Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

Abstract

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and ultimately affect viral replication.

Baculovirus IE2 Interacts with Viral DNA through Daxx To Generate an Organized Nuclear Body Structure for Gene Activation in Vero Cells

“…15A). Since IE2 nuclear bodies have been well reported to diminish after 8 h after virus infection (hpi) in insect cells (22,(57)(58)(59), we cannot study later association and disassociation events among these proteins as we have done for the mammalian system. We reason that the reduction of IE2 nuclear bodies in insect cells may arise from the effects of other viral genes, e.g., IE1 (59,60) or other gene products, during baculovirus infection.…”

“…Cell lines and cell culture. Vero E6 (ATCC CRL1586) derived from vervet monkey (Cercopithecus aethiops) kidney cells and Spodoptera frugiperda IPLB-Sf21 (Sf21) cells were cultured as previously described (23,57). Briefly, Vero E6 cells were cultured with MEM-alpha medium (Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) and maintained in a humidified incubator with 5% CO 2 at 37°C.…”

“…Plasmid and virus construction. The ie2 gene was amplified from the AcMNPV genome and cloned into pTriEx-3 plasmid (Novagen), which contains p10, CMV, and T7 promoters for expression in insect, mammalian, and bacterial cells (23,57). Recombinant baculoviruses vAcIE2, vAcC230S, and vAcEGFP, which express wt IE2, RING domain mutant IE2, and EGFP, respectively, by the TriEx promoter (Novagen) were generated as previously described (23).…”

“…All IE2 mutants contained His tags at their C-terminal regions and were cloned into the pTriEx-3 vector to generate pAcSIM-66, pAcSIM-108, and pAcSIM-DM, respectively. The luciferase gene was obtained by PCR from pTre-luc plasmid as previously described (23,57) and inserted into pTriEx-3 to generate the pAcLuc vector. The pAcSIM-66, pAcSIM-108, pAcSIM-DM, and pAcLuc vectors were cotransfected with FlashBAC (a modified AcMNPV baculovirus genome; Mirus) into Sf21 cells by Cellfectin (Thermo Fisher; 10362100).…”

“…The pAcSIM-66, pAcSIM-108, pAcSIM-DM, and pAcLuc vectors were cotransfected with FlashBAC (a modified AcMNPV baculovirus genome; Mirus) into Sf21 cells by Cellfectin (Thermo Fisher; 10362100). The resulting recombinant baculoviruses-vAcSIM-66, vAcSIM-108, vAcSIM-DM, and vA-cLuc-were isolated through endpoint dilutions as described previously (23,57). For the insect experiment, the baculovirus enhancer sequence hr1 (homologous region 1) was obtained by PCR using 2ϫ Ultra Hi-Fi PCR Master Mix (TTC-PE15, TOOLS) as previously described (23,57) and inserted into the pAB-6ϫHis vector (Invitrogen).…”