The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

中文翻译：

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黄病毒NS2B-NS3蛋白酶活性的可激活荧光的报告基因使单细胞和病毒斑块中感染的实时成像成为可能。

黄病毒科的黄病毒属包括许多医学上重要的病毒，例如登革热病毒（DENV），寨卡病毒（ZIKV）和黄热病病毒。寻找抗黄病毒感染的治疗靶标需要更好地了解天然病毒株感染期间病毒-宿主相互作用的动力学。然而，这被当前用于监测活细胞中黄病毒感染的基于细胞的系统的局限性所阻止。在本研究中，我们报告了荧光激活传感器的构建，以检测活细胞中黄病毒NS2B-NS3丝氨酸蛋白酶的活性。该系统由基于GFP的报道分子组成，该报道分子在体外被重组DENV-2 / ZIKV蛋白酶裂解后即发荧光。此传感器的一个包含黄病毒内部NS3裂解位点接头的传感器报道，在DENV-2稳定转导的哺乳动物细胞中，荧光激活最高/ ZIKV感染。此外，荧光的发作与病毒蛋白酶活性有关。这种黄病毒传感器的远红外版本在荧光Dulbecco噬菌斑测定中具有最佳的信噪比，从而导致构建了一个结合了黄病毒传感器和报告染料的多报告平台，用于检测染色质浓缩和细胞死亡，从而实现了单细胞分辨率的病毒斑形成研究。最后，该平台的应用使得能够研究DENV-2，ZIKV和黄热病毒感染和死亡的细胞动力学。我们预计，使用此报告系统进行的病毒感染动力学的未来研究将使病毒-宿主相互作用的基础研究成为可能，并促进抗病毒药物研究中将来用于管理黄病毒感染的应用。