Characterization of the trans-activation properties of equine herpesvirus 1 EICP0 protein

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, gamma1 late, and gamma2 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, gamma1 late, and gamma2 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 gamma1 late promoters. A panel of EICP0 truncation and deletion mutant plasmids was generated and used in experiments to define the domains of the 419-amino-acid (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 gamma1 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 gamma2 late class of promoters.     

A negative regulatory element (base pairs -204 to -177) of the EICP0 promoter of equine herpesvirus 1 abolishes the EICP0 protein's trans-activation of its own promoter

The early EICP0 protein is a powerful trans-activator that activates all classes of equine herpesvirus 1 (EHV-1) promoters but, unexpectedly, trans-activates its own promoter very weakly. Transient transfection assays that employed constructs harboring deletions within the EICP0 promoter indicated that EICP0 cis-acting sequences within bp -224 to -158 relative to the first ATG abolished the EICP0 protein's trans-activation of its own promoter. When inserted into the promoters of other EHV-1 genes, this sequence also downregulated activation of the immediate-early IE(-169/+73), early thymidine kinase TK(-215/+97), and late glycoprotein K gK(-83/+14) promoters, indicating that the cis-acting sequence (-224 to -158) downregulated expression of representative promoters of all classes of EHV-1 genes and contains a negative regulatory element (NRE). To define the cis-acting element(s), three synthetic oligonucleotides (Na [bp -224 to -195], Nb [bp -204 to -177], and Nc [bp -185 to -156]) were synthesized and cloned upstream of the EICP0(-157/-21) promoter. Of the three synthetic sequences, only the Nb oligonucleotide caused the downregulation of the EICP0 promoter. The NRE was identified as a 28-bp element to lie at -204 to -177 that encompassed the sequence of ([-204]AGATACAGATGTTCGATAAATTGGAACC[-177]). Gel shift assays performed with mouse L-M, rabbit RK-13, and human HeLa cell nuclear extracts and gamma-(32)P-labeled wild-type and mutant NREs demonstrated that a ubiquitous nuclear protein(s) (NRE-binding protein, NREBP) binds specifically to a sequence (bp -193 to -183) in the NRE. The NREBP is also present in the nucleus of EHV-1-infected cells; however, the amount of NREBP in EHV-1-infected L-M cells that bound to the Nb oligonucleotide was reduced compared to that in uninfected L-M cells. Transient transfection assays showed that deletions or mutations within the NREBP-binding site abolished the NRE activity of the EICP0 promoter. These results suggested that the NREBP may mediate the NRE activity of the EICP0 promoter and may function in the coordinate expression of EHV-1 genes.     

The EICP22 protein of equine herpesvirus 1 physically interacts with the immediate-early protein and with itself to form dimers and higher-order complexes

The EICP22 protein (EICP22P) of Equine herpesvirus 1 (EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.     

Regulatory function of the equine herpesvirus 1 ICP27 gene product.

The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides -123 to +20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.     

The extreme carboxyl terminus of the equine herpesvirus 1 homolog of herpes simplex virus VP16 is essential for immediate-early gene activation.

Gene 12 of equine herpesvirus 1 (EHV-1), the homolog of herpes simplex virus (HSV) VP16 (alpha TIF, Vmw65), was cloned into a eukaryotic expression vector by PCR and used in transactivation studies of both the EHV-1 and HSV-1 IE1 promoters. Results demonstrated that the product of gene 12 is a potent transactivator of immediate-early gene expression of both viruses, which requires sequences in the upstream HSV-1 promoter for activity. Mutational analysis of the gene 12 open reading frame indicated that removal of the C-terminal 7 amino acids, which contain a short region of homology with the extreme C terminus of VP16, inactivated the protein. Within this region, only a single methionine residue appeared to be essential for activity, implying that gene 12 may have a modular array of organization similar to that of VP16. However, fusion of the gene 12 C terminus to a truncated form of VP16, which contained the complex formation domain, did not restore activity to the HSV-1 protein. These data demonstrate that the EHV-1 immediate-early transactivator may not be functionally colinear with VP16, with transactivation requiring both the C terminus and another region(s) present within the N-terminal portion.