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首页> 外文期刊>Journal of Virology >Influenza Virus Temperature-Sensitive Cap (m7GpppNm)-Dependent Endonuclease
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Influenza Virus Temperature-Sensitive Cap (m7GpppNm)-Dependent Endonuclease

机译：流感病毒温度敏感帽（M7GPPPNM） - 依赖性内切核酸酶

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The first step in influenza viral mRNA synthesis is the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m7GpppNm) structures to generate capped primers that are 10 to 13 nucleotides long, which are then elongated to form the viral mRNA chains. We examined the temperature sensitivity of these steps in vitro by using two WSN virus temperature-sensitive mutants, ts1 and ts6, which have a defect in the genome RNA segment coding for the viral PB2 protein. For these experiments, it was necessary to employ purified viral cores rather than detergent-treated virions to catalyze transcription, as preparations of detergent-treated virions contain destabilizing or inhibitory activities which render even the transcription catalyzed by wild-type virus temperature sensitive. Using purified wild-type viral cores, we found that the rates of endonucleolytic cleavage of capped primers and of overall transcription were similar at 39.5 and 33°C, the in vivo nonpermissive and permissive temperatures, respectively. In contrast, the activities of the cap-dependent endonucleases of ts1 and ts6 viral cores at 39.5°C were only about 15% of those at 33°C. The steps in transcription after endonucleolytic cleavage of the capped RNA primer were largely, if not totally, temperature insensitive, indicating that the mutations in the PB2 protein found in ts1 and ts6 virions affect only the endonuclease step. The temperature-sensitive defect is most likely in the recognition of the 5′-terminal cap 1 structure that occurs as a required first step in the endonuclease reaction: the cap-dependent binding of a specific capped primer fragment to ts1 viral cores was temperature sensitive under conditions in which binding to wild-type viral cores was not affected by increasing the temperature from 33 to 39.5°C. Thus, our results establish that the viral PB2 protein functions in cap recognition during the endonuclease reaction.
机译：流感病毒MRNA合成的第一步是含有帽1（m 7℃> gpppnm）结构的异源RNA的内核裂解，以产生10至13个核苷酸长的封端引物，然后伸长以形成病毒mRNA链。通过使用两个WSN病毒温度敏感突变体， TS 1和 Ts 6，在体外检查这些步骤的温度敏感性，这些步骤在基因组RNA段编码中具有缺陷用于病毒PB2蛋白。对于这些实验，需要使用纯化的病毒芯而不是洗涤剂处理的病毒粒以催化转录，因为洗涤剂处理的病毒粒子的制剂含有稳定或抑制活动，使得甚至通过野生型病毒温度敏感的转录甚至转录。使用纯化的野生型病毒芯，我们发现封端引物和总转录的内啡酰基裂解的速率分别在39.5和33℃，体内非智能温度下相似。相反，在39.5℃下在39.5℃下的 Ts 1和 Ts 6病毒芯的依赖性内切核酸酶的活性仅为33℃的约15％。在封端的RNA引物的内核核酸裂解后转录的步骤很大程度上是（如果不是完全，那么温度不敏感，表明在 TS 1和 TS 中发现的PB2蛋白中的突变6病毒群落仅影响内切核酸酶的步骤。温度敏感缺陷最有可能在核酸内切酶反应中所需的第一步骤中识别5'-端子帽1结构：特定封端的底漆片段与 Ts的盖子依赖性结合 1病毒核心是在与野生型病毒核的结合的条件下温度敏感，不受33至39.5℃的温度的影响。因此，我们的结果确定了在内切核酸酶反应期间的病毒PB2蛋白在帽识别中起作用。

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