Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping
Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping
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DOI：
10.1128/jvi.74.19.8803-8811.2000
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发表时间：
2000-10-01
影响因子：
5.4
通讯作者：
Ahlquist, P
中科院分区：
文献类型：
--
作者：
Ahola, T;den Boon, JA;Ahlquist, P
雀麦花叶病毒（BMV）编码两种RNA复制蛋白：1a，含有RNA帽和解旋酶样结构域; 2a，与聚合酶相关。BMV 1a和2a可以在酿酒酵母中指导病毒特异性RNA复制，其再现了植物细胞中BMV复制的已知特征。我们在预测的1a的加帽和解旋酶活性位点处构建了单个氨基酸点突变，并分析了它们对BMV RNA 3在酵母中复制的影响。解旋酶突变体在所使用的任何测定中均未显示出功能：如通过la诱导的RNA 3稳定化所测量的，它们在RNA复制的模板募集方面存在强烈缺陷，并且它们合成了不可检测的负链或亚基因组RNA。加帽结构域突变体分为两组。第一个表现出增加的模板募集，但只允许低水平的负链和亚基因组mRNA的合成。第二个在模板募集方面有很大缺陷，产生非常低水平的负链，并且没有可检测的亚基因组。为了区分RNA合成和加帽缺陷，我们删除了染色体基因XRN 1，该基因编码降解未加帽mRNA的主要外切核酸酶，XRN 1缺失抑制了第二组但不是第一组加帽突变体，允许大量未加帽的亚基因组mRNA的合成和积累，从而为病毒RNA加帽功能的重要性提供了直接证据。解旋酶和加帽酶突变体搁置没有互补，相反，在高水平的表达，解旋酶突变体显性干扰野生型蛋白质的功能。正链RNA病毒复制的不同步骤所需的相互关联的功能，这些结果进行了讨论。
Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast, The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by la-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1, encoding the major exonuclease that degrades uncapped mRNAs, XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants shelved no complementation, Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.