COUPLED TRANSLATION AND REPLICATION OF POLIOVIRUS RNA INVITRO - SYNTHESIS OF FUNCTIONAL 3D POLYMERASE AND INFECTIOUS VIRUS
COUPLED TRANSLATION AND REPLICATION OF POLIOVIRUS RNA INVITRO - SYNTHESIS OF FUNCTIONAL 3D POLYMERASE AND INFECTIOUS VIRUS
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DOI：
10.1128/jvi.67.2.822-831.1993
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发表时间：
1993-02-01
影响因子：
5.4
通讯作者：
FLANEGAN, JB
中科院分区：
文献类型：
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作者：
BARTON, DJ;FLANEGAN, JB
脊髓灰质炎病毒 RNA 聚合酶和感染性病毒颗粒是通过 HeLa S10 提取物中的病毒体 RNA 体外翻译来合成的。体外翻译反应针对受感染细胞中发现的病毒蛋白的合成，特别是病毒聚合酶 3d(pol) 的合成进行了优化。在反应的前 6 小时内，合成的标记蛋白的量呈线性增加。由于前体蛋白的蛋白水解加工需要额外的时间，翻译产物中 3D(pol) 的出现被延迟。 1 小时时首次在聚丙烯酰胺凝胶中观察到 3D(pol)，并在 6 小时及之后检测到大量的 3D(pol)。最初尝试直接在翻译反应中测定聚合酶活性并未成功。然而，通过将少量（3μl）翻译产物添加到含有脊髓灰质炎病毒RNA的标准聚合酶测定中，可以轻松检测聚合酶活性。在oligo(U) 引物存在下合成全长负链RNA。在没有oligo(U)的情况下，这些反应中合成的产物RNA大约是病毒颗粒RNA大小的两倍。 RNA 稳定性研究和噬菌斑测定表明，翻译反应中输入的病毒体 RNA 的很大一部分非常稳定，可以保持完整 20 小时或更长时间。空斑测定表明感染性病毒是在体外翻译反应中合成的。在最佳条件下，体外翻译反应产生的感染性病毒滴度大于100,000 PFU/ml。病毒在 6 小时时首次检测到，并在 12 小时时增加到最高水平。总体而言，在 HeLa S10 起始因子体外翻译反应中观察到的脊髓灰质炎病毒复制动力学（蛋白质合成、聚合酶活性和病毒产生）与在感染细胞中观察到的相似。
Poliovirus RNA polymerase and infectious virus particles were synthesized by translation of virion RNA in vitro in HeLa S10 extracts. The in vitro translation reactions were optimized for the synthesis of the viral proteins found in infected cells and in particular the synthesis of the viral polymerase 3d(pol). There was a linear increase in the amount of labeled protein synthesized during the first 6 h of the reaction. The appearance of 3D(pol) in the translation products was delayed because of the additional time required for the proteolytic processing of precursor proteins. 3D(pol) was first observed at 1 h in polyacrylamide gels, with significant amounts being detected at 6 b and later. Initial attempts to assay for polymerase activity directly in the translation reaction were not successful. Polymerase activity, however, was easily detected by adding a small amount (3 mul) of translation products to a standard polymerase assay containing poliovirion RNA. Full-length minus-strand RNA was synthesized in the presence of an oligo(U) primer. In the absence of oligo(U), product RNA about twice the size of virion RNA was synthesized in these reactions. RNA stability studies and plaque assays indicated that a significant fraction of the input virion RNA in the translation reactions was very stable and remained intact for 20 h or more. Plaque assays indicated that infectious virus was synthesized in the in vitro translation reactions. Under optimal conditions, the titer of infectious virus produced in the in vitro translation reactions was greater than 100,000 PFU/ml. Virus was first detected at 6 h and increased to maximum levels by 12 b. Overall, the kinetics of poliovirus replication (protein synthesis, polymerase activity, and virus production) observed in the HeLa S10-initiation factor in vitro translation reactions were similar to those observed in infected cells.