The baculovirus expression system (BEVS) was established in the 1980s (Maeda et al., 1985, Smith et al., 1983), together with Escherichia coli, yeast, and mammalian cell as the four prevalent expression systems (Cox, 2021). Compared with the other expression systems, the BEVS has the advantage of high levels of expression and post-translational modification capabilities, in addition, multiple large gene fragments can be expressed simultaneously with high biosafety (Grünewald et al., 1996, Jarvis and Summers, 1989, Medin et al., 1990, Yee et al., 2018). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda cell including Sf9 cells are one of the most commonly used baculoviruses and host cells in BEVS. In 2009, the U.S. Food and Drug Administration (FDA) approved the Cervarix®, the first bivalent virus-like particles (VLPs) vaccine for human papillomavirus produced by BEVS (Paavonen et al., 2007). Recently, BEVS has also been used to produce adeno-associated viruses (AAV) vectors and has the highest per-cell yield of viral particles, promising to be one of the general methods for AAV production (Aponte-Ubillus et al., 2018). To sum up, it is increasingly used in the production of biopharmaceuticals, such as antibodies, vaccines, and VLPs, to prevent and treat animal and human diseases.

During protein production using BEVS, expression cassettes of the foreign protein were inserted into the viral genome, subsequently, baculoviruses infected host cells and expressed proteins using the transcriptional and translational elements of the cells (Nobiron et al., 2003). However, a large number of late and very late proteins of baculoviruses are expressed and aggregated intracellularly, which leads to the expressed protein products being misfolded in an insoluble inactive form (Pichard et al., 2022). Meanwhile, these proteins cause a significant adverse impact on the intracellular environment and organelles (Thiem, 2009). In addition, when the BEVS is used to produce recombinant particulate products such as VLPs and AAV vectors (Aucoin et al., 2007, Wu et al., 2019, Zhang et al., 2010), baculovirus particles need to be removed in the final product, which is a challenge. Regulators require VLPs and AAV products to be free of unnecessary nucleic acids and/or proteins, including baculovirus particles (Possee et al., 2020). Marek et al. established a virion-free baculovirus expression system by deleting vp80, a crucial structural protein of AcMNPV. Foreign genes driven by the very late viral promoter polh were successfully expressed in this system without baculovirus particles (Marek et al., 2011).

Baculovirus late expression factors (LEFs) have regulatory functions on many late gene expressions in baculovirus, affecting viral DNA replication or directly involved in viral late gene transcription (Hefferon and Kathleen, 2004). LEF5 is a late transcription initiation factor necessary for baculovirus infection and affects the expression of structural genes such as vp80 (Guarino et al., 2002, Harwood et al., 1998). In the lef5-deficient AcMNPV, late transcript levels were significantly decreased without a reduction in early gene expression and viral DNA replication. Meanwhile, no progeny virus was detected after infection of Sf9 cells with the defective virus (Su et al., 2011). The deletion of lef5 in the AcMNPV did not affect viral DNA replication, but greatly reduced the expression of late and very late genes by preventing the late transcriptional cascade, resulting in no progeny virus production. Therefore, lef5 is a promising knockout target for the establishment of virion-free BEVS.

In this study, a lef5-deficient baculovirus insect cell expression system was developed for the production of exogenous protein in insect cells without baculovirus virion contamination (Fig. 1). In this system, a lef5-deficient baculovirus (Ac-Δlef5) was used as the vector. In contrast to conventional BEVS, the defective baculovirus removed the ability of virus packaging and budding while retaining the ability of viral transduction into host cells and viral DNA replication. To package the Ac-Δlef5 virus, a packaging cell line expressing lef5 was developed and several alternative promoters were screened to complement lef5. Various promoters controlling foreign proteins were tested to improve the efficiency of the new system. For availability, Ac-Δlef5-Sf9 and conventional BEVS (Ac-wt-Sf9) were used to express TPO and examine differences between protein secretion and production. In the Ac-Δlef5-Sf9 expression system, the replication of viral DNA provides a rich template for heterologous gene expression, and the reduction of late and very late gene expression removes contamination of progeny viruses.