An attenuating mutation in nsP1 of the Sindbis-group virus SAAR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis
An attenuating mutation in nsP1 of the Sindbis-group virus SAAR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis
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DOI：
10.1128/jvi.77.2.1149-1156.2003
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发表时间：
2003-01-01
影响因子：
5.4
通讯作者：
Johnston, RE
中科院分区：
文献类型：
--
作者：
Heise, MT;White, LJ;Johnston, RE
辛德毕斯组甲病毒 S.A.AR86 在非结构蛋白 1 (nsP1) 538 处编码苏氨酸，该苏氨酸与成年小鼠的神经毒力相关。在非神经毒力 Sindbis 组 α 病毒中发现的 nsP1 538 Thr 向共有 Ile 的突变减弱了成年小鼠 S.A.AR86 的神经毒力，而在非神经毒力 Sindbis 病毒背景中在位置 538 处引入 Thr 则赋予神经毒力增加（M. T. Heise 等人，J. Virol. 74:4207-4213， 2000）。由于病毒非结构区域的变化可能会影响病毒复制，因此进行了研究以评估 nsP1 538 处的 Thr 或 Ile 对病毒生长、非结构蛋白加工和 RNA 合成的影响。 Neuro2A 和 BHK-21 细胞中的多步生长曲线表明，减毒 s51 (nsP1 538 Ile) 病毒比野生型 s55 (nsP1 538 Thr) 病毒具有轻微但可重复的生长优势。 nsP1 538 位于 nsP1 和 nsP2 之间的切割识别域内，并且 nsP1 538 处减毒 Ile 的存在加速了 S.A.AR86 非结构蛋白在体外和感染细胞中的加工。由于已知非结构蛋白加工可调节甲病毒 RNA 合成，因此进行了实验以评估 nsP1 538 处的 Ile 或 Thr 对病毒 RNA 合成的影响。 S.A.AR86 衍生的报告基因检测和 RNase 保护检测的组合确定，nsP1 538 处 Ile 的存在导致病毒 26S 启动子更早表达，而不影响病毒负链或正链合成。这些结果表明，野生型 S.A.AR86 感染中非结构蛋白加工速度减慢和 26S RNA 合成延迟可能导致 S.A.AR86 成年小鼠神经毒力表型。
The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.