An in vitro system for human cytomegalovirus immediate early 2 protein (IE2)-mediated site-dependent repression of transcription and direct binding of IE2 to the major immediate early promoter.

Abstract

In vivo, negative autoregulation of the strong major immediate early promoter (MEEP) of human cytomegalovirus requires the viral immediate early 2 protein (IE2) and a cis element located from position -13 through position -1 relative to the transcription start site. We have established an in vitro transcription system that reproduces the specificity of EE2-mediated negative autoregulation. [12][13][14].The precise mechanism of IE2-mediated repression is not yet understood. We have expressed the carboxyl-terminal 290 amino acids of IE2 as a bacterial fusion protein and have established an in vitro system that reproduces the specificities of repression as defined by in vivo studies. In addition, using conditions functionally tested by the in vitro transcription system, we have performed gel mobility-shift assays that provide evidence that the carboxyl-terminal 290 amino acids of IE2 are sufficient to mediate direct and site-dependent binding to the MIEP. Our data suggest that the mechanism for IE2-mediated repression of the MIEP involves direct interaction between the wild-type IE2 and the cis-acting repressor element.MATERIALS AND METHODS Recombinant Plasmids. To produce the pKS760E (wildtype promoter) and pKSd1REE (mutant promoter) templates used in run-off transcription assays, the 1-kilobase (kb) EcoRI fragment from pCAT760 (2) or pCAT760d1RE (12), respectively, was subcloned into the EcoRI site of the pBluescript II phagemid vector KS+ (Stratagene).Constructs used to produce recombinant proteins were based on the plasmid vector pMAL-c (New England Biolabs), which expresses the cloned sequence of interest fused to the Escherichia coli maltose binding protein by induction with isopropyl P-D-thiogalactoside (IPTG). Plasmid pMALcXS has a 1.4-kb Xho I (filled-in with Klenow fragment)/Sal I fragment from exon 5 of IE2 inserted into the Stu I/Sal I cloning sites of pMAL-c. Plasmid pMALcHL was constructed in the same manner as pMALcXS except that the 1.4-kb insert was isolated from the mutant plasmid HL446,452 (K. C. Yeung, M. Leatham, and M.F.S., unpublished work).Purification of Recombinant Proteins. The protein fusion and purification system was obtained from New England Biolabs and used according to manufacturer instructions with the following modifications: (i) batch purification was performed and (ii) the elution buffer was 10 mM Hepes, pH 8.0/10%o (vol/vol) glycerol/0.1 mM EDTA/30 mM maltose.Preparation of HeLa Cell Nuclear Extracts. Nuclear extracts were prepared by a modification of the method of Lee and Green (15). Extracts were diluted, rather than dialyzed, with an equal volume of buffer D-at the final step (16). In some experiments, the HeLa cell nuclear extracts were obtained from Promega.In

Small Internal Deletions in the Human Cytomegalovirus IE2 Gene Result in Nonviable Recombinant Viruses with Differential Defects in Viral Gene Expression

“…The aa 356 to 359 deletion removes a region that has been shown to be important for the activation of the viral early promoter for the UL54 gene (65), and the arginine-toalanine substitution mutant similarly disrupts this region without introducing a deletion that might result in a large-scale alteration of the IE2 86 structure. Removal of aa 427 to 435 disrupts a predicted zinc finger, which when altered in other studies resulted in a protein that can activate transcription of the MIE region but cannot bind DNA or autorepress transcription (34,42). Finally, the aa 505 to 511 deletion mutant is missing part of a leucine-rich region that is predicted to form a helix-loop-helix structure (21).…”

“…IE2 86 is thought to bind DNA through interactions with the minor groove, a notable example being its binding to the 14-bp cis-repression signal (CRS) between the TATAA box and the transcription start site in the MIEP. It has been shown that this binding is the source of IE2 86's negative regulatory effect on its own transcription (13,31,39,40,42,50). In addition, IE2 86 binds to similar 14-bp sites upstream of the TATAA box in early promoters, including the UL112-113 (2.2-kb RNA) and 1.2-kb RNA promoters (5,12,59,60).…”

The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation

“…Our data indicate that the regions of IE2 that are important for binding TBP and TFIIB lie within this essential residues 195 to 579 region of exon 5, whereas the residues 98 to 194 region of IE2 which is not required for trans-activation also does not contribute to binding of TBP or TFIIB. Moreover, it has recently been shown that the C-terminal half of IE2 (amino acids 290 to 579) is sufficient to mediate specific repression of transcription from the HCMV major IE promoter in vitro (Macias & Stinski, 1993). This IE2 construct resembles the late isoform of IE2 (comprising amino acids 242 to 579) which possesses autoregulation but not trans-activation function (Malone et al, 1990;Pizzorno et al, 1991).…”

“…Additionally, the recent observation that IF2 can bind DNA directly at the cis repression signal between the TATA box and cap site of the HCMV major IE promoter-enhancer, thereby effecting negative autoregulation (Lang & Stamminger, 1993 ;Macias & Stinski, 1993), might suggest that trans-regulation of other promoters by IF2 may also be mediated by direct binding of IF2 to DNA. …”

“…These proteins, the 72K IE1 and the 86K IE2, are known to autoregulate their expression. IE1 positively regulates its own expression via 18 base pair repeats in the IE enhancer (Cherrington & Mocarski, 1989), whereas IE2 negatively autoregulates (Pizzorno et al, 1988) by binding directly to the cis repression signal between the cap site and TATA box of the IE promoter (Lang & Stamminger, 1993;Macias & Stinski, 1993). In adition, both proteins are independently able to trans-activate a number of minimal homologous (i.e.…”