RNASE H ACTIVITY ENCODED BY HEPADNAVIRUSES

这个应用程序的主要长期目标是提供 申请者实习生(迈克尔·天丝，D.D.S.)以优秀的研究为基础 基础分子病毒学实验室培训将导致IS 获得微生物学系博士学位和 北卡罗来纳大学教堂山分校的免疫学教授。焦点 对于拟议培训方案的研究部分将是 由约翰·纽博尔德博士领导的庚型核糖核酸病毒小组。拟议的研究将 尝试鉴定一种病毒编码的RNAseH酶活性 在未成熟的核心粒子中活跃，亚病毒核衣壳 这是传染性子代病毒粒子的前驱物质，它在 感染的肝细胞的细胞质。RNAseH Will的识别 通过使用丙烯酰胺凝胶的原位凝胶分析(活性凝胶) 含有放射性标记的酶底物(DNA.RNA杂交物)的铸型 P32在RNA链上。这项研究将使用部分和高度纯化的 日本血吸虫肝组织中分离的DHBVc核心颗粒的制备 先天感染的鸭子。RNAseH被广泛认为是必不可少的 这是复制庚型肝炎病毒基因组的关键一步 发生在反转录之后的岩心内。蛋白 用计算机程序对庚型核糖核酸病毒的序列分析已鉴定出一种 位于庚型病毒POL ORF 3‘端的RNAseH样结构域，但直接 身份识别仍然缺乏。在活性凝胶分析中，浓缩物 DHBV核心颗粒被十二烷基硫酸钠破坏，在十二烷基硫酸钠-聚丙烯酰胺凝胶中电泳化， 在稀释十二烷基硫酸钠的缓冲液中漂洗几次，使 蛋白质在凝胶基质中改变其天然构象。RNAseH 在漂洗过程中，蛋白质开始在原位降解模板并 产生用于放射性标记衬底的耗尽的带。此方法具有 已成功地用于MoMLV的RNAseH检测。到那时，这些研究将 开发一组针对DHBVPOL序列的抗肽抗体。 蛋白质印迹和免疫沉淀分析与RNAseH联用 活性凝胶分析可以将该酶与POL ORF的一个结构域联系起来。 最后，这个假定的RNAseH结构域将被亚克隆到大肠杆菌中 表达载体(M13 Mptac18)及其在大肠杆菌中检测到的酶 同样的凝胶分析。子克隆将扩展到相应的 HBVPOL的结构域，从而使该酶可用于研究和 作为药物治疗的抗病毒靶点的评价。登革热病毒的亚克隆 和HBVRNAseH将接受定向突变分析，以 确定这种病毒酶中的关键氨基酸。

The primary long-term objective of this application is to provide the applicant trainee (Michael Tencza, D.D.S.) with an excellent research-based laboratory training in basic molecular virology that would lead to is obtaining the Ph.D. degree from the Department of Microbiology and Immunology at the University of North Carolina at Chapel Hill. The focus for the research component of the proposed training program will be the hepadnavirus group headed by Dr. John Newbold. The proposed research will attempt to identify a viral encoded RNaseH enzyme activity that is believed to be active within the immature core particle, a subviral nucleocapsid that is a precursor to infectious progeny virions and which is abundant in the cytoplasm of infected liver cells. Identification of the RNaseH will be by in situ gel assay (activity gels) using acrylamide gels which are cast to contain the enzyme substrate (a DNA.RNA hybrid) radiolabeled with P32 in the RNA strand. The studies will use partially and highly purified preparations of DHBV core particles isolated from the livers of congenitally infected ducks. The RNaseH is widely believed to be essential for a crucial step in the replication of the hepadnavirus genome and which occurs within the cores subsequent to reverse transcription. Protein sequence analyses of hepadnaviruses by computer programs have identified an RNaseH-like domain at the 3' end of the hepadnaviral POL ORF, but direct identification remains lacking. In the activity gel assay the concentrated DHBV core particles are disrupted by SDS, electrophoresed in SDS-PAGE gels, rinsed several times in a buffer that dilutes away the SDS and allows the proteins to reform their native conformation within the gel matrix. RNaseH proteins, during the rinsing, begin to degrade the template in situ and produce a band depleted for the radiolabeled substrate. This method has been used successfully to assay the RNaseH of MoMLV. The studies will then exploit a panel of antipeptide antibodies specific for DHBV POL sequences. Western blot and immunoprecipitation analysis coupled with the RNaseH activity gel assay could associate the enzyme with a domain of the POL ORF. Finally this putative RNaseH domain will be subcloned into an E. coli expression vector (M13 mptac18) and the enzyme detected in E. coli by the same gel assay. The subcloning will be extended to the corresponding domain for HBV POL, thus making this enzyme available for study and evaluation as an antiviral target for drug therapy. The subclones of DHBV and HBV RNaseH will be subjected to directed mutagenesis analysis to identify the critical amino acids in this viral enzyme.