Generation of West Nile Virus Infectious Clones Containing Amino Acid Insertions Between Capsid and Capsid Anchor
Generation of West Nile Virus Infectious Clones Containing Amino Acid Insertions Between Capsid and Capsid Anchor
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DOI：
10.3390/v6041637
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发表时间：
2014-04-01
期刊：
影响因子：
4.7
通讯作者：
Fredericksen, Brenda L.
中科院分区：
文献类型：
--
作者：
Vandergaast, Rianna;Hoover, Lisa I.;Fredericksen, Brenda L.
西尼罗河病毒（WNV）是一种正链RNA虫媒病毒，导致美国和欧洲最近爆发的严重神经系统疾病。由于缺乏足够的WN报告病毒，抑制病毒复制的抗病毒化合物的大规模分析受到限制。先前尝试将报告基因插入到WNV的3'非翻译区产生不稳定的病毒，表明该区域不容纳额外的核苷酸。在此，我们工程化了两个在病毒多蛋白的衣壳（C）/衣壳锚（CA）连接处含有插入的WNV感染性克隆。成功回收了在该位置含有达特（1-67）或Gaussia荧光素酶（GLuc）基因的重组病毒。然而，两种病毒的报告基因序列的大部分（如果不是全部的话）快速丢失，表明报告基因病毒不稳定。虽然GLuc病毒主要恢复到野生型WNV长度，但达特病毒保留了报告序列的多达75个额外核苷酸。这些额外的核苷酸在至少五代内是稳定的，并且没有显著改变WNV适应性。因此，西尼罗河病毒的C/CA连接可以容忍额外的核苷酸，虽然插入受到某些限制。
West Nile virus (WNV) is a positive-sense RNA arbovirus responsible for recent outbreaks of severe neurological disease within the US and Europe. Large-scale analyses of antiviral compounds that inhibit virus replication have been limited due to the lack of an adequate WN reporter virus. Previous attempts to insert a reporter into the 3' untranslated region of WNV generated unstable viruses, suggesting that this region does not accommodate additional nucleotides. Here, we engineered two WNV infectious clones containing insertions at the Capsid (C)/Capsid Anchor (CA) junction of the viral polyprotein. Recombinant viruses containing a TAT(1-67) or Gaussia Luciferase (GLuc) gene at this location were successfully recovered. However, rapid loss of most, if not all, of the reporter sequence occurred for both viruses, indicating that the reporter viruses were not stable. While the GLuc viruses predominantly reverted back to wild-type WNV length, the TAT viruses retained up to 75 additional nucleotides of the reporter sequence. These additional nucleotides were stable over at least five passages and did not significantly alter WNV fitness. Thus, the C/CA junction of WNV can tolerate additional nucleotides, though insertions are subject to certain constraints.