Structure and Function of Hepatitis Delta Virus RNA-Protein Complexes

 描述(申请人提供)：丁型肝炎病毒(HDV)是一种重要的人类病原体，可导致严重的急性和慢性肝病。这种病毒有一个独特的复制周期，目前还没有有效的许可治疗方法，这一点还不完全清楚。HDV是一种RNA病毒，它编码一种蛋白质HDAg，其功能类似于核蛋白；它与病毒(-)和(+)RNA形成核糖核蛋白复合体。这些复合体在宿主RNA聚合酶复制病毒RNA的过程中起着至关重要的作用，但由于在实验室中很难组装，它们的特性还不是很好。最近的发展表明，现在有可能通过使用HDV 3型毒株的HDAg在体外创建离散的RNP复合体。这项提议旨在利用这一最新发展，并更充分地描述HDV RNPs的特征。有两个目标，每个目标都有子目标。使用显微注射技术，目标1的第一个子目标将确定必须在RNA上组装多少HDAg多聚体才能启动合成-首先是整个RNA复制周期，然后是单独的RNA合成。目标1的第三部分将利用前两个分目标收集的信息，合理地设计一种尝试，以观察POL II体外从头转录HDV RNA的尝试。目标2将确定RNPs结构的各个方面，特别关注目标1中显示的具有RNA复制功能的RNPs。一个子目标将使用原子力显微镜来可视化单独的RNP复合体，以了解蛋白质如何沿着RNA分布，以及单独的蛋白质多聚体是否相互作用。另外两个子目标将确定哪些特定的RNA序列与细胞、病毒粒子和体外组装的RNPs中的HDAg密切相关。总之，这项提议将极大地扩大我们对HDV RNPs中结构-功能关系的理解，以及病毒如何选择宿主RNA聚合酶进行复制。

 DESCRIPTION (provided by applicant): Hepatitis delta virus (HDV) is a significant human pathogen that causes serious acute and chronic liver disease. There is no effective licensed therapy for this virus, which has a unique replication cycle that is not fully understood. HDV is a RNA virus that encodes a single protein, HDAg, that functions as a nucleoprotein; it forms ribonucleoprotein complexes with the viral (-) and (+) RNAs. These complexes play essential roles in replication of the viral RNA by host RNA polymerase but they are not well characterized because it has been difficult to assemble them in the laboratory. Recent developments have shown that it is now possible to create discrete RNP complexes in vitro by using HDAg from a genotype 3 strain of HDV. This proposal aims to take advantage of this recent development and to more fully characterize the HDV RNPs. There are two aims, each with sub-aims. Using microinjection techniques, the first sub-aims of Aim 1 will determine how many HDAg multimers must assemble on the RNA in order to initiate synthesis - first of the entire RNA replication cycle, then of the individual RNAs independently. The third part of Aim 1 will use the information gleaned from the first two sub-aims to rationally design an attempt to observe de novo HDV RNA transcription by Pol II in vitro. Aim 2 will determine aspects of the structure of the RNPs, with a particular focus on those RNPs shown in Aim 1 to be functional for RNA replication. One sub-aim will employ atomic force microscopy to visualize individually RNP complexes to see how the protein is distributed along the RNA and whether separate protein multimers interact with each other. The other two sub-aims will determine what specific sequences of the RNA are closely associated with HDAg in RNPs from cells, virions and from those assembled in vitro. Altogether, the proposal will substantially expand our understanding of the structure-function relationships in the HDV RNPs and how the virus coopts the host RNA polymerase for its replication.