Analysis of luciferase dsRNA production during baculovirus infection of Hi5 cells: RNA hairpins expressed by very late promoters do not trigger gene silencing
نویسندگان
چکیده
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.
منابع مشابه
In vitro transcription of pe38/polyhedrin hybrid promoters reveals sequences essential for recognition by the baculovirus-induced RNA polymerase and for the strength of very late viral promoters.
In vitro transcription was used to analyze the promoter specificity of the alpha-amanitin-resistant RNA polymerase that is induced late during infection of Autographa californica multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The viru...
متن کاملHow does RNA editing affect dsRNA-mediated gene silencing?
In general, double-stranded RNA (dsRNA)-binding proteins (dsRBPs) are not sequence-specific. A dsRNA molecule in a cell will interact with any dsRBP it comes in contact with, suggesting that different dsRNA-mediated pathways intersect and affect each other. This paper analyzes evidence that the ADAR RNA editing enzymes, which act on dsRNA, affect dsRNA-mediated gene silencing pathways. Examples...
متن کاملSilencing of the baculovirus Op-iap3 gene by RNA interference reveals that it is required for prevention of apoptosis during Orgyia pseudotsugata M nucleopolyhedrovirus infection of Ld652Y cells.
The Op-iap3 gene from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) inhibits apoptosis induced by a mutant of Autographa californica MNPV (AcMNPV) that lacks the antiapoptotic gene p35, as well as apoptosis induced by a wide range of other stimuli in both mammalian and insect cells. However, the role of Op-iap3 during OpMNPV infection has not been previously examined. To ...
متن کاملA 30-kDa host protein binds to two very-late baculovirus promoters.
A 30-kDa host factor (polyhedrin-promoter-binding protein; PPBP) specifically binds to sequences critical for transcription from the baculovirus polyhedrin (p29) gene initiator promoter [Burma, S., Mukherjee, B., Jain, A., Habib, S. & Hasnain, S. E. (1994) J. Biol. Chem. 269, 2750-2757; Mukherjee, B., Burma, S. & Hasnain, S. E. (1995) J. Biol. Chem. 270, 4405-4411]. A host factor also binds, in...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
ژورنال
عنوان ژورنال: Frontiers in insect science
سال: 2022
ISSN: ['2673-8600']
DOI: https://doi.org/10.3389/finsc.2022.959077