The role of carboxypeptidase B1 in modulating DENV replication and release from Aedes aegypti midgut cells Tham, Hong Wai 10.4225/03/58b75e38089ff https://bridges.monash.edu/articles/thesis/The_role_of_carboxypeptidase_B1_in_modulating_DENV_replication_and_release_from_Aedes_aegypti_midgut_cells/4711627 The female Aedes aegypti mosquito, the principal vector for the transmission of Dengue virus (DENV), is able to support DENV propagation within its tissues, especially the midgut and salivary gland. Understanding host-virus relationships may lead to the discovery of new vector control strategies for better virus controls. In this study, preliminary biological interaction screens between DENV2 and female Ae. aegypti, were conducted using the yeast two-hybrid (Y2H) assays. A cDNA library of the female adult Ae. aegypti was constructed and used in several Y2H screens with DENV2 E, prM, M and NS1 proteins. Among the many host interacting proteins identified, carboxypeptidase B1 (CPB1) shown to bind to the E protein was selected for further studies. The interaction was also demonstrated using in silico docking analyses. Further analyses to verify the CPB1-E protein-protein interactions were conducted using mammalian two-hybrid, co-immunoprecipitation and double immunofluorescent co-localisation assays. It was shown that CPB1 was present in the endoplasmic reticulum (ER) of Ae. aegypti primary midgut cells and binds to DENV2 in the ER. The function of CPB1 with respect to the binding of virus in the ER was further investigated by the up- and down-regulation of CPB1 expression. CPB1 overexpression in mosquito C6/36 cells resulted in intracellular accumulation of DENV2 genomic RNA or virus particles, with only small amounts of virus released from the cells. In contrast, induced expression of CPB1 in Vero cells exhibited a different outcome. Therefore, it was suggested that CPB1 functions by regulating DENV2 replication exclusively in mosquito midgut cells. Based on the DENV replication cycle, we postulated that, in Ae. aegypti midgut cells, CPB1 binds to the E protein of the virus and regulates DENV2 genome encapsulation into the ER lumen, or inhibits immature virus particle transportation to the Golgi complexes, which subsequently reduces the amount of mature DENV2 released from the midgut cells to other tissues. The small amount of virus released from the midgut cells could be sufficient to infect the salivary gland cells for further replication and transmission to humans, but not enough to fully infect other tissues that cause adverse effects on the health of the mosquito. 2017-03-01 23:50:14 Carboxypeptidase thesis(doctorate) DENV2 DENV ethesis-20150430-162018 Open access 2015 monash:155407 Aedes aegypti mosquito 1959.1/1173653 CPB1-E protein-protein