Transcription factor Sp1 mediates cell-specific trans-activation of the human cytomegalovirus DNA polymerase gene promoter by immediate-early protein IE86 in glioblastoma U373MG cells

Abstract

Human cytomegalovirus (HCMV) gene expression is highly cell and tissue specific. Cell factor-mediated regulatory interactions are involved in regulating the restricted expression of the HCMV major immediate-early (IE) gene (J. F. Baskar, P. P. Smith, G. Nilaver, R. A. Jupp, S. Hoffmann, N. J. Peffer, D. J. Tenney, A. M. Colberg-Poley, P. Ghazal, and J. A. Nelson, 70:3207-3213, 1996). To gain an understanding of HCMV early gene activation, we studied the effect of each of the three major IE proteins, IE72, IE86, and IE55, on the HCMV DNA polymerase gene (pol; UL54) promoter. In transient-expression assays, the IE86 protein alone was able to transactivate the pol promoter, but IE72 and IE55 were not, in permissive U373MG cells. However, we were unable to detect IE86-mediated transactivation in nonpermissive HeLa or C33-A cells. Using electrophoretic mobility shift assays (EMSAs), we found that expression of the IE86 protein in U373MG cells resulted in specific binding of a DNA complex to an inverted-repeat element, IR1, of the pol promoter. Antibody supershifting and EMSA-Western blotting experiments further showed that IE86 and the cellular transcription factor Sp1 were components of the IR1 DNA-binding complex. Furthermore, we found that binding of DNA by Sp1 was dramatically increased in the presence of IE86. Interestingly, this IE86-induced DNA-binding activity of Sp1 was inhibited by a repressor activity presented in HeLa cells. In summary, our study suggests that a viral regulatory protein can modulate the DNA binding activity of a cellular transcription factor, resulting in cell-specific transactivation of viral genes.

FIG. 1

The IE86 protein is essential for the activation of the HCMV DNA polymerase (pol) gene promoter. U373MG cells were cotransfected with the pol-luciferase reporter and increasing amounts of the IE gene expression vectors RSVIE86, RSVIE72, and RSVIE55 and a β-galactosidase control vector. Luciferase activity was normalized to β-galactosidase activity. The data represent the results of three independent experiments.

FIG. 2

Transactivation of the pol promoter by IE86 in the permissive U373MG and primary HFF cells. The pol-luciferase (pol-luc) and β-galactosidase reporters were cotransfected with increasing amounts of the RSVIE86 expression vector in permissive U373MG (A) and HFF (B) cells and in nonpermissive HeLa (C) and C33-A (D) cells as indicated. Luciferase activities were normalized to β-galactosidase activity. Units are fold activation. The data represent the results of three independent experiments.

FIG. 3

Transactivation of the UL112 promoter by IE86. The UL112-luciferase (UL112-luc) and β-galactosidase reporters were cotransfected with increasing amounts of the RSVIE86 expression vector in permissive U373MG (A) and HFF (B) cells and in nonpermissive HeLa (C) and C33-A (D) cells as indicated. Luciferase activities were normalized to β-galactosidase activity. Units are fold activation. The data represent the results of three independent experiments.

FIG. 4

Activation of the pol promoter in the U373MG stable cell line expressing IE86. The U373MG parental line and U373-IE86 stable cell line (A) or the HeLa parental line and HeLa-IE86 stable cell line (B) were transfected with increasing amounts of the pol-luciferase (pol-luc) reporter. Cells were harvested, and luciferase activity was assayed. The levels of expression of IE86 protein in the stable cell lines (C and D) were determined by Western blotting with a monoclonal antibody specific for the HCMV IE proteins.

FIG. 5

Activation of the UL112 promoter in U373-IE86 and HeLa-IE86 stable cell lines. The U373MG parental cell line and U373-IE86 stable cell line (A) or the HeLa parental line and HeLa-IE86 stable cell line (B) were transfected with increasing amounts of the UL112-luciferase (UL112-luc) reporter. Cells were harvested, and luciferase activity was assayed. The levels of expression of IE86 protein in the stable cell lines (C and D) were determined by Western blotting with a monoclonal antibody specific for the HCMV IE proteins.

FIG. 6

Identification of an IR1-specific DNA-binding complex in U373MG cells expressing IE86. Five-microgram quantities of nuclear extract from U373MG and U373-IE86 (A) or HeLa and HeLa-IE86 (B) cell lines were incubated with ³²P-labeled IR1 oligonucleotide. IR1 binding was analyzed by EMSA. (C) U373MG-IE86 nuclear extract was incubated with labeled IR1 oligonucleotide and a 50-fold excess of either unlabeled wild-type (IRwt) or mutant (IRmut) IR1 oligonucleotides. The arrows indicate the specific complex. NS, nonspecific complexes.

FIG. 7

IE86 antibody recognizes the IR1-DNA complex-bound IE86 protein. (A) U373-IE86 nuclear extract was incubated with labeled IR1 oligonucleotide and either a monoclonal antibody specific for the HCMV IE proteins (MAb 810) or polyclonal antibodies specific for NF-κB p65 and p50 subunits (p65 Ab and p50 Ab, respectively). U373-IE86 with the IR1 probe was used as a control. (B to D) EMSA was done as a control (see panel A, lane 1), and then the gel was blotted onto DEAE (B) and nitrocellulose (C and D) membranes. The nitrocellulose membranes were subsequently probed with a monoclonal antibody specific for IE86 (see Materials and Methods). U373-IE86 extracts and recombinant IE86 protein (rIE86) alone were used as controls in EMSA-Western blot analysis (D). Arrows indicate the IR1 DNA-binding complex (A and B) and IE86 protein present in the IR1 DNA-binding complex (C and D).

FIG. 8

Neither CRS nor EAIE2 IE86 cis elements can compete with the IR1-DNA complex. Nuclear extracts isolated from the U373-IE86 stable cell line were incubated with a radiolabeled IR1 oligonucleotide and a 50-fold excess of unlabeled CRS or EAIE2 competitor oligonucleotides as indicated. The arrow indicates the IR1 complex. wt, wild type; mut, mutant.

FIG. 9

The cellular transcription factor Sp1 binds to the IR1 element. U373-IE86 nuclear extracts were incubated with a radiolabeled IR1 oligonucleotide and a 50-fold excess of unlabeled IR1, CREB, or Sp1 competitor oligonucleotides (panel A, lanes 2 to 4, respectively) or 1 μg of polyclonal antibodies specific for ATF, CREB, or Sp1 (panel B, lanes 2 to 4, respectively). U373-IE86 nuclear extract alone was used as a control (lanes 1). The unlabeled arrows indicate specific complexes. SS, supershifted complex.

FIG. 10

IE86 protein enhances the DNA binding activity of Sp1. (A) U373MG (lane 2), U373-IE86 (lane 3), HeLa (lane 5), and HeLa-IE86 (lane 6) cell nuclear extracts were incubated with a radiolabeled Sp1 consensus oligonucleotide. (B) U373-IE86 nuclear extracts were incubated with the same Sp1 probe and a 50-fold excess of an unlabeled oligonucleotide (oligo) or polyclonal antibodies (Ab) as indicated. The unlabeled arrows indicate the Sp1 DNA-binding complex. SS, supershifted complex.

FIG. 11

The factor(s) present in HeLa cells inhibits IE86-mediated DNA-binding activity of Sp1. U373-IE86 nuclear extract (2.5 μg) was incubated with radiolabeled IR1 (A) or Sp1 (B) consensus oligonucleotides, respectively. Increasing amounts of HeLa (lanes 2 to 4) or HeLa-IE86 (lanes 5 to 7) nuclear extract were added as indicated. U373-IE86 nuclear extract plus probe was used as a control (Lanes 1). The arrows indicate Sp1 DNA-binding complex.