Interactions Among Sequences, Structures and Proteins Involved in Viral Replication

9630191西蒙病毒编码的RNA依赖性RNA聚合酶（RdRp）负责两阶段复制过程：RdRp合成互补的负链，负链反过来成为正链合成的模板。许多不同的结构元件涉及复制，包括假结，茎环和tRNA样结构。较少了解的病毒，其基因组RNA末端为3 '-末端羟基，如芜菁皱缩病毒（TCV），将被用作研究复制中涉及的序列和结构的模型。TCV是最小和最简单的植物RNA病毒之一，并与亚病毒卫星RNA和DI RNA相关，使这些简单的非蛋白质编码RNA作为复制研究的模板成为可能。已经开发了原生质体和体外系统来研究基因组和亚病毒RNA的复制，并且已经使用杆状病毒载体在昆虫细胞中表达病毒编码的RdRp亚基。在正链satC（356个碱基）的3'端的29个碱基序列（包括在所有TCV相关RNA的末端发现的稳定发夹和CCUGCCC基序）是体外负链合成所需的并且足够。 诱变研究表明，茎的稳定性，而不是茎或环的序列，对启动子功能很重要。发现负链satC具有两个独立的启动子，一个在3'端49个核苷酸内，第二个在310至334位附近。 将进行以下实验：（1）将使用进一步诱变和体外选择策略（SELEX）确定satC的正链和负链启动子的序列/结构要求，其中如果随机多核苷酸序列包含功能性启动子，则“选择”大量随机多核苷酸序列用于进一步扩增。(2)将使用缺失和定点诱变来确定涉及satD的体内复制和体外转录的顺式信号。(3)将检查CCUGCCC 3 '末端基序相对于satC和TCV的3'发夹的序列和位置的重要性。此外，我们将确定TCV和satC的3'末端的缺失是否也导致体内“新”RNA的形成，其中CCUGCCC末端基序被替换，如在satD中发现的那样。(4)将使用凝胶阻滞分析、过滤结合和羟基自由基足迹法，使用TCV编码的RdRp亚基p28和p88与satC和satD相关的启动子序列进行结合研究。(5)将使用野生型TCV和具有外壳蛋白开放阅读框缺失的TCV（TCV（CP））来确定外壳蛋白对原生质体中TCV亚病毒RNA正链和负链复制的影响。此外，我们还将利用接种TCV（CP）的原生质体的RdRp活性提取物，测定不同量的高度纯化的TCV（CP）对satC和satD正链和负链体外转录的影响。 植物RNA病毒通过复制其基因来复制自身的过程非常复杂。 芜菁皱缩病毒RNA的复制可以在试管中进行。 这些研究将集中在RNA的特定序列与产生新病毒RNA拷贝的酶活性相互作用的贡献上。 这些研究可能会导致理解如何操纵和控制RNA病毒在植物和动物系统中的活动。 ***

9630191 Simon Virus-encoded RNA-dependent RNA polymerases (RdRp) are responsible for a two-stage replication process: the RdRp synthesizes complementary minus-strands that in turn become templates for plus-strand synthesis. A number of different structural elements have been implicated in replication including pseudoknots, stem-loops and tRNA-like structures. Less understood viruses whose genomic RNA(s) end in a 3'-terminal hydroxyl group such as turnip crinkle virus (TCV) will be utilized as a model to study sequences and structures involved in replication. TCV is among the smallest and simplest of the plant RNA viruses and is associated with subviral sat-RNAs and DI RNAs, making possible the use of these simple, non-protein-coding RNAs as templates for replication studies. Protoplast and in vitro systems have been developed to study replication of genomic and subviral RNAs and virus-encoded RdRp subunits have been expressed in insect cells using baculovirus vectors. A 29 base sequence at the 3' end of plus-strand satC (356 bases), which includes a stable hairpin and the CCUGCCC motif found at the ends of all TCV-associated RNAs, is required and sufficient for minus-strand synthesis in vitro. Mutagenesis studies indicated that the stability of the stem, and not the sequence of the stem or loop, is important for promoter function. Minus-strand satC was found to have two independent promoters, one within the 3' terminal 49 nucleotides and a second near positions 310 to 334. The following experiments will be performed: (1) The sequence/structural requirements for plus-strand and minus-strand promoters of satC will be determined using further mutagenesis and an in vitro selection strategy (SELEX), where a large pool of random polynucleotide sequences are "selected" for further amplification if they comprise a functional promoter. (2) Cis-signals involved in replication in vivo and transcription in vitro for satD will be determined using deletions and site-directed mutagenesis. (3) The importa nce of the sequence and location of the CCUGCCC 3'-terminal motif in relation to the 3' hairpin for satC and TCV will be examined. In addition, we will determine if deletion of the 3' end of TCV and satC also result in the formation of "new" RNAs in vivo with replacement of the CCUGCCC terminal motif as was found for satD. (4) Binding studies using the TCV-encoded RdRp subunits p28 and p88 to promoter sequences associated with satC and satD will be performed using gel retardation analysis, filter binding, and hydroxyl radical footprinting. (5) The effect of coat protein on replication of TCV subviral RNA plus and minus strands in protoplasts will be determined using wild-type TCV and TCV with a coat protein open reading frame deletion (TCV(CP). In addition, using RdRp-active extracts from protoplasts inoculated with TCV(CP, we will assay for the effect of different amounts of highly purified TCV(CP on in vitro transcription of satC and satD plus- and minus -strands. %%% The process by which a plant RNA virus can reproduce itself by copying its gene is very complex. Turnip crinkle virus RNA replication can be carried out in a test tube. These studies will focus on the contribution of specific sequences of the RNA interacting with the enzymatic activities that produce new viral RNA copies. These studies may lead to understanding how to manipulate and control RNA virus activities in plant and animal systems. ***