PDF(Pubmed)
Abstract：
Papain-like protease PLpro, a domain within a large polyfunctional protein, nsp3, plays key roles in the life cycle of SARS-CoV-2, being responsible for the first events of cleavage of a polyprotein into individual proteins (nsp1-4) as well as for the suppression of cellular immunity. Here, we developed a new genetically encoded fluorescent sensor, named PLpro-ERNuc, for detection of PLpro activity in living cells using a translocation-based readout. The sensor was designed as follows. A fragment of nsp3 protein was used to direct the sensor on the cytoplasmic surface of the endoplasmic reticulum (ER) membrane, thus closely mimicking the natural target of PLpro. The fluorescent part included two bright fluorescent proteins-red mScarlet I and green mNeonGreen-separated by a linker with the PLpro cleavage site. A nuclear localization signal (NLS) was attached to ensure accumulation of mNeonGreen into the nucleus upon cleavage. We tested PLpro-ERNuc in a model of recombinant PLpro expressed in HeLa cells. The sensor demonstrated the expected cytoplasmic reticular network in the red and green channels in the absence of protease, and efficient translocation of the green signal into nuclei in the PLpro-expressing cells (14-fold increase in the nucleus/cytoplasm ratio). Then, we used PLpro-ERNuc in a model of Huh7.5 cells infected with the SARS-CoV-2 virus, where it showed robust ER-to-nucleus translocation of the green signal in the infected cells 24 h post infection. We believe that PLpro-ERNuc represents a useful tool for screening PLpro inhibitors as well as for monitoring virus spread in a culture.
摘要：
木瓜蛋白酶样蛋白酶PLpro,大型多功能蛋白质中的一个结构域，nsp3在SARS-CoV-2的生命周期中起关键作用，负责将多蛋白裂解成单个蛋白（nsp1-4）以及抑制细胞免疫的第一个事件。这里,我们开发了一种新的基因编码荧光传感器，名叫PLpro-ERNuc,用于使用基于易位的读数检测活细胞中的PLpro活性。传感器设计如下。nsp3蛋白的片段用于将传感器引导到内质网（ER）膜的细胞质表面，从而密切模仿PLpro的自然目标。荧光部分包括两个明亮的荧光蛋白-红色mScarletI和绿色mNeonGreen-由具有PLpro切割位点的接头分离。连接核定位信号(NLS)以确保mNeonGreen在切割时积累到核中。我们在HeLa细胞中表达的重组PLpro模型中测试了PLpro-ERNuc。在不存在蛋白酶的情况下，传感器显示了红色和绿色通道中预期的细胞质网状网络，以及绿色信号有效易位到表达PLpro的细胞中的细胞核中(细胞核/细胞质比率增加14倍)。然后，我们在感染SARS-CoV-2病毒的Huh7.5细胞模型中使用PLpro-ERNuc，在感染后24小时，它显示出感染细胞中绿色信号的ER到核的强烈易位。我们认为PLpro-ERNuc是筛选PLpro抑制剂以及监测培养物中病毒传播的有用工具。
