Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP: identification of the viral RNA polymerase.
Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP: identification of the viral RNA polymerase.
复制标题
用 8-叠氮基-ATP 进行轮状病毒 VP1 的光亲和标记：病毒 RNA 聚合酶的鉴定。
DOI：
10.1128/jvi.65.7.3964-3967.1991
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发表时间：
1991
影响因子：
5.4
通讯作者：
Spencer,E
中科院分区：
文献类型：
--
作者：
Valenzuela,S;Pizarro,J;Sandino,AM;Vasquez,M;Fernandez,J;Hernandez,O;Patton,J;Spencer,E
轮状病毒单壳颗粒具有多种酶活性，在体内和体外都与合成被封顶的mRNAs有关。由于单壳颗粒必须结构完整才能进行转录，因此事实证明，很难识别这种颗粒中具有相关RNA聚合酶活性的蛋白质。一种表征单壳颗粒中单个蛋白质功能的方法是使用核苷酸类似物来专门标记那些与核苷酸有亲和力的蛋白质，如病毒RNA聚合酶。在这项研究中，8-叠氮基-ATP(叠氮-ATP)，一种光反应的核苷酸类似物，被用来鉴定病毒RNA聚合酶，基于类似物在紫外光下抑制与轮状病毒颗粒相关的转录活性的能力。当单壳颗粒在[α-32P]叠氮三磷酸腺苷存在下用紫外光处理时，由于核苷酸类似物的交联，结构蛋白VP1被放射性标记，颗粒合成mRNA的能力相应降低。在没有暴露在紫外光下的单壳颗粒的平行实验中，VP1没有被放射性标记，并且颗粒成功地使用叠氮-ATP作为底物来合成病毒mRNAs。综上所述，这些结果只与VP1是轮状病毒RNA依赖的RNA聚合酶的结论一致。
Rotavirus single-shelled particles have several enzymatic activities that are involved with the synthesis of capped mRNAs both in vivo and in vitro. Because single-shelled particles must be structurally intact to carry out transcription, it has proven to be difficult to identify the protein within such particles that possesses associated RNA polymerase activity. One approach for characterizing the function of the individual proteins within single-shelled particles is to use nucleotide analogs to specifically label those proteins, such as the viral RNA polymerase, that have affinity for nucleotides. In this study, 8-azido-ATP (azido-ATP), a photoreactable nucleotide analog, was used to identify the viral RNA polymerase on the basis of the ability of the analog to inhibit transcription activity associated with rotavirus particles on exposure to UV light. When single-shelled particles were treated with UV light in the presence of [alpha-32P]azido-ATP, the structural protein VP1 became radiolabeled because of cross-linking of the nucleotide analog, and there was a corresponding decrease in the ability of the particles to synthesize mRNA. In parallel experiments in which single-shelled particles were not exposed to UV light, VP1 was not radiolabeled and the particles successfully used azido-ATP as a substrate for the synthesis of viral mRNAs. Taken together, these results are consistent only with the conclusion that VP1 is the rotavirus RNA-dependent RNA polymerase.