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首页> 外文期刊>PLoS One >Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins
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Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

机译：编码分泌型高斯荧光素酶的甲型流感病毒是分析病毒复制及其被抗病毒化合物和细胞蛋白抑制的有用工具

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Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.
机译：插入病毒基因组中的报告基因可以轻松快速地量化病毒复制，这对于有效地体外筛选抗病毒化合物或体内分析病毒传播和发病机制非常重要。基于已发布的设计，我们已经生成了几种带有荧光蛋白或高斯荧光素酶的能复制的甲型流感病毒。记者的活动可以很容易地在感染的培养物中定量，但是编码高斯荧光素酶的病毒比带有荧光蛋白的病毒更稳定，因此进行了详细分析。感染培养物上清液中高斯荧光素酶活性的定量可以方便，高度灵敏地检测病毒传播，并且酶活性与感染细胞释放的感染颗粒数量有关。此外，编码高斯荧光素酶的病毒可以灵敏定量神经氨酸酶抑制剂（NAI）扎那米韦和宿主细胞干扰素诱导的跨膜（IFITM）蛋白1-3的抗病毒活性，已知它们可以抑制流感病毒的进入。最后，该病毒被用来证明甲型流感病毒感染对内体胆固醇的调节剂敏感，这与IFITM通过改变内体膜中的胆固醇水平抑制病毒进入的概念保持一致。总而言之，我们报告了一种新型A型流感报道基因病毒的特征，该病毒可快速，灵敏地检测病毒传播及其抑制作用，并且我们证明A型流感病毒的进入对内体胆固醇水平的变化敏感。

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