Assessing specificity and selectivity of flavivirus target sequences by NS3 proteases in Aedes aegypti cells

Aedes aegypti transmits flaviviruses such as dengue, Zika, and yellow fever. The flavivirus genome is translated as a single polyprotein and cleaved by both cellular proteases and the virally encoded NS3 protease. Our long-term goal is to express an insect-specific neurotoxin in the mosquito that is tethered to the endoplasmic reticulum via an NS3 target sequence, only to be released by the protease in systemically infected mosquitos. In this study, we seek to determine the cleavage efficiency of three target sequences by flavivirus proteases in Ae. aegypti cells, with the most widely recognized sequence being 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 co-transfected with plasmids encoding an active or inactive DENV2-NS3. For six out of eight cleavage sites, imaging at 96 hours post-transfection was consistent with viral protease-induced cleavage, as significant increases in fluorescence were observed when the reporters and active protease were expressed in cells. The same was not true for cells co-transfected with the plasmid expressing the inactive protease. Cleavage of the reporters was also confirmed and quantified via Western blot. Finally, the ability of DENV1-4, ZIKV, YFV, AEFV, and CFAV proteases to cleave the three selected DENV2-derived target sites was confirmed. These results both increase our basic understanding of constraints on flavivirus NS3 target sites and provide a tool for the development of transgenic mosquito strains programmed to die upon infection with a number of flaviviruses.