a decrease in [32P]DNA in the DNA-RNA hybrid region of the Cs2SO4density gradient and a proportional increase in [32P]DNA in the DNA region of the gradient was observed. This effect was seen most dramatically with particles isolated from freshly obtained ground squirrel hepatitis virus-infected livers in which 90% of the pulse labeled DNA in the hybrid species at the buoyant density of 1.57 g/cm3appeared to be converted to a form with the buoyant density of pure DNA (1.42 g/cm3). Storage of virus particles at 4°, or prior freezing of infected ground squirrel liver almost completely abolished the ability of the endogenous DNA polymerase activity to incorporate32P-deoxynucleotides into hybrid molecules, while incorporation into DNA molecules was apparently unaffected. These results suggest that different enzymatic activities catalyze synthesis of the viral DNA in DNA-RNA hybrids and in molecules with buoyant density of pure DNA. Thus particles from infected liver synthesize DNA of DNA-RNA hybrid molecules which can be converted in the particles into molecules with the buoyant density of pure DNA. This indicates that DNA-RNA hybrids may be intermediates in viral DNA replication and that the mechanism of hepatitis B virus (and closely related viruses of ground squirrels and ducks) DNA replication differs from that known for other DNA viruses.