A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques
Fecha
2020-01-09
Tipo
artículo original
Autores
Arias Arias, Jorge Luis
MacPherson, Derek J.
Hill, Maureen E.
Hardy, Jeanne A.
Mora Rodríguez, Rodrigo Antonio
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Resumen
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 thera-
peutic 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 flavivi-
rus 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 fluo-
rescent upon cleavage by recombinant DENV-2/ZIKV proteases
in vitro. A version of this sensor containing the flavivirus inter-
nal 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 Dulbec-
co’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 flavivi-
rus infections.
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cell death, dengue virus (DENV), flavivirus, fluorescence, internal cleavage, NS2B-NS3 protease, plaque assay, cell-based reporter, Zika virus (ZIKV), yellow fever virus (YFV)