Oberhaus S M, Newbold J E
Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill 27599.
J Virol. 1993 Nov;67(11):6558-66. doi: 10.1128/JVI.67.11.6558-6566.1993.
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.
嗜肝DNA病毒的复制涉及中间RNA分子的逆转录。人们普遍认为，这种复制机制是由病毒编码的多功能聚合酶来完成的，该聚合酶具有依赖DNA的DNA聚合酶、逆转录酶和核糖核酸酶H活性。聚合酶蛋白的生化研究一直受到限制，因为无法从病毒颗粒中纯化出足够量的功能酶，而且直到最近，也无法在异源系统中表达具有酶活性的聚合酶蛋白。M.R. 巴万德和O. 劳布（《病毒学杂志》62:626 - 628，1988年）使用了一种活性凝胶分析法，该方法在部分变性聚丙烯酰胺凝胶中电泳后原位检测DNA聚合酶的催化活性，以显示与体外产生的乙型肝炎病毒颗粒相关的依赖DNA和RNA的DNA聚合酶活性。该分析方法是检测与酶活性相关的嗜肝DNA病毒聚合酶蛋白的唯一手段。由于传统方法无法纯化出足够量的具有酶活性的聚合酶蛋白，我们设计了一种方案，用于将大量鸭乙型肝炎病毒（DHBV）核心颗粒纯化至近乎均一。如内源性DNA聚合酶分析所示，这些未成熟病毒颗粒含有依赖DNA和RNA的DNA聚合酶活性。我们使用活性凝胶分析法检测与这些纯化的DHBV核心颗粒相关的多种依赖DNA和RNA的DNA聚合酶蛋白。这些具有酶活性的蛋白看起来比从聚合酶开放阅读框预测的未修饰DHBV聚合酶蛋白大、大小大致相同或小。这是首次报道从天然宿主中检测到具有活性的嗜肝DNA病毒核心相关DNA聚合酶蛋白。
