(307b) Quantitative Assessment of in Vivo HIV Protease Activity Using Genetically Engineered QD-Based FRET Probes

Inhibitors targeting the HIV protease are a major component of anti-HIV therapy. Genetic drift in the virus due to constantly accumulating mutations leads to resistance and failure of therapy. The resistance in case of protease inhibitors is a combined effect of mutations accumulating in the protease and its cleavage sites. We have developed several quantum dot (QD)-based FRET probes sensitive to HIV protease. These FRET probes consisted of known HIV protease cleavage site sequences and employed QD as the donor and alexa as the acceptor moiety. Upon introduction of this probe into the HIV infected cells, protease cleaves the probe and disrupts FRET. The changes in the FRET can be assessed both qualitatively and quantitatively. The FRET changes quantified in terms of probe cleaved in vivo over time helps us capture the kinetics of HIV protease. We have used different protease wild type substrates, reported to have varied processing rate and successfully demonstrated the kinetic differences in their processing with our assay.  Apart from FRET probes with wild type cleavage sequences, we have constructed probes with mutated cleavage sequences found in drug resistant HIV and captured their reported differences in their cleavage rates. We will extend this study to include commonly reported drug resistant protease mutations along with the cleavage site mutations. The complete system displaying both protease mutations and cleavage site mutations, reflective of drug resistant HIV will be used as a screen to identify newer inhibitors.