In recent years, baculovirus has emerged as a tool for high-efficiency gene transfer into mammalian cells. However, the level of gene expression is often limited by the strength of the mammalian promoter used. Here, we show that the baculovirus RING protein IE2 is a strong, promiscuous trans-activator in mammalian cells, dramatically upregulating the cytomegalovirus (CMV) promoter in both Vero E6 and U-2OS cells. Further study of the cellular mechanism for the activation led to the discovery of a novel IE2 nuclear body structure which contains a high concentration of G-actin and closely associates with RNA polymerase II, PML, and SUMO1. IE2 mutagenesis studies indicated that the RING and coiled-coil domains of IE2 were necessary for nuclear body formation, as well as for strong activation of the CMV promoter in mammalian cells. Overall, this study shows that the IE2 trans-activator could significantly advance the use of baculovirus in mammalian gene transfer and protein production.

The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

Since the discovery of mammalian cells susceptible to baculovirus transduction in 1995, baculovirus has emerged as a safe, economical and convenient tool for introducing foreign genetic material into mammalian systems. The feasibility of baculovirus as a novel gene transfer vehicle for large scale mammalian protein expression was not obvious at first, as the baculovirus genome is unable to amplify within mammalian cells and expression of the transferred foreign gene does not usually last more than 20 days. However, several recent articles have demonstrated that baculovirus contains both the elements - hr sequences, and the factor - IE2 protein, which can function in mammalian cells to dramatically boost baculovirus-transduced gene expression. The longevity of genes transferred by baculovirus can also be extended by inserting a replication ori with the necessary factors and induce self-replicating episomes. Lastly, peptide-based baculovirus surface modification and two groups of drugs: histone deacetylase inhibitors and microtubule depolymerizing agents, which improve baculovirus gene expression were described. In this article we summarized ways to activate, enhance, and prolong baculovirus-mediated foreign protein expression. The mechanism behind IE2 activation through its unique nuclear body structure was given special emphasis, including our most current data which suggested PML NBs could be disrupted by IE2. Finally, strategies to maximizing mammalian protein production through baculovirus gene transfer are proposed in prospect to current breakthroughs.

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