Development of a stable Sf9 insect cell line to produce VSV-G pseudotyped baculoviruses.

Baculoviruses have shown great potential as gene delivery vectors in mammals, although their effectiveness in transferring genes varies across different cell lines. A widely employed strategy to improve transduction efficiency is the pseudotyping of viral vectors. In this study, we aimed to develop a stable Sf9 insect cell line that inducibly expresses the G-protein of the vesicular stomatitis virus to pseudotype budded baculoviruses. It was obtained by inserting the VSV-G gene under the control of the very strong and infection-inducible pXXL promoter and was subsequently diluted to establish oligoclonal lines, which were selected by the fusogenic properties of VSV-G and its expression levels in infected cells and purified budded virions. Next, to enhance the performance of the cell line, the infection conditions under which functional pseudotyped baculoviruses are obtained were optimized. Finally, different baculoviruses were pseudotyped and the expression of the transgene was quantified in mammalian cells of diverse origins using flow cytometry. The transduction efficiency of pseudotyped baculovirus consistently increased across all tested mammalian cell lines compared with control viruses. These findings demonstrate the feasibility and advantages of improving gene delivery performance without the need to insert the pseudotyping gene into the baculoviral genomes.

P26 enhances baculovirus gene delivery by modulating the mammalian antiviral response.

Poxins are poxviral proteins that act by degrading 2´3´-cGAMP, a key molecule of cGAS-STING axis that drives and amplifies the antiviral response. Previous works have described some poxin homologous among lepidopteran and baculoviral genes. In particular, P26, a poxin homologous from AcMNPV retains the 2´3´-cGAMP degradation activity in vitro. In this work, we demonstrated that the antiviral activity triggered by baculovirus was disrupted by the transient expression of P26 in murine and human cell lines, and the effect of this action is not only on IFN-ß production but also on the induction of IFN-λ. Besides, we proved P26 functionality in a stable-transformed cell line where the protein was constitutively expressed, preventing the production of IFN-ß induced by baculovirus and resulting in an improvement in the transduction efficiency by the attenuation of the antiviral activity. Finally, we incorporated P26 into budded virions by capsid display or passive incorporation, and the results showed that both strategies resulted in an improvement of 3-17 times in the efficiency of transgene expression in murine fibroblasts. Our results suggest that the incorporation of P26 to budded baculoviral vectors is a very promising tool to modulate negatively the innate antiviral cellular response and to improve the efficiency of gene delivery in mammalian cells. KEY POINTS: • P26 affects baculovirus-induced IFN-ß and IFN-λ production in mammalian cells. • Murine fibroblasts expressing P26 are more susceptible to transduction by baculovirus. • Incorporation of P26 into the virion improves gene delivery efficiency of baculovirus.