trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication
trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication
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DOI：
10.1128/jvi.71.12.9608-9617.1997
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发表时间：
1997-12-01
影响因子：
5.4
通讯作者：
Rice, CM
中科院分区：
文献类型：
--
作者：
Lindenbach, BD;Rice, CM
对黄热病病毒 (YF) 非结构蛋白 1 (NS1) 的突变分析表明它与病毒 RNA 复制有关。为了进一步探索这一观察结果，我们寻求一种将 NS1 功能与 NS1 表达和加工作为大 YF 多蛋白一部分解偶联的方法。在这里，我们描述了一种利用非细胞病变的辛德比斯病毒载体反式提供 NS1 的策略。含有大量 NS1 框内缺失的缺陷 YF 基因组的复制依赖于 NS1 的功能表达。回收的突变病毒显示含有缺失并被 YF 特异性抗血清中和。补体突变病毒滴度增加，其动力学与亲本 YF 17D 相似，但滴度达到峰值。反式互补使我们能够获得 NS1 缺陷型 YF 的高滴度、无辅助细胞库存，从而进一步表征该基因产物在 RNA 复制中的作用。通过使用敏感的链特异性 RNase 保护测定来分析 RNA 复制的第一个周期。我们记录了突变型和野生型病毒在存在或不存在互补的情况下的这些事件。这些数据强烈表明 NS1 在负链合成之前或初始负链合成时发挥作用。
Mutational analysis of the nonstructural protein 1 (NS1) of yellow fever virus (YF) has implicated it in viral RNA replication. To further explore this observation, we sought a method for uncoupling NS1 function from NS1 expression and processing as part of the large YF polyprotein. Here we describe a strategy for providing NS1 in trans, utilizing a noncytopathic Sindbis virus vector. Replication of a defective YF genome containing a large in-frame deletion of NS1 was dependent on functional expression of NS1. Recovered mutant virus was shown to contain the deletion and was neutralized by YF-specific antiserum. Complemented mutant virus increased in titer with kinetics similar to those of parental YF 17D but peaked at lower titers. trans complementation has allowed us to derive high-titer, helper-free stocks of YF defective in NS1 with which to further characterize the role of this gene product in RNA replication. The first cycles of RNA replication were analyzed by using a sensitive strand-specific RNase protection assay. We document these events for mutant and wild-type viruses in the presence or absence of complementation. These data strongly suggest a role for NS1 prior to or at initial minus-strand synthesis.