Conserved structural motifs at the C-terminus of baculovirus protein IE0 are important for its functions in transactivation and supporting hr5-mediated DNA replication

Abstract

IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

Keywords: IE0 transactivator; baculovirus; functions; mutagenesis.

Figure 1

IE0 transactivation of the 39K promoter. Sf9 1 × 10⁵ cells were transfected with 250 ng pQ39lacZ and increasing amounts of phspie0M (indicated at the bottom). All the transfections were normalized to 750 ng by addition of pBSK. LacZ activity (OD 405 nm) in the cell extracts was determined by standard methods. Results represent duplicate transfections. Bars: standard error.

Figure 2

IE0-mediated replication of the hr5-containing plasmid pQ35. Sf9 cells were co‑transfected with the replication library containing 500 ng of each plasmid without (lane 1), or with 250 ng of phspie0M (lane 2) or phspie1 (lane 3). Total intracellular DNA was isolated at 72 h posttransfection, digested with DpnI, linearized with XhoI, electrophoresed on a 0.7% agarose gel, and transferred to a GeneScreen Hybridization Transfer Membrane. Hybridization was performed with a Fluorescein dUTP-labeled pQ35 probe. The replicated complexes were detected by chemiluminescence. The samples were digested with XhoI and DpnI. Asterisk, replicated pQ35.

Figure 3

IE0 localizes to the cell nucleus. (a) and (b) Mock-infected (m.i.) lysed at 24 h- and AcMNPV-infected Sf9 cell extracts collected and lysed at 4, 8 and 24 h post infection. The polypeptides were separated by PAGE and subjected to immunoblot analysis with anti-IE0 (panel a) or anti-IE1/IE0 antiserum (panel b) as described in Experimental section. (c) and (d) Confocal microscopy analysis of AcMNPV- and mock-infected Sf9 cells, respectively, fixed at 16 h post treatment, incubated with anti-IE0 antiserum and decorated with anti-rabbit IgG fraction Alexa Fluor 488 (F, green); DAPI staining (blue). B F, bright field. Bars, 10 µm. (e) Biochemical fractionation of nuclei and cytoplasm from Sf9 cells infected with AcMNPV, vAcGSTIE1 or vAcGFP: cell extracts subjected to Western blot analysis utilizing anti-IE0, anti-GST and anti-GFP antiserum, respectively. Molecular markers are indicated on the left.

Figure 4

Cellular localization of C-terminal mutants of IE0. (a) Western blot analysis of Sf9 cells transfected with the plasmid constructs phspIE0M, p578/580, p591/592, p597/601 or p604/608 (indicated above the figure). (b) Confocal microscopy analysis. Sf9 cells were transfected with the plasmids p578/580, p591/592, p597/601 or p604/608 (arrows) or mock-transfected (Sf9) and treated as described in Figure 3. The fluorescence and bright light channels are indicated at the bottom. Bars, 10 µm.

Figure 5

Transactivation of the 39K promoter by C-terminal mutants of IE0. Fold of transactivation of lacZ expression from cells cotransfected with 250 ng pQ39lacZ and 80 ng of each the constructs indicated at the bottom. The samples were processed as indicated in Figure 1. Results represent duplicate transfections. Bars: standard error.

Figure 6

The ability of the various C-terminal mutants of IE0 to mediate replication of the hr5-containing plasmid pQ35. Sf9 cells were co-transfected with the replication library plasmids as described in Figure 1 (500 ng of each plasmid) with 250 ng of phspie0M or each of the mutants indicated above the figure. Total intracellular DNA was isolated at 72 h posttransfection, digested with DpnI, linearized with XhoI, electrophoresed on a 0.7% agarose gel, and transferred to a GeneScreen Hybridization Transfer Membrane. Hybridization was performed with a Fluorescein dUTP-labelled pQ35 probe. The replicated complexes were detected by chemiluminescence. The samples were digested with XhoI and DpnI. Asterisk, replicated pQ35.

Figure 7

Effect of co-transfection of C-terminal mutants of IE0 and IE0 on lacZ expression from the 39K promoter. Sf9 cells were transfected with pQ39lacZ (250 ng) and phspie0M alone (160 ng), or with phspie0M (80 ng) and each one of the constructs indicated below the figure (80 ng). All the transfections were treated as described above. Results represent duplicate transfections. Error bars represent standard errors.