RING and Coiled-Coil Domains of Baculovirus IE2 Are Critical in Strong Activation of the Cytomegalovirus Major Immediate-Early Promoter in Mammalian Cells

Abstract

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.

Baculovirus IE2 stimulates the expression of heat shock proteins in insect and mammalian cells to facilitate its proper functioning

“…With the use of baculovirus IE2, expression of a foreign gene can be significantly boosted in some mammalian systems to further advance the application of baculoviruses [4]. We have identified the first viral factor that induces HSP expression upon baculovirus infection.…”

“…Baculovirus IE2-an immediate early gene product-is responsible for turning on early and late genes during virus infection in insect cells [15][16][17]. Our previous study also found that IE2 forms novel nuclear body structures where transcription-related factors, such as RNA Pol II and actin, are concentrated for the strong activation of promoters in mammalian cells [4]. In this study, by purifying the IE2 nuclear bodies, we identified heat shock proteins as IE2 interaction proteins, which are crucial for the proper functioning of IE2.…”

“…Because we had previously determined that the formation of intact nuclear bodies was crucial for trans-activation activity [4] and that HSPs are usually important for maintenance of target protein conformations, we conducted immunofluorescence studies of the IE2 structure in the nucleus. Interestingly, treatment with 1 μM KNK437 was the lowest concentration at which we could still clearly observe the formation of regular IE2 nuclear The results show that IE2 trans-activation activity on the CMV promoter was down-regulated in a dose-dependent manner.…”

Identification of a High-Efficiency Baculovirus DNA Replication Origin That Functions in Insect and Mammalian Cells

“…Since 1995 (34), baculovirus has been used as a valuable and efficient tool for gene expression, tissue engineering, and gene therapy in mammalian cells or organisms (25,35). Further enhancement of baculovirus gene expression in these cells could be very useful for applications.…”

“…The PCR fragments were inserted downstream of the mammalian cytomegalovirus (CMV) immediate early 1 promoter (p-CMV) on plasmid pAcL (25) to generate plasmids pAcLhr1, pAcLp143-3, pAcLp143-3.2, and pAcLp143-3.2a. All the constructs were verified by sequencing of the entire inserted fragments.…”