IN VITRO TRANSLATION AS A PROBE FOR THE MECHANISM OF POLIOVIRUS ATTENUATION

脊髓灰质炎病毒神经毒力的减弱是由于 脊髓灰质炎病毒基因组中核苷酸变化的数量。 一个主要的网站， 3型脊髓灰质炎病毒（472位）的减毒发生在非编码 基因组中与病毒调节有关的区域 翻译. 翻译中的变化是如何（以及是否）导致 脊髓灰质炎病毒衰减仍有待确定。 莫拉保罗和维默 Science，254，1647-1651（1991）报道了体外翻译 来源于未感染的HeLa细胞的系统翻译添加的脊髓灰质炎病毒RNA 以接近地模拟人类中的完整病毒复制周期的方式 细胞 最近，巴顿和Flanegan，J. of Virology，67，822- 831， （1993），提供了关于该系统的更多信息。 我们 用这个系统来确定基因组变化是否会减少 神经毒力对病毒粒子的体外翻译有影响 核糖核酸 HeLa细胞提取物对添加野生型 正在对脊髓灰质炎病毒RNA进行评估，并将用于比较 对来自减毒Sabin株的RNA的应答。 的目标 该项目旨在获得有关以下方面的进一步信息： 导致脊髓灰质炎病毒减毒的核苷酸变化 基因组 了解这些序列与病毒的相互作用， 宿主因素可能导致减毒基因组的进一步变化， 将导致减毒表型的稳定性增加。 法国 研究人员最近发现了一种以前未被发现的， 病毒RNA中核苷酸472周围区域的重要作用 合成. 这一观察提供了几种可能的解释， 我们的实验结果表明抑制脊髓灰质炎病毒RNA 蛋白质合成抑制剂抑制核糖体合成 当使用蛋白质合成抑制剂时， 允许核糖体活性，同时阻止功能性 proteins. 我们相信我们的结果证明了一种病毒的存在 反馈调节机制将脊髓灰质炎病毒RNA合成与 核糖体活性 再加上法国的成绩，表明一个双重的 作用的调节区周围的核苷酸472，这些结果 表明脊髓灰质炎病毒神经毒力的减弱可能是由于 影响病毒反馈调节机制的核苷酸变化。

Attenuation of poliovirus neurovirulence is the result of a limited number of nucleotide changes in the poliovirus genome. A major site for attenuation of Type 3 poliovirus (position 472) occurs in a noncoding region of the genome that is involved in the regulation of viral translation. Exactly how (and if) changes in translation result in poliovirus attenuation remain to be established. Mola, Paul, and Wimmer, Science, 254, 1647-1651(1991) have reported that an in vitro translation system derived from uninfected HeLa cells translated added poliovirus RNA in a manner that closely mimics the full viral replicative cycle in human cells. More recently, Barton and Flanegan, J. of Virology, 67, 822- 831, (1993), have provided additional information about this system. We are using this system to determine whether the genomic changes that decrease neurovirulence exhibit an effect on the in vitro translation of virion RNA. The response of HeLa cell extracts to the addition of wild-type poliovirus RNA are being evaluated and will be used to compare the response to RNA derived from the attenuated Sabin strain. The goal of this project is to obtain further information concerning the function of the nucleotide changes that result in attenuation of the poliovirus genome. Understanding the interaction of these sequences with viral and host factors could lead to further changes in the attenuated genome that would result in increased stability of the attenuated phenotype. French researchers have recently demonstrated a previously undetected and essential role of the region surrounding nucleotide 472 in viral RNA synthesis. This observation provides several possible explanations for our experimental results indicating that inhibition of poliovirus RNA synthesis seen with protein synthesis inhibitors that inhibit ribosomal activity is not seen when protein synthesis inhibitors are used that allow ribosomal activity while preventing the synthesis of functional proteins. We believe our results demonstrate the existence of a viral feedback regulatory mechanism linking poliovirus RNA synthesis to ribosomal activity. Coupled with the French results, indicating a dual role for the regulatory region surrounding nucleotide 472, these results suggest that attenuation of poliovirus neurovirulence may result from nucleotide changes that affect the viral feedback regulatory mechanism.