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...

An amplified colliding pulse modelocked laser system is described which is capable of subpicosecond resolution flash-photolysis studies. As an application of this system we used the anisotropic absorption technique (polarization spectroscopy) to measure the rotational reorientation of trans-stilbene. These measurements give a more accurate estimate of the microscopic friction involved in the isomerization dynamics of this molecule. When the friction is assumed proportional to the reorientation times, the isomerization rates in alkane solvents are adequately described by activated barrier-crossing theory.

For an A + BC -+ AB+C reaction, the partitioning of the total angular momentum J between initial and final orbital momenta of the collision partners, 1 and l', and rotation of the reactant and product molecules, j and j ' , often imposes distinctive dynamical features. Many examples are known that approach one or another of two very different limiting cases: ( I ) J = 1 = j ' or (11) J = I = 1'. In a quasiclassical trajectory study of the H + Li2 -+ LiH + Li reaction, we find the distributions of the product scattering angle and the rotational orientation of j ' relative to the initial velocity conform to case 11. However, the dihedral orientation of j ' relative to the plane of initial and final velocity vectors conforms to case I. Also, the opacity function has an unusual bimodal form and the entropy deficiency for the triple-angle correlation is extremely large. The cu1prit.k vector addition: both j ' and I' are large and largely opposed, so the reaction appears schizophrenic.

The equine herpesvirus 1 immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and trans-represses its own promoter. Transient-transfection assays showed that the IE protein trans-represses the gamma2 late gK promoter. Gel shift and DNase I footprinting assays demonstrated that the IE protein binds to the gK promoter sequences from -42 to -26 and from -13 to +12 that overlap the transcription initiation site (+1). These results indicated that the IE protein binds to the transcription initiation site of the gK promoter sequences, thereby repressing transcription. On the other hand, the EICP0 protein trans-activates the gamma2 late gK promoter [Bowles, D. E., Holden, V. R., Zhao, Y., and O'Callaghan, D. J. (1997). The ICP0 protein of equine herpesvirus 1 is an early protein that independently transactivates expression of all classes of viral promoters. J. Virol. 71, 4904-4914]. Overall, the EICP0 protein is able to release the gK promoter from the repressive effects of the IE protein. It has not been previously demonstrated that the major immediate-early transcriptional regulator of a herpesvirus represses expression of a late gene during infection.

No abstract

The EICP0 protein of equine herpesvirus 1 (EHV-1) is an early, viral regulatory protein that independently trans-activates EHV-1 immediate-early (IE), early, ␥1 late, and ␥2 late promoters. To assess whether this powerful trans-activator functions in conjunction with three other EHV-1 regulatory proteins to activate expression of the various classes of viral promoters, transient cotransfection assays were performed in which effector plasmids expressing the EICP22, EICP27, and IE proteins were used either singly or in combination with an EICP0 effector construct. These analyses revealed that (i) independently, the EICP0 and IE proteins are powerful trans-activators but do not function synergistically, (ii) the IE protein inhibits the ability of the EICP0 protein to trans-activate the IE, ␥1 late, and ␥2 late promoters, (iii) the EICP22 and EICP0 proteins do not function together to significantly trans-activate any EHV-1 promoter, and (iv) the EICP27 and EICP0 proteins function synergistically to trans-activate the early and ␥1 late promoters. A panel of EICP0 truncation and deletion mutant plasmids was generated and used in experiments to define the domains of the 419-aminoacid (aa) EICP0 protein that are important for the trans-activation of each class of EHV-1 promoters. These studies revealed that (i) carboxy-terminal truncation mutants of the EICP0 protein exhibited a progressive loss of trans-activating ability as increasing portions of the carboxy terminus were removed, (ii) the amino terminus of the EICP0 protein containing the RING finger (aa 8 to 46) and the acidic region (aa 71 to 84) was necessary but not sufficient for activation of all classes of EHV-1 promoters, (iii) the RING finger was absolutely essential for activation of EHV-1 promoters, since deletion of the entire RING finger motif (aa 8 to 46) or a portion of it (aa 19 to 30) completely abrogated the ability of these mutants to activate any promoter tested, (iv) the acidic region contributed to the ability of the EICP0 protein to activate the early and ␥1 late promoters, and deletion of the acidic region enhanced the ability of this mutant to activate the IE promoter, (v) the carboxy terminus (aa 325 to 419), which is rich in glutamine residues, was dispensable for the EICP0 trans-activation function, (vi) a motif resembling a nuclear localization signal (aa 289 to 293) was unnecessary for the EICP0 protein to trans-activate promoters of any temporal class, and (vii) the EICP0 protein was phosphorylated during infection, and deletion of the serine-rich region (aa 210 to 217), a potential site for phosphorylation, reduced by more than 70% the ability of the EICP0 protein to activate the ␥2 late class of promoters.