Role of the IE62 consensus binding site in transactivation by the varicella-zoster virus IE62 protein

Abstract

The varicella-zoster virus (VZV) IE62 protein is the major transcriptional activator. IE62 is capable of associating with DNA both nonspecifically and in a sequence-specific manner via a consensus binding site (5'-ATCGT-3'). However, the function of the consensus site is poorly understood, since IE62 efficiently transactivates promoter elements lacking this sequence. In the work presented here, sequence analysis of the VZV genome revealed the presence of 245 IE62 consensus sites throughout the genome. Some 54 sites were found to be present within putative VZV promoters. Electrophoretic mobility shift assay (EMSA) experiments using an IE62 fragment containing the IE62 DNA-binding domain and duplex oligonucleotides that did or did not contain the IE62 consensus binding sequence yielded K(D) (equilibrium dissociation constant) values in the nanomolar range. Further, the IE62 DNA binding domain was shown to have a 5-fold-increased affinity for its consensus site compared to nonconsensus sequences. The effect of consensus site presence and position on IE62-mediated activation of native VZV and model promoters was examined using site-specific mutagenesis and transfection and superinfection reporter assays. In all promoters examined, the consensus sequence functioned as a distance-dependent repressive element. Protein recruitment assays utilizing the VZV gI promoter indicated that the presence of the consensus site increased the recruitment of IE62 but not Sp1. These data suggest a model where the IE62 consensus site functions to down-modulate IE62 activation, and interaction of IE62 with this sequence may result in loss or decrease of the ability of IE62 to recruit cellular factors needed for full promoter activation.

FIG. 1.

Schematic of the VZV genome indicating the positions of all IE62 consensus sites. Boxes represent regions with sparse or dense consensus sites. Sites were determined to be potentially within promoter regions when positioned within 200 to 300 bp upstream of the translational start site of an open reading frame.

FIG. 2.

Expression of the IE62 DBD and characterization of its DNA-binding Activity. (A) Schematic showing the five regions spanning the length of the IE62 protein and the amino acid sequence of the tagged recombinant IE62-DBD. The region of homology with the helix-turn-helix motif of the Drosophila AntP homeodomain is underlined. (B) Coomassie-stained SDS-PAGE gels of fractions from a typical elution of a nickel-Sepharose column with 300 mM imidazole. (C) Sequence of the 62 consensus, gI promoter, and random oligonucleotide probes used in gel shift assays. Underlining indicates the partial IE62 consensus site within the gI promoter region and the full consensus site within the 62 consensus probe. (D, E, and F) Typical results from EMSAs for the 62 consensus, gI promoter, and random oligonucleotide probes, respectively. The data (•) were fit to a single-site-binding model (○) using the SigmaPlot (Systat) program. DNA binding is shown as the fraction of total probe bound. K_Ds for each probe were calculated using the SigmaPlot program from data obtained from three independent experiments.

FIG. 3.

Insertion of an IE62 consensus site lowers transactivation of the gI promoter in transfection and superinfection experiments. (A) Sequences of the native (top) and mutated (bottom) gI promoter regions. Underlining indicates the positions of the wild-type partial consensus site, the mutated full consensus site, and the atypical gI promoter TATA sequence ATAAA. (B) Results of reporter assays in MeWo cells transfected with 1 μg of the pgI-Luc and pgI-Luc/ATCGT reporter plasmids and 0.01 μg, 0.02 μg, or 0.04 μg of pCMV62. (C) Results of reporter assays in MeWo cells transfected with 1 μg of the pgI-Luc and pgI-Luc/ATCGT reporter plasmids followed by superinfection with VZV-infected cells 24 h following the initial transfection. The luciferase activity from the reporter in the absence of infection or cotransfection was normalized to 1, and experimental activities are reported as n-fold activation over this level.

FIG. 4.

The IE62 consensus site has a position-dependent inhibitory effect on the activity of a minimal model promoter containing only an IE62-responsive TATA box. (A) Consensus site positions in model promoter constructs relative to the TATA box. (B) Results from reporter assays in MeWo cells transfected with 1 μg of the pTA-Luc, p5DS-Luc, p10US-Luc, p20US-Luc, p50US-Luc and p100US-Luc reporter plasmids and 0.01 μg, 0.02 μg or 0.04 μg of pCMV62. (C) Results of reporter assays in MeWo cells transfected with 1 μg of the reporter plasmids followed by superinfection with VZV infected cells 24 h following the initial transfection. The luciferase activity from the reporter in the absence of infection or cotransfection was normalized to 1 and experimental activities are reported as n-fold activation over this level.

FIG. 5.

Tandem consensus sites exacerbate the inhibitory effect seen on IE62 transactivation of a model TATA promoter in transfection experiments. (A) Schematic showing position of the IE62 consensus site relative to the TATA box in the model promoter constructs. (B) Results of reporter assays. One microgram of pTA-Luc, p10US-Luc, p2xUS-Luc, or p3xUS-Luc reporter plasmid was cotransfected with 0.01 μg, 0.02 μg, or 0.04 μg of pCMV62 into MeWo cells. The luciferase activity from the reporters in the absence of infection or cotransfection was normalized to 1, and experimental activities are reported as n-fold activation over this level. (C) Results of reporter assays in MeWo cells transfected with 1 μg of the reporter plasmids followed by superinfection with VZV infected cells 24 h following the initial transfection. The luciferase activity from the reporter in the absence of infection or cotransfection was normalized to 1, and experimental activities are reported as n-fold activation over this level.

FIG. 6.

The ORF 62 promoter sequence. Binding sites for the following viral and cellular factors are underlined: GA-binding protein, CCAAT-binding proteins, ATF/CRE factors, Sp1, and the VZV IE enhancer core (EC) complex composed of Oct-1/HCF-1/ORF10. The three consensus sites are located 267 (1), 252 (2), and 72 (3) base pairs upstream of the reported TATA box. Site 2 is in the opposite orientation relative to sites 1 and 3.

FIG. 7.

Consensus sites suppress transactivation of the ORF62 promoter. The native ORF62 promoter consensus binding sequences (1, 2, and 3) were mutated alone or in unison to generate the m1, m2, m1-2, m3, and m1-2-3 mutant promoters. One microgram of wild-type or mutant reporter plasmid was transfected into MeWo cells with 0.01 μg, 0.02 μg, or 0.04 μg of the pCMV62 expression plasmid (B) or followed by superinfection 24 h posttransfection (A). The luciferase activity from the reporters in the absence of cotransfection was normalized to 1. In superinfection experiments, activity from the wild-type promoter in the presence of infection was set to 1, and experimental activities are reported as n-fold activation over these levels.

FIG. 8.

Consensus sites suppress IE62-mediated transactivation of the ORF62 promoter in primary rat dorsal root ganglion neurons. Two micrograms of either the wild-type or m1-2-3 reporter plasmid was cotransfected with 0.16 μg of pCMV62 expression plasmids into primary DRG neurons. The luciferase activity from the reporters in the absence of cotransfection was normalized to 1. Experimental activities are reported as n-fold activation over this level.

FIG. 9.

Mutation of a native consensus site increases IE62-mediated transactivation of the gC promoter. (A) Position of the IE62 consensus site relative to the gC TATA box. One microgram of wild-type or mutant reporter plasmid was transfected into MeWo cells with 0.01 μg, 0.02 μg, or 0.04 μg of the pCMV62 expression plasmid (C) or followed by superinfection 24 h posttransfection (B). The luciferase activity from the reporters in the absence of infection or cotransfection was normalized to 1, and experimental activities are reported as n-fold activation over this level.

FIG. 10.

(A) Model of IE62-mediated transactivation in the absence of a consensus site. (B) Model of simple increased occupancy of the promoter in the presence of a consensus site, resulting in either increased or decreased transcription depending on whether the site properly positions IE62 to make its protein-protein contacts. (C) Model of increased occupancy coupled with a conformational change which interrupts IE62 protein-protein interactions upon binding to the consensus site, resulting in the reduced activity observed in almost all tested positions within native and model promoters. (D) Results from magnetic bead recruitment assays examining IE62 and Sp1 binding to the wild-type gI and gI62con promoter elements. The presence of Sp1 and IE62 in eluants of bound proteins was detected by immunoblotting with rabbit polyclonal antibodies specific for the two proteins.