This study describes a non-infectious in-cell imaging assay for
HIV-1 protease inhibitor screening. It is based on re-distribution of a fluorescence reporter
protein upon
protease cleavage and the fact that HIV-infected cells undergo apoptosis. The in-cell assay utilizes fluorescent reporter
proteins consisting of an intracellular translocation
signal sequence, a caspase-3-specific cleavage sequence, and a fluorescent tagging
protein. The reporter
proteins are designed to change their intracellular localization upon cleavage, either from the cytosol to a subcellular organelle (type I) or from a subcellular organelle to the cytosol (type II). Inhibition of
protease activity can be monitored at the single cell level. Interestingly, the expression of
HIV-1 protease induced endogenous
caspase-3 activation; thus, the fluorescence reporter
protein containing the
caspase-3 cleavage sequence translocalized upon cleavage. This is the first time that
HIV-1 protease expression, not whole
virus infection of the cell, was observed to trigger the apoptotic pathway, including caspse-3 activation. A validation of this assay was performed with a known
HIV-1 protease inhibitor, Ac-Leu-Val-
phenylalanine. The clear cellular change in fluorescence pattern makes this system an ideal tool for various types of life science and
drug discovery research, including high throughput and high content screening applications.