Interactions Among Sequences, Structures and Proteins Involved in Viral Replication

9630191西蒙病毒编码的核糖核酸依赖的核糖核酸聚合酶(RdRp)负责两个阶段的复制过程：RdRp合成互补的负链，然后成为正链合成的模板。许多不同的结构元件参与了复制，包括伪结、茎环和tRNA样结构。基因组核糖核酸(S)末端为3‘端羟基的病毒，如萝卜皱缩病毒(TCV)，将被用作研究复制过程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，与亚病毒SAT-RNAs和DI RNAs有关，使得使用这些简单的、非蛋白质编码的RNAs作为复制研究的模板成为可能。已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，并利用杆状病毒载体在昆虫细胞中表达了病毒编码的RdRp亚单位。正链SATC 3‘端的29个碱基序列(356个碱基)，包括一个稳定的发夹和在所有TCV相关RNA末端发现的CCUGCCC基序，是体外合成负链所必需的，也是足够的。诱变研究表明，对启动子功能重要的是茎的稳定性，而不是茎或环的序列。负链SATC被发现有两个独立的启动子，一个在3‘端49个核苷酸内，另一个在310到334位附近。将进行以下实验：(1)将使用进一步的突变和体外选择策略(SELEX)来确定SATC的正链和负链启动子的序列/结构要求，其中，如果包含功能启动子，则选择大量随机多核苷酸序列用于进一步扩增。(2)sATD的体内复制和体外转录所涉及的顺式信号将通过缺失和定点突变来确定。(3)CCUGCCC 3‘-末端基序的序列和位置与SATC和TCV 3’发夹的关系将被研究。此外，我们还将确定TCV和SATC的3‘端缺失是否也会导致体内“新”RNA的形成，取代CCUGCCC末端基序，就像对SatD所发现的那样。(4)使用TCV编码的RdRp亚基P28和P88与SATC和SatD相关的启动子序列的结合研究将使用凝胶延迟分析、过滤器结合和羟基自由基足迹来进行。(5)利用野生型TCV和带有外壳蛋白开放阅读框缺失(TCV(CP))的TCV，研究了外壳蛋白对TCV亚病毒RNA正链和负链在原生质体中复制的影响。此外，使用RdRp活性提取物从接种TCV(CP)的原生质体中，我们将检测不同数量的高纯度TCV(CP)对SATC和SatD正负链体外转录的影响。植物RNA病毒通过复制其基因进行自我复制的过程非常复杂。萝卜皱缩病毒的RNA复制可以在试管中进行。这些研究将集中在特定的RNA序列与产生新的病毒RNA拷贝的酶活性相互作用的贡献。这些研究可能有助于理解如何在植物和动物系统中操纵和控制RNA病毒的活动。***

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. ***