Structure and Function of Hepatitis Delta Virus RNA-Protein Complexes

 描述（由申请方提供）：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。对于这种病毒没有有效的许可疗法，它具有尚未完全了解的独特复制周期。HDV是一种RNA病毒，其编码单一蛋白HDAg，其作为核蛋白发挥功能;它与病毒（-）和（+）RNA形成核糖核蛋白复合物。这些复合物在宿主RNA聚合酶复制病毒RNA中发挥重要作用，但由于难以在实验室中组装它们，因此尚未对其进行充分表征。最近的发展表明，现在可以通过使用来自HDV基因型3株的HDAg在体外产生离散的RNP复合物。该提案旨在利用这一最新发展，并更充分地表征HDV RNP。有两个目标，每个目标都有次级目标。使用显微注射技术，目标1的第一个子目标将确定有多少HDAg多聚体必须组装在RNA上以启动合成-首先是整个RNA复制周期，然后是独立的单个RNA。目标1的第三部分将使用从前两个子目标收集的信息来合理地设计一种尝试，通过Pol II在体外观察HDV RNA的从头转录。目标2将确定RNP结构的各个方面，特别关注目标1中显示的对RNA复制有功能的RNP。其中一个子目标将采用原子力显微镜来单独观察RNP复合物，以观察蛋白质如何沿着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RNA的哪些特定序列与来自细胞、病毒体和体外组装的RNP中的HDAg密切相关。总之，该提案将大大扩展我们对HDV RNP结构-功能关系以及病毒如何选择宿主RNA聚合酶进行复制的理解。

 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.