D3027: Determining the specificity and selectivity of flavivirus cleavage sites by NS3 proteases in Aedes aegypti A20 cells

Aedes aegypti transmits viruses such as dengue, Zika, and yellow fever, all members of the Flavivirus genus. The flavivirus genome is a single stranded positive sense RNA that is translated as one polyprotein and cleaved by both cellular proteases and the virally-encoded NS3 protease. We seek to exploit NS3 by developing a transgene that will result in the virus-induced death of Aedes aegypti. Our overall goal is to express an insect specific neurotoxin that will be tethered to the endoplasmic reticulum via an NS3 cleavage sequence, only to be released by the protease in systemically infected mosquitos. In this study, our goal is to determine the cleavage efficiency of cleavage sites by several flavivirus proteases in Ae. aegypti cells. The most widely recognized sequence will be incorporated into our final construct. We designed a reporter plasmid to separately express an ER-tethered, eGFP-mCherry fusion protein and NS3. DENV2 derived NS3 cleavage sites were inserted at the eGFP-mCherry junction. A20 cells were transfected with each test plasmid and imaged, with the resulting images being analyzed for colocalization. Critically, we have observed a decrease in colocalization with at least one target site. Results indicate that we can successfully observe NS3 activity in vivo, therefore making this method effective for evaluating NS3 cleavage. This should allow us to create a transgene with the potential to kill mosquitos infected with any flavivirus.