Dual-fluorescent reporter for live-cell imaging of the ER during DENV infection.
Dual-fluorescent reporter for live-cell imaging of the ER during DENV infection.
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DOI：
10.3389/fcimb.2022.1042735
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发表时间：
2022
影响因子：
5.7
通讯作者：
中科院分区：
文献类型：
--
作者：
黄病毒感染导致内质网（ER）的急剧重塑。病毒复制发生在ER膜中病毒诱导的囊泡内陷内。黄病毒感染的标志是ER膜的扩张，这可以在感染后的特定时间点观察到。然而，这一过程在整个感染过程中的活细胞中并没有以单细胞分辨率有效地可视化。在这项研究中，我们开发了一种基于质粒的报告系统，以监测黄病毒感染和同时病毒诱导的操纵单细胞在整个感染过程中的实时。该系统需要病毒蛋白酶切割以释放与核定位信号（NLS）融合的ER锚定的荧光蛋白感染报告基因。这种蛋白水解切割允许感染报告信号转移到细胞核，而ER特异性荧光标记物仍然位于管腔中。因此，该构建体允许在整个感染过程中观察ER的病毒依赖性变化。在这项研究中，我们表明，我们的报告基因在多种黄病毒蛋白酶表达时被有效切割，包括登革病毒（DENV），我们还发现，我们的ER锚定报告基因的DENV蛋白酶依赖性切割表现出比先前用生物化学测定所显示的更严格的切割序列特异性。使用该系统对感染DENV的活细胞进行长时间的延时成像，我们观察到报告信号的核转位开始于感染后约8小时，并且在整个时间过程中持续增加。有趣的是，我们发现报告信号转位的增加与ER信号强度的增加相关，这表明DENV感染和ER扩增之间存在时间依赖性的正相关。总体而言，该报告表明FlavER平台提供了一种有用的工具，用于监测黄病毒感染，同时观察宿主细胞ER的病毒依赖性变化，从而研究病毒-宿主相互作用的时间性质。
Infection by flaviviruses leads to dramatic remodeling of the endoplasmic reticulum (ER). Viral replication occurs within virus-induced vesicular invaginations in the ER membrane. A hallmark of flavivirus infection is expansion of the ER membrane which can be observed at specific time points post infection. However, this process has not been effectively visualized in living cells throughout the course of infection at the single cell resolution. In this study, we developed a plasmid-based reporter system to monitor flavivirus infection and simultaneous virus-induced manipulation of single cells throughout the course of infection in real-time. This system requires viral protease cleavage to release an ER-anchored fluorescent protein infection reporter that is fused to a nuclear localization signal (NLS). This proteolytic cleavage allows for the translocation of the infection reporter signal to the nucleus while an ER-specific fluorescent marker remains localized in the lumen. Thus, the construct allows for the visualization of virus-dependent changes to the ER throughout the course of infection. In this study, we show that our reporter was efficiently cleaved upon the expression of multiple flavivirus proteases, including dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). We also found that the DENV protease-dependent cleavage of our ER-anchored reporter exhibited more stringent cleavage sequence specificity than what has previously been shown with biochemical assays. Using this system for long term time-lapse imaging of living cells infected with DENV, we observed nuclear translocation of the reporter signal beginning approximately 8 hours post-infection, which continued to increase throughout the time course. Interestingly, we found that increased reporter signal translocation correlated with increased ER signal intensity, suggesting a positive association between DENV infection and ER expansion in a time-dependent manner. Overall, this report demonstrates that the FlavER platform provides a useful tool for monitoring flavivirus infection and simultaneously observing virus-dependent changes to the host cell ER, allowing for study of the temporal nature of virus-host interactions.
影响因子：
3.1
作者：
Medin CL;Valois S;Patkar CG;Rothman AL
通讯作者：
Rothman AL