Abstract
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.
Highlights
The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus
After careful analysis and avoiding the formation of cleavage sites for other cellular proteases within the resulting protein sequence of the sensor,3 we selected the cleavage sequences that define the linker. Three variants of this reporter were constructed by changing the linker sequence: ZIKVA-GFP (ZIKV polyprotein NS2B/NS3 cleavage site linker), DENV2A-GFP (DENV-2 polyprotein NS2B/NS3 cleavage site linker), and FlaviA-GFP with the internal NS3 cleavage site present in many members of the Flavivirus genus [3, 5, 30]
The present study reports the construction of fluorescent protein-based sensors of flavivirus NS2B–NS3 serine proteases activity that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro (Fig. 1)
Summary
A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques. Reporter replicons and viral genomes allow kinetic studies in living cells but are limited to molecular clones and not suitable to study clinical isolates or native virus strains In this respect, genetically encoded molecular reporters monitoring the flavivirus NS2B–NS3 proteolytic activity upon infection are an advantageous approach that is suitable for live-cell imaging studies of native flavivirus strains. We developed genetically encoded flavivirus molecular reporters by inserting a flaviviral NS2B–NS3 cleavage site into our caspase-activatable (CA) GFP [26] or CA-mNeptune [28], giving rise to the flavivirus-activatable (FlaviA) GFP and FlaviA-mNeptune reporters, respectively To our knowledge, this is the first fluorescence-activatable molecular reporter system for live-cell imaging of the infection by both reference and native strains of flaviviruses like DENV, ZIKV, and YFV
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