RNASE H ACTIVITY ENCODED BY HEPADNAVIRUSES

本申请的主要长期目标是提供 申请人实习生（迈克尔·巴扎，D.D.S.）一个优秀的基于研究的 基础分子病毒学的实验室培训， 获得博士学位微生物学系学位， 查佩尔山的北卡罗来纳州大学免疫学。 重点 拟议培训计划的研究部分将是 由John Newbold博士领导的嗜肝DNA病毒组。 拟议的研究将 试图鉴定一种病毒编码的RNaseH酶活性， 在未成熟的核心颗粒中是活跃的，亚病毒核衣壳 它是感染性子代病毒体的前体， 感染肝细胞的细胞质。 RNaseH的鉴定将 通过使用丙烯酰胺凝胶的原位凝胶测定（活性凝胶）， 浇铸以包含用放射性标记的酶底物（DNA.RNA杂合体）， RNA链中的P32。 这些研究将使用部分和高度纯化的 制备从肝脏分离的DHBV核心颗粒， 先天感染的鸭子 RNaseH被广泛认为是必不可少的 是嗜肝DNA病毒基因组复制的关键步骤， 发生在逆转录后的核心内。 蛋白 通过计算机程序对嗜肝DNA病毒的序列分析已经鉴定出一种 RNA酶H样结构域位于嗜肝DNA病毒POL ORF的3'端，但直接 身份识别仍然缺乏。 在活性凝胶测定中， 用SDS破坏DHBV核心颗粒，在SDS-PAGE凝胶中电泳， 在缓冲液中冲洗几次，以稀释SDS并允许 蛋白质在凝胶基质内改革它们的天然构象。 RNaseH 蛋白质在冲洗过程中开始原位降解模板， 产生放射性标记底物耗尽的条带。 该方法具有 成功地用于MoMLV的RNaseH测定。 这些研究将 利用一组对DHBV POL序列特异的抗肽抗体。 与RNaseH偶联的Western印迹和免疫沉淀分析 活性凝胶分析可以将酶与POL ORF的结构域相关联。 最后，将该推定的RNaseH结构域亚克隆到E.杆菌 表达载体（M13 mptac 18）和在E.大肠杆菌， 相同的凝胶分析。 子克隆将扩展到相应的 结构域的HBV POL，从而使这种酶可用于研究， 评价作为药物治疗的抗病毒靶点。 DHBV的亚克隆 和HBV RNaseH将进行定向诱变分析， 找出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.