Bosch V, Bartenschlager R, Radziwill G, Schaller H
Zentrum für Molekulare Biologie, Universität Heidelberg, Federal Republic of Germany.
Virology. 1988 Oct;166(2):475-85. doi: 10.1016/0042-6822(88)90518-1.
A number of antisera, elicited against different segments of the duck hepatitis B virus (DHBV) P-gene translation product, were used to immunoprecipitate the protein that is covalently bound to the 5'-end of the DHBV DNA minus strand. For monitoring purposes, a small DNA minus-strand fragment, carrying this protein, was radioactively labeled. All of the P-specific antisera specifically immunoprecipitated this DNA fragment demonstrating that the protein species attached to the immunoprecipitated DNA fragment were products of the DHBV P-gene. The electrophoretic behavior, in SDS gels, of the DNA minus-strand fragment-protein complex indicated that it was present mostly in the form of aggregates. However, a small fraction consisted of DNA minus-strand fragments carrying P-gene proteins, encoded solely within the 5'-region of the P-gene. This indicated that different P-gene proteins, presumably covalently bound at a common region and subsequently processed, were bound to the 5'-end of the DHBV DNA minus strand. The DHBV P-gene presumably codes for the virus-associated reverse transcriptase and DNA polymerase activities. Using the P-gene-specific antisera, it was not possible to detect putative P-gene-coded polymerase proteins in a free form, i.e., not bound to viral DNA. This may be due to insufficient sensitivity or to the polymerase protein(s) being heterogeneous and/or aggregated. In addition, it is possible that the genome-bound protein itself may have polymerase activity.
针对鸭乙型肝炎病毒(DHBV)P基因翻译产物的不同片段产生的多种抗血清,被用于免疫沉淀与DHBV DNA负链5′端共价结合的蛋白质。为便于监测,携带该蛋白质的一小段DNA负链片段被放射性标记。所有P特异性抗血清均能特异性免疫沉淀此DNA片段,这表明与免疫沉淀的DNA片段相连的蛋白质是DHBV P基因的产物。DNA负链片段 - 蛋白质复合物在SDS凝胶中的电泳行为表明,它大多以聚集体形式存在。然而,一小部分由携带仅在P基因5′区域编码的P基因蛋白质的DNA负链片段组成。这表明不同的P基因蛋白质,可能在一个共同区域共价结合并随后加工,与DHBV DNA负链的5′端相连。DHBV P基因可能编码与病毒相关的逆转录酶和DNA聚合酶活性。使用P基因特异性抗血清,无法检测到游离形式(即不与病毒DNA结合)的假定P基因编码的聚合酶蛋白。这可能是由于灵敏度不足,或者聚合酶蛋白具有异质性和/或聚集体化。此外,有可能与基因组结合的蛋白质本身可能具有聚合酶活性。