Isolation and characterization of dengue virus membrane-associated replication complexes from Aedes aegypti.

摘要

Ultrastructural studies of flavivirus replication have long observed proliferation of host membranes. Membrane-bound replication compartments have recently been isolated and characterized from flavivirus-infected mammalian cells, providing insight into the morphology, organelle of origin, and protein components of the flavivirus membrane-associated replication complex. Our laboratory has proposed that a balance exists between dengue virus (DENV) replication in Aedes aegypti and the mosquito's RNA interference (RNAi) based antiviral response. Here, we have isolated and characterized membrane-bound replication compartments from mosquito cell culture and Ae. aegypti to evaluate the role that these membranes may play in shielding DENV double-stranded RNA (dsRNA) from RNAi.;Membrane isolation techniques and immunofluorescent staining techniques for dsRNA identification were developed to isolate and characterize membrane-associated replication complexes in DENV-infected mosquito cell culture and Ae. aegypti. Here we show that double-membrane vesicles arise from the endoplasmic reticulum (ER) and are associated with DENV dsRNA in mosquitoes. These data suggest that DENV dsRNA replicative intermediates may be shielded from the RNAi response in the mosquito.;DENV membrane-associated replication complexes were characterized in mosquito cell culture and Ae. aegypti using immunofluorescent staining for dsRNA, confocal microscopy, sucrose gradient cellular fractionation, and electron microscopy. In addition, we compared immunofluorescent staining for dsRNA between DENV and Sindbis virus (SINV). We also evaluated replication of DENV mutants in the DENV-resistant transgenic mosquito strain known as Carb77 and whether mutations in DENV genome sequence lead to evasion of the enhanced RNAi response of Carb77 mosquitoes.;This is the first isolation of membrane-associated replication complexes and first characterization of dsRNA staining from DENV-infected mosquito cell culture and Ae. aegypti, providing knowledge which can be used to develop improved RNAi-based control strategies for DENV in mosquitoes.