Title: Engineering Sf9 host cells with AcMNPV genes to control baculovirus infection dynamics and heterologous gene expression
Abstract:
The baculovirus–insect cell expression system is extensively used for recombinant protein production and vaccine development; however, interactions between viral genes and their host cells critically influence infection efficiency, heterologous gene expression, and virus yield. In this study, we established stable Spodoptera frugiperda (Sf9) cell lines, a permissive host for Autographa californica multiple nucleopolyhedrovirus (AcMNPV), constitutively expressing ORF35, ORF141, and ORF66, to investigate their roles during baculovirus infection.
These stable Sf9 cell lines were infected with AcMNPV displaying the SARS-CoV-2 receptor-binding domain (RBD) fused to SfGFP at different multiplicities of infection (MOI). Infection efficiency, fluorescence intensity, viral replication, and budded virus (BV) titers were quantitatively evaluated and compared with parental Sf9 cells.
Sf9 cells stably expressing ORF35 showed a significant enhancement in SfGFP fluorescence intensity across all tested MOIs, accompanied by a marked increase in viral replication and BV titers relative to parental Sf9 cells. In contrast, ORF141-expressing Sf9 cells exhibited a pronounced reduction in fluorescence intensity, viral replication, and BV production, indicating an inhibitory role for ORF141 during AcMNPV infection. Sf9 cells expressing ORF66 displayed modest, MOI-dependent effects with no substantial improvement over control cells.
Collectively, these results demonstrate that specific AcMNPV genes distinctly modulate infection dynamics and heterologous gene expression in their native insect host when expressed prior to infection. Engineering Sf9 cells with selected viral genes, particularly ORF35, represents a promising strategy to enhance baculovirus-based platforms for vaccine development and recombinant protein production.
