Evidence for a Genetic and Physical Interaction between Nonstructural Proteins NS1 and NS4B That Modulates Replication of West Nile Virus
Evidence for a Genetic and Physical Interaction between Nonstructural Proteins NS1 and NS4B That Modulates Replication of West Nile Virus
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DOI：
10.1128/jvi.00157-12
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发表时间：
2012-07-01
影响因子：
5.4
通讯作者：
Diamond, Michael S.
中科院分区：
文献类型：
--
作者：
Youn, Soonjeon;Li, Tuo;Diamond, Michael S.
黄病毒NS1是一种非结构糖蛋白，在细胞表面表达并分泌到细胞外空间。尽管NS1通过分泌途径转运，但它是与早期病毒RNA复制相关的重要基因。考虑到NS1和病毒RNA复制复合体的不同定位（后者存在于内质网（ER）的细胞质面），这种情况如何发生仍然是一个谜。我们最近在NS1中发现了一个n端二氨基酸基序，它调节蛋白质靶向并影响病毒复制。登革病毒（DENY）与西尼罗病毒（WNV） NS1 （RQ10NK）交换第10和第11位的两个氨基酸，改变了其相对表面表达和分泌，降低了其传染性。然而，含有NS1 RQ10NK的西尼罗河病毒的表型是不稳定的，因为在两个传代中观察到异质斑块变异。在这里，利用编码NS1 RQ10NK突变的突变型WNV，我们在NS4B中发现了一个抑制突变（F86C）， NS4B是一种病毒编码的跨膜蛋白，在内质膜的管腔和细胞质两侧都有环。引入NS4B F86C特异性地挽救了突变型西尼罗病毒的RNA复制，但对野生型病毒没有影响。质谱和共免疫沉淀研究建立了NS1和NS4B之间新的物理相互作用，提示了管腔NS1向细胞质传递信号调控RNA复制的机制。
Flavivirus NS1 is a nonstructural glycoprotein that is expressed on the cell surface and secreted into the extracellular space. Despite its transit through the secretory pathway, NS1 is an essential gene linked to early viral RNA replication. How this occurs has remained a mystery given the disparate localization of NS1 and the viral RNA replication complex, as the latter is present on the cytosolic face of the endoplasmic reticulum (ER). We recently identified an N-terminal di-amino acid motif in NS1 that modulates protein targeting and affected viral replication. Exchange of two amino acids at positions 10 and 11 from dengue virus (DENY) into West Nile virus (WNV) NS1 (RQ10NK) changed its relative surface expression and secretion and attenuated infectivity. However, the phenotype of WNV containing NS1 RQ10NK was unstable, as within two passages heterogeneous plaque variants were observed. Here, using a mutant WNV encoding the NS1 RQ10NK mutation, we identified a suppressor mutation (F86C) in NS4B, a virally encoded transmembrane protein with loops on both the luminal and cytoplasmic sides of the ER membrane. Introduction of NS4B F86C specifically rescued RNA replication of mutant WNV but did not affect the wild-type virus. Mass spectrometry and coimmunoprecipitation studies established a novel physical interaction between NS1 and NS4B, suggesting a mechanism for how luminal NS1 conveys signals to the cytoplasm to regulate RNA replication.