Inhibition of gene expression in mosquitoes mediated by RNA delivered by the double subgenomic Sindbis virus expression systems

Abstract

The double subgenomic Sindbis (dsSIN) virus expression system was used to deliver RNA transcripts complementary to endogenous mosquito mRNA to inhibit gene expression in vivo. dsSIN has been used to stably express genes in Aedes aegypti salivary glands and to inhibit flavivirus replication by expression of RNA transcripts complementary to the flavivirus genome. The dsSIN viruses TE/3'2J and MRE/3'2J were assessed for their ability to inhibit the expression of endogenous mosquito genes expressed exclusively in the salivary glands. Experiments were conducted to inhibit expression of a reporter gene in a germline transformed Ae. aegypti line (APY-LUC43). These mosquitoes constituitively express luciferase from the mosquito apyrase promoter. APY-LUC43 mosquitoes were intrathoracically inoculated with TE/3'2J/α-luc virus, which transcribed RNA complementary to the 5' end of the luciferase mRNA. Luciferase activity was monitored over time in mosquitoes infected with either TE/3'2J/α-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with TE/3'2J/α-luc virus exhibited 90% reduction in luciferase activity compared to uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. Apyrase is a salivary gland protein with anti-platelet aggregating properties. TE/3'2J/α-apyrase virus was engineered to contain 375 bases from the 5' coding region of the Ae. aegypti apyrase gene, inserted in antisense orientation. TE/3'2J/α-apyrase was intrathoracically inoculated into female adult mosquitoes, and apyrase activity and protein production was assessed. TE/3'2J/α-apyrase virus effectively infected salivary gland tissue; however, no diminishment of apyrase production was observed. An orally infective dsSIN chimeric virus was engineered that contained the structural genes of MRE16 and the nonstructural genes and second subgenomic promoter of TE/3'2J/α-apyrase. MRE/3'2J/α-apyrase was tested for its ability to infect larvae per os and inhibit expression of apyrase in adults. Infection rates of up to 42% were obtained, but inhibition of apyrase expression was not observed. These studies support the overall hypothesis of this dissertation, that expression of an endogenous mosquito gene can be specifically inhibited by RNA delivered by the SIN virus expression system. The dsSIN antisense RNA expression system provides an important tool for studying gene expression in vivo, and may lead to defining genetic determinants of mosquito vector competence.