Structure and Replication of Hepatitis Delta Virus

HDV是一种人类病原体，通常与更具破坏性的肝脏感染有关。这些研究的长期目标是了解HDV复制的新机制，重点关注在其他动物病毒复制过程中迄今未知的特征。提出了四个目标：(I)启动HDV RNA指导的RNA合成：将使用标记的复制能力基因组进行竞争分析，以衡量RNA构象(环状与线形)、RNA长度(单体与多聚体)和RNA极性(基因组与反基因组)在体内启动HDV RNA转录的重要性。它还将被用于测试RNA亚稳态在转录启动中的作用，而不是预测的未分枝的杆状折叠。另一种方法是测试非复制型RNA诱饵的表达对报告基因组复制的影响。(Ii)Polv(A)对RNA指导的反基因组RNA转录本的添加：实验将检验这一处理RNA指导的转录本的独特例子的机制，并询问是否需要这种处理来转录大于单位长度的反基因组RNA。一 策略是突变HDV RNA上的顺式作用信号，另一个策略是共表达流感NS1A蛋白，并利用其干扰聚(A)-加工的能力。(Iii)HDV RNA和蛋白质与宿主成分之间的相互作用：进行HDV复制的细胞将用甲醛固定，作为一种检测Delta蛋白与HDV和宿主RNA相互作用的方法。人类cDNA阵列将被用来识别与Delta蛋白相互作用的宿主RNA。此外，还将使用交联法检测Delta蛋白和RNA与宿主蛋白的相互作用。这些宿主蛋白将通过二维凝胶电泳和质谱学相结合的方法进行鉴定。(4)转录后基因沉默作用(S)。将寻找转录后基因沉默的指标。 (PTGS)在HDV复制过程中。此外，将改变HDV RNA结构，以确定这是否可以诱导PTGS，最后，将应用特定的外源小干扰RNA来确定HDV复制对这种抑制的可及性程度。综上所述，提出了四个具体目标的研究，这些研究将提供关于HDV生命周期的独特方面的信息，并且由于HDV如此依赖宿主功能，因此关于该宿主机制如何通过与HDV的RNA和蛋白质的相互作用而被重定向的信息。

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.