Baumstark T, Ahlquist P
Institute for Molecular Virology and Howard Hughes Medical Institute, University of Wisconsin-Madison, 53706, USA.
RNA. 2001 Nov;7(11):1652-70.
The genome of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, consists of three capped, messenger-sense RNAs. RNA1 and RNA2 encode viral replication proteins 1a and 2a, respectively. RNA3 encodes the 3a movement protein and the coat protein, which are essential for systemic infection in plants but dispensable for RNA3 replication in plants and yeast. A subset of the 250-base intergenic region (IGR), the replication enhancer (RE), contains all cis-acting signals necessary for a crucial, early template selection step, the 1a-dependent recruitment of RNA3 into replication. One of these signals is a motif matching the conserved box B sequence of RNA polymerase III transcripts. Using chemical modification with CMCT, kethoxal, DMS, DEPC, and lead, we probed the structure of the IGR in short, defined transcripts and in full-length RNA3 in vitro, in yeast extracts, and in whole yeast cells. Our results reveal a stable, unbranched secondary structure that is not dependent on the surrounding ORF sequences or on host factors within the cell. Functional 5' and 3' deletions that defined the minimal RE in earlier deletion studies map to the end of a common helical segment. The box B motif is presented as a hairpin loop of 7 nt closed by G:C base pairs in perfect analogy to the TpsiC-stem loop in tRNA(Asp). An adjacent U-rich internal loop, a short helix, and another pyrimidine-rich loop were significantly protected from base modifications. This same arrangement is conserved between BMV and cucumoviruses CMV, TAV, and PSV. In the BMV box B loop sequence, uridines corresponding to tRNA positions T54 and psi55 were found to be modified in yeast and plants to 5mU and pseudouridine. Together with the aminoacylated viral 3'-end, this is thus the second RNA replication signal within BMV where the virus has evolved a tRNA structural mimicry to a degree that renders it a substrate for classical tRNA modification reactions in vivo.
雀麦花叶病毒（BMV）是甲病毒样超家族中的一种正链RNA病毒，其基因组由三条加帽的信使意义RNA组成。RNA1和RNA2分别编码病毒复制蛋白1a和2a。RNA3编码3a运动蛋白和外壳蛋白，它们对植物中的系统感染至关重要，但对植物和酵母中RNA3的复制并非必需。250个碱基的基因间隔区（IGR）的一个子集，即复制增强子（RE），包含了关键的早期模板选择步骤所需的所有顺式作用信号，即1a依赖的将RNA3招募到复制过程中。其中一个信号是与RNA聚合酶III转录本保守的盒B序列匹配的基序。我们使用CMCT、乙二醛、DMS、DEPC和铅进行化学修饰，在体外、酵母提取物和完整酵母细胞中探测了短的、特定转录本以及全长RNA3中IGR的结构。我们的结果揭示了一种稳定的、无分支的二级结构，该结构不依赖于周围的开放阅读框序列或细胞内的宿主因子。在早期缺失研究中确定最小RE的功能性5'和3'缺失定位到一个共同螺旋段的末端。盒B基序呈现为一个由G:C碱基对封闭的7个核苷酸的发夹环，与tRNA（Asp）中的TpsiC茎环完全类似。一个相邻的富含尿苷的内环、一个短螺旋和另一个富含嘧啶的环对碱基修饰有显著的保护作用。这种相同的排列在BMV和黄瓜花叶病毒CMV、TAV和PSV之间是保守的。在BMV盒B环序列中，发现对应于tRNA位置T54和psi55的尿苷在酵母和植物中被修饰为5 - 甲基尿苷和假尿苷。因此，连同氨基酰化的病毒3'末端，这是BMV内的第二个RNA复制信号，在该信号中病毒已经进化出一种tRNA结构模拟，其程度使其成为体内经典tRNA修饰反应的底物。
