The equine herpesvirus type 1 (EHV-1) immediate-early (IE) gene encodes a phosphoprotein that is essential for the activation of transcription from viral early and late promoters and that regulates the transcription from its own promoter. Employment of EHV-1 IE promoter DNA probes and glutathione S-transferase fusion proteins harboring truncated portions of the IE gene product in gel shift assays, super shift assays with anti-IE monoclonal antibodies, and DNase I footprinting analyses revealed: (1) amino acid residues 422 to 597 within the 1487-amino-acid IE protein are sufficient for sequence-specific DNA binding; (2) the IE protein binds to EHV-1 DNA at sequences from -11 to +14 that overlap the transcription initiation site (+1); (3) the conserved pentanucleotide 5'-ATCGT-3' in the IE promoter located at nucleotides (nt) -6 to -2, relative to the transcription initiation site (+1), is critical for IE protein binding; (4) a weak binding site for the IE protein is also present at nt -92 to -82 of the IE gene within the sequence (-86)ATCGA(-82) in which four of the five nt in the consensus binding sequence are conserved; (5) the IE protein binds to sequences in EHV-1 early and late promoters that contain a degenerate version of the consensus sequence 5'-ATCGT-3'; and (6) mutation of the C or G nt in the pentanucleotide 5'-ATCGT-3' prevents sequence-specific binding of the IE protein, whereas mutation of each of the other three nt only reduces binding. These results suggest that the IE protein can recognize the sites which differ slightly from the proposed consensus sequence. Overall, these findings suggest that formation of a specific complex between an IE protein and its own gene promoter may be a common mechanism used by Alphaherpesvirinae to autoregulate transcription of an essential IE gene. In addition, the finding that the DNA binding domain of the IE protein maps within amino acids 422 to 597, a domain conserved in the IR2 early protein that is a truncated form of the IE protein, suggests that the IR2 protein plays a role in the regulation of the IE gene expression.
Rotational reorientation times were obtained for Zranr-stilbene in the series of «-alkanes over a wide temperature range by using picosecond anisotropic absorption measurements and fluorescence depolarization measurements. The data show that the coupling of solute and solvent decreases as the size of the solvent increases. However, in a given solvent the reorientation depends linearly on tj/ T over our temperature range. The results are discussed in the context of the free space model of Dote et al. The new data are used to discuss the applicability of current models for activated barrier crossing to the isomerization of stilbene.
The IR2 protein (IR2P) is a truncated form of the immediate-early protein (IEP) lacking the essential acidic transcriptional activation domain (TAD) and serine-rich tract and yet retaining binding domains for DNA and TFIIB and nuclear localization signal (NLS). Analysis of the IR2 promoter indicated that the IR2 promoter was upregulated by the EICP0P. The IR2P was first detected in the nucleus at 5 h postinfection in equine herpesvirus 1 (EHV-1)-infected HeLa and equine NBL6 cells. Transient-transfection assays revealed that (i) the IR2P by itself downregulated EHV-1 early promoters (EICP0, TK, EICP22, and EICP27) in a dosedependent manner; (ii) the IR2P abrogated the IEP and the EICP27P (UL5) mediated transactivation of viral promoters in a dose-dependent manner; and (iii) the IR2P, like the IEP itself, also downregulated the IE promoter, indicating that the IEP TAD is not necessary to downregulate the IE promoter. In vitro interaction assays revealed that the IR2P interacts with TATA box-binding protein (TBP). The essential domain(s) of the IR2P that mediate negative regulation were mapped to amino acid residues 1 to 706, indicating that the DNA-binding domain and the NLS of the IR2P may be important for the downregulation. In transienttransfection and virus growth assays, the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector. Overall, these studies suggest that the IR2P downregulates viral gene expression by acting as a dominant-negative protein that blocks IEP-binding to viral promoters and/or squelching the limited supplies of TFIIB and TBP.The equine herpesvirus 1 (EHV-1) genome is comprised of 78 genes that are coordinately regulated and temporally expressed as immediate-early (IE), early (E), and late (L) ␥1 and true late ␥2 genes (10,16,17). The coordinated transcription of EHV-1 genes is regulated by six regulatory proteins that are expressed as one IE protein (IEP), four early proteins (EICP22P, EICP27P, EICP0P, and IR2P), and the late protein ETIF (5-7, 10, 13, 16-18, 20, 22, 24-26, 34-37). In addition, our ongoing work indicated that the IR3 transcript that is antisense to a portion of the IE mRNA negatively regulates IE gene expression (21; B. C. Ahn, S. K. Kim, and D. J. O'Callaghan, unpublished data).During a lytic infection, two transcripts arise from the IE open reading frame: a single, spliced 6.0-kb IE mRNA and a 3Ј-coterminal 4.4-kb early IR2 mRNA (20). The IEP (1,487 amino acids [aa]) is the major regulatory protein of EHV-1 and mediates the activation of transcription from E and some L viral promoters (34, 35) and is, thus, essential for replication (15). At the same time, the IEP downregulates its own promoter (34) and represses transcription of the true late glycoprotein K (gK) gene by binding to the transcription initiation site of the gK promoter (24). The IEP binds to the consensus binding sequence 5Ј-ATCGT-3Ј that overlaps the transcription initiation site of the IE promoter and to degenerate versions of this cognate...
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