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首页> 外文期刊>Virus Research: An International Journal of Molecular and Cellular Virology >Visualization of the N~(pro) protein in living cells using biarsenically labeling tetracysteine-tagged classical swine fever virus
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Visualization of the N~(pro) protein in living cells using biarsenically labeling tetracysteine-tagged classical swine fever virus

机译：使用双砷标记的四半胱氨酸标记的经典猪瘟病毒可视化活细胞中的N〜（pro）蛋白

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摘要

Real-time fluorescence imaging of viral proteins in living cells is a valuable means to study virus-host interactions, and tetracysteine (TC)-biarsenical technology has been used in several viruses but not in classical swine fever virus (CSFV). Here, we generated CSFV mutants vSMTC385 or vSMTC412 bearing the small TC tag (CCPGCC) in the N-terminal region of the N~(pro) protein. The mutants showed growth characteristics indistinguishable from that of the wild-type virus, and retained similar N~(pro) subcellular localization to that of the parent virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in the fluorescent N~(pro) protein in the context of virus infection. Finally, we showed that N~(pro) was localized in the cytoplasm of CSFV-infected cells at 27 h post-infection (hpi) and present in the nucleus at 48 hpi, and the nuclear import and export was clearly observed from 36.5 to 37 hpi. Interestingly, our results demonstrated that N~(pro) transported across the nuclear pores by passive diffusion, which might be prevented by exogenous interferon regulatory factor 3 interacting with N~(pro). Taken together, biarsenical labeling allows real-time visualization of the nucleus import and export of the fluorescent N~(pro) protein in CSFV-infected living cells.
机译：活细胞中病毒蛋白的实时荧光成像是研究病毒与宿主相互作用的重要手段，四半胱氨酸（TC）-双酚A技术已用于几种病毒中，但在经典猪瘟病毒（CSFV）中并未使用。在这里，我们生成了CSFV突变体vSMTC385或vSMTC412，在N〜（pro）蛋白的N端区域带有小TC标签（CCPGCC）。突变体的生长特性与野生型病毒没有区别，并且保留了与亲本病毒相似的N〜（pro）亚细胞定位。此外，在病毒感染的情况下，用可渗透膜的双砷染料标记会产生荧光N〜（pro）蛋白。最后，我们表明N〜（pro）在感染后27 h（hpi）定位于CSFV感染细胞的细胞质中，并在48 hpi存在于核中，并且从36.5至37马力。有趣的是，我们的结果表明N〜（pro）通过被动扩散穿过核孔，这可能被外源干扰素调节因子3与N〜（pro）相互作用所阻止。综上所述，双砷标记可以实时观察CSFV感染活细胞中荧光N〜（pro）蛋白的细胞核导入和导出。

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