Early to Late VSV-G Expression in AcMNPV BV Enhances Transduction in Mammalian Cells but Does Not Affect Virion Yield in Insect Cells.

Baculoviruses represent promising gene delivery vectors for mammalian systems, combining high safety profiles with substantial cargo capacity. While pseudotyping with vesicular stomatitis virus G-protein (VSV-G) enhances transduction efficiency, optimal expression strategies during the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection cycle remain unexplored. This study investigates how VSV-G expression timing affects pseudotype incorporation into budded virions (BVs) and subsequent transduction efficacy. Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (, , and ) and GFP via a CMV promoter. VSV-G incorporation was verified by Western blot, while transduction efficiency was quantified in mammalian cell lines (fluorescence microscopy/flow cytometry) and rat hind limbs. Viral productivity was assessed through production kinetics and plaque assays. All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The promoter drove the highest VSV-G expression and transduction efficiency. Crucially, BV production yields and infectivity remained unaffected by VSV-G expression timing. The in vivo results mirrored the cell culture findings, with p10-driven constructs showing greater GFP expression at low doses (10 virions). Strategic VSV-G expression via very late promoters (particularly ) maximizes baculoviral transduction without compromising production yields. This study establishes a framework for optimizing pseudotyped BV systems, demonstrating that late-phase glycoprotein expression balances high mammalian transduction with preserved insect-cell productivity-a critical advancement for vaccine vector development.