Evaluating specificity and selectivity of flavivirus target sequences by NS3 proteases in Aedes aegypti cells using a quenched-GFP reporter

Aedes aegypti transmits viruses such as dengue, Zika, and yellow fever, all members of the Flavivirus genus. The flavivirus genome is translated directly as a single polyprotein and cleaved by both cellular proteases and the virally encoded NS3 protease. Our long-term goal is to express an inactive insect-specific neurotoxin in the mosquito that can be subsequently activated via NS3 cleavage to specifically kill systemically infected mosquitos. In this study, we seek to determine the cleavage efficiency of eight target sequences by flavivirus proteases in Ae. aegypti cells, with the most widely recognized sequence incorporated into our final transgene construct. Reporter plasmids that express a quenched or non-fluorescing eGFP with DENV2 derived NS3 target sequences inserted at the eGFP-quenching peptide junction were individually transfected into A20 cells as well as co-transfected with a plasmid encoding DENV2-NS3. For at least two cleavage sites, imaging at 48, 72, and 96 hours was consistent with viral protease induced cleavage, as a significant increase in fluorescence was observed when both the reporter and protease were expressed in cells. Cleavage of the reporter was also confirmed via Western blot. These results indicate that we can successfully quantitate NS3 activity in vivo, therefore making this method effective for evaluating the cleavage of NS3 target sequences by a range of viral proteases not of the same genome. This should allow us to develop a transgene with the potential to kill mosquitos infected with any flavivirus.