Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme.
Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme.
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DOI：
10.1016/j.fob.2015.02.001
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发表时间：
2015
期刊：
影响因子：
2.6
通讯作者：
Roy, Polly
中科院分区：
文献类型：
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作者：
Stewart, Meredith E.;Roy, Polly
VP 4加帽酶2′-O MTase活性与上游功能无关。催化四分体K-D-K-E内的单个突变消除了2 '-O MTase活性。2′-O MTase活性对病毒复制至关重要。cap 0结合口袋的突变影响2′-O MTase活性和病毒复制。蓝舌病毒（BTV）编码一个加帽蛋白VP 4，它催化在新生病毒转录本上产生cap 1结构所需的所有反应。此外，通过X射线晶体学的结构分析表明，每个催化反应被安排为一个离散的结构域，包括核苷-2 ′-O-甲基转移酶（2′-O MTase）。在这项研究中，我们已经开发的结构信息，以确定的残基是重要的2′-O MTase的VP 4的催化活性和它们对BTV复制的影响。这些突变对GMP结合，鸟苷酰转移酶（GT酶）和甲基化酶活性的影响进行了分析，通过一系列的体外生化试验，使用重组突变蛋白，随后通过引入相同的突变，复制病毒基因组中使用反向遗传学系统的病毒复制的影响进行了评估。我们的数据表明，催化四联体K-D-K-E中的单取代突变足以在体外消除2′-O MTase活性并完全消除BTV在细胞中的复制;尽管这些突变体保留了上游GMP结合、GTase和鸟嘌呤-N7-甲基转移酶活性。周围底物结合口袋的突变（预测招募cap 0）对体外VP 4加帽活性有不同的影响。基因组中这些残基的三重取代突变而非单重取代突变导致病毒复制动力学降低。这是第一份研究2′-O MTase功能对呼肠孤病毒科任何成员的重要性的报告，并强调了K-D-K-E四分体及其周围残基对2′-O MTase活性效率的重要性，进而对病毒适应性的重要性。
The VP4 capping enzyme 2′-O MTase activity is independent of upstream functions. A single mutation within the catalytic tetrad K-D-K-E abolishes 2’-O MTase activity. 2′-O MTase activity is critical for virus replication. Mutations at cap0 binding pocket affect 2′-O MTase activity and virus replication. Bluetongue virus (BTV) encodes a single capping protein, VP4, which catalyzes all reactions required to generate cap1 structures on nascent viral transcripts. Further, structural analysis by X-ray crystallography indicated each catalytic reaction is arranged as a discrete domain, including a nucleoside-2′-O-methyltransferase (2′-O MTase). In this study, we have exploited the structural information to identify the residues that are important for the catalytic activity of 2′-O MTase of VP4 and their influence on BTV replication. The effect of these mutations on GMP binding, guanylyltransferase (GTase) and methylase activities were analysed by a series of in vitro biochemical assays using recombinant mutant proteins; subsequently their effects on virus replication were assessed by introducing the same mutations in replicating viral genome using a reverse genetics system. Our data showed that single substitution mutations in the catalytic tetrad K-D-K-E were sufficient to abolish 2′-O MTase activity in vitro and to completely abrogate BTV replication in cells; although these mutants retained the upstream GMP binding, GTase and guanine-N7-methyltransferase activities. Mutations of the surrounding substrate-binding pocket (predicted to recruit cap0) had variable effects on in vitro VP4 capping activity. Only triple but not single substitution mutations of these residues in genome resulted in reduced virus replication kinetics. This is the first report investigating the importance of 2′-O MTase function for any member of the Reoviridae and highlights the significance of K-D-K-E tetrad and surrounding residues for the efficiency of 2′-O MTase activity and in turn, for virus fitness.
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影响因子：
5.4
作者：
Boyce, Mark;Celma, Cristina C. P.;Roy, Polly
通讯作者：
Roy, Polly
影响因子：
64.8
作者：
Grimes, JM;Burroughs, JN;Stuart, DI
通讯作者：
Stuart, DI
影响因子：
5.4
作者：
Li, Shi-Hua;Dong, Hongping;Shi, Pei-Yong
通讯作者：
Shi, Pei-Yong
DOI：
10.1073/pnas.95.23.13537
发表时间：
1998-11-10
影响因子：
11.1
作者：
Ramadevi, N;Burroughs, NJ;Roy, P
通讯作者：
Roy, P
病毒mRNA帽的2'-O甲基化通过IFIT家族成员逃避了宿主的限制。
影响因子：
64.8
作者：
通讯作者：
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