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，重点关注迄今为止尚不知道在其他动物病毒复制过程中发生的特征。提出了四个目标：(i)启动HDV RNA定向RNA合成：使用标记复制能力基因组的竞争分析将用于测量RNA构象（圆形与线性），RNA长度（单体与多聚体）和RNA极性（基因组与抗基因组）在体内启动HDV RNA转录的重要性。它还将被应用于测试RNA亚稳态的作用，而不是预测的未分枝的棒状折叠，在转录的起始。另一种方法是测试非复制性RNA诱饵的表达对报告基因组复制的影响。（ii） Polv(A)加入rna定向抗。基因组RNA转录本：实验将检验这种独特的转录本加工的机制，这些转录本是RNA导向的，并且还将询问这种加工是否需要转录大于单位长度的抗基因组RNA。一个

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.