Analysis of luciferase dsRNA production during baculovirus infection of Hi5 cells: RNA hairpins expressed by very late promoters do not trigger gene silencing

The baculovirus expression vector system (BEVS) has become an important platform for the of recombinant proteins and is especially useful production large protein complexes such as virus-like particles (VLPs). An application VLPs their use vehicles targeted delivery drugs or toxins which requires development methods efficient loading with intended cargo. Our research intends to employ BEVS insecticidal dsRNA molecules insect pests (as “dsRNA-VLPs”). A convenient strategy would be co-expression long dsRNAs viral capsid simultaneous encapsulation during VLP assembly but capacity not been assessed so far. In this study, efficiency RNA hairpins targeting luciferase gene (“dsLuc”) by polyhedrin promoter infection was evaluated. However, RNAi reporter assays could detect significant amounts dsLuc in Hi5 cells infected baculovirus, even presence co-expressed dsRNA-binding B2-GFP employment MS2-MCP system. Nevertheless, dot blot analyses using anti-dsRNA antibody revealed that baculovirus-mediated resulted increases levels may correspond hybridized complementary transcripts. Using a genetically encoded sensor, foci were detected nuclei partially co-localized DAPI staining, consistent localization at virogenic stroma. Co-localization experiments vp39, Ac93, ODV-E25 gp64 indicated limited overlap between ring zone compartment where nucleocapsids virions occurs. Stability showed exogenous resistant degradation extracts non-infected it proposed strong unwinding activity stroma neutralize annealing strands block dsRNAs. Because stability dsRNA, transfection can explored alternative method cargo dsRNA-VLPs baculovirus-infected cells.