Structure and Replication of Hepatitis Delta Virus

HDV是一种人类病原体，通常与肝脏的更具破坏性的感染有关。这些研究的长期目标是了解HDV复制的新机制，重点是迄今为止在其他动物病毒复制过程中不知道发生的特征。提出了四个目标：（i）HDV RNA指导的RNA合成的起始：将应用使用标记的复制能力基因组的竞争测定来测量RNA构象（圆形与线形）、RNA长度（单体与多聚体）和RNA极性（基因组与反基因组）对于HDV RNA转录的体内起始的重要性。它也将被应用于测试的作用，RNA亚稳态，而不是预测的无分支棒状折叠，在转录的起始。另一种方法是测试非复制RNA诱饵的表达对报告基因组复制的影响。(ii)Polv（A）添加到RNA指导的反基因组RNA转录物中：实验将检查RNA指导的转录物的这种独特加工实例的机制，并且还询问这种加工对于大于单位长度的反基因组RNA的转录是否是必需的。一 另一种策略是诱变HDV RNA上的顺式作用信号，另一种策略是共表达流感NS 1A蛋白并应用其干扰poly（A）加工的能力。(iii)HDV RNA和蛋白质与宿主组分之间的相互作用：经历HDV复制的细胞将用甲醛固定，作为检测δ蛋白与HDV和宿主RNA相互作用的方法。人类cDNA阵列将用于鉴定与δ蛋白相互作用的宿主RNA。此外，交联将用于检测δ蛋白和RNA与宿主蛋白的相互作用。这些宿主蛋白将通过2-D凝胶电泳和质谱的组合来鉴定。(iv)转录后基因沉默的作用。将搜索转录后基因沉默的指标 HDV复制期间的PTGS。此外，HDV RNA结构将被改变以确定这是否可以诱导PTGS，最后，特异性外源性小干扰RNA将被应用以确定HDV复制对这种抑制的可及性程度。总之，提出了四个具体目标的研究，将提供HDV生命周期的独特方面的信息，因为HDV是如此依赖于宿主功能，信息如何通过与HDV的RNA和蛋白质的相互作用，这种主机机器被重定向。

HDV is a human pathogen typically associated with more damaging infections of the liver. The long-range goal of these studies is to understand the novel mechanism of HDV replication, focussing on features so far not known to occur during the replication of other animal viruses. Four aims are proposed: (i) Initiation of HDV RNA-directed RNA synthesis: A competition assay using marked replication-competent genomes will be applied to measure the importance of RNA conformation (circles vs. linears), RNA length (monomers vs. multimers) and RNA polarity (genomic vs. antigenomic) for in vivo initiation of HDV RNA transcription. It will also be applied to test the role of RNA metastable states, other than the predicted unbranched rod-like folding, in the initiation of transcription. An alternative approach will be to test the effect of expression of non-replicating RNA decoys on the replication of a reporter genome. (ii) Polv(A) addition to RNA-directed anti.qenomic RNA transcripts: Experiments will examine the mechanism of this unique example of processing of transcripts that are RNA-directed, and also ask if this processing is needed for transcription of greater than unit-length antigenomic RNAs. One strategy will be to mutagenize cis-acting signals on the HDV RNA, and another will be to co-express the influenza NS1A protein and apply its ability to interfere with poly(A)-processing. (iii) Interactions between HDV RNAs and proteins with host components: Cells undergoing HDV replication will be fixed with formaldehyde as an approach to detect interactions of delta protein with HDV and host RNAs. Human cDNA arrays will be used to identify the host RNAs that interact with the delta protein. Also, cross-linking will be used to detect interaction of delta protein and RNA with host proteins. These host proteins will be identified by a combination of 2-D gel electrophoresis and mass spectrometry. (iv) Role(s) of post-transcriptional gene silencing. A search will be made for indicators of post-transcriptional gene silencing (PTGS) during HDV replication. Also, HDV RNA structures will be altered to determine if this can induce PTGS and finally, specific exogenous small interfering RNAs will be applied to determine the extent of accessibility of HDV replication to such inhibition. In summary, studies are proposed in four specific aims that will provide information on unique aspects of the HDV life cycle and, because HDV is so dependent on host functions, information on how this host machinery is redirected via interactions with the RNAs and proteins of HDV.