Bluetongue virus VP1 polymerase activity in vitro: template dependency, dinucleotide priming and cap dependency.
Bluetongue virus VP1 polymerase activity in vitro: template dependency, dinucleotide priming and cap dependency.
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DOI：
10.1371/journal.pone.0027702
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Roy P
中科院分区：
文献类型：
--
作者：
Matsuo E;Roy P
众所周知，蓝舌病病毒（BTV）蛋白VP1具有内在的聚合酶功能，而轮状病毒VP1则需要衣壳蛋白VP2才能发挥催化活性。然而，与呼肠孤病毒科其他成员的聚合酶相比，BTV VP1尚未被详细表征。利用体外聚合酶测定系统，我们证实BTV VP1可以在一次体外反应中同时从BTV核心衍生的ssRNA模板合成10个dsRNA，也可以从轮状病毒核心衍生的ssRNA模板合成与BTV没有序列相似性的基因组dsRNA片段。相反，VP1不能从非病毒ssRNA模板合成dsRNAs，除非它们与特定的BTV序列融合。此外，我们发现从带帽的ssRNA模板中合成的dsRNAs明显高于从未带帽的ssRNA模板中合成的dsRNAs，并且添加二核苷酸可以增强活性，只要二核苷酸的最后一个碱基与ssRNA模板的3 '端核苷酸互补。我们发现，聚合酶的活性受到两种不同因素的刺激：帽结构（可能是由于变构效应）和二核苷酸（由于引物）。我们的结果还表明呼肠孤病毒科成员中可能存在ssrna共有的顺式作用元件。
Bluetongue virus (BTV) protein, VP1, is known to possess an intrinsic polymerase function, unlike rotavirus VP1, which requires the capsid protein VP2 for its catalytic activity. However, compared with the polymerases of other members of the Reoviridae family, BTV VP1 has not been characterized in detail. Using an in vitro polymerase assay system, we demonstrated that BTV VP1 could synthesize the ten dsRNAs simultaneously from BTV core-derived ssRNA templates in a single in vitro reaction as well as genomic dsRNA segments from rotavirus core-derived ssRNA templates that possess no sequence similarity with BTV. In contrast, dsRNAs were not synthesized from non-viral ssRNA templates by VP1, unless they were fused with specific BTV sequences. Further, we showed that synthesis of dsRNAs from capped ssRNA templates was significantly higher than that from uncapped ssRNA templates and the addition of dinucleotides enhanced activity as long as the last base of the dinucleotide complemented the 3′ -terminal nucleotide of the ssRNA template. We showed that the polymerase activity was stimulated by two different factors: cap structure, likely due to allosteric effect, and dinucleotides due to priming. Our results also suggested the possible presence of cis-acting elements shared by ssRNAs in the members of family Reoviridae.
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