Characterization of the trans -Activation Properties of Equine Herpesvirus 1 EICP0 Protein

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.

Mapping the Sequences That Mediate Interaction of the Equine Herpesvirus 1 Immediate-Early Protein and Human TFIIB

“…7B). Consistent with our previous findings (3,32), the wild-type IE protein activated expression of the IR5 promoter approximately 3.2-fold alone and 12.2-fold in combination with the auxiliary proteins over basal levels (pIR5-CAT alone). Expression of pIR5-CAT was not observed in the presence of the IE⌬407-757 mutant, which lacks the TFIIB-binding domain.…”

“…The EHV-1 IE gene (i) is located within each invertedrepeat region and encodes a polypeptide of 1,487 amino acids (aa) with a predicted molecular mass of approximately 155 kDa (19,21,27), (ii) has a product with a high degree of homology with HSV-1 ICP4 and the varicella-zoster virus ORF62 gene products (21), and (iii) is transcribed as a 6.0-kb spliced mRNA (19,27,51) that gives rise to both structurally and antigenically related protein species ranging from 125 to 200 kDa (7,8,51). In transient-cotransfection assays, the IE protein is a bifunctional regulatory protein capable of (i) negatively autoregulating its own promoter (55), (ii) independently activating EHV-1 early and heterologous viral promoters (55, 56), (iii) cooperating synergistically with two early auxiliary regulatory proteins (EICP22 and EICP27) to activate EHV-1 early and ␥1 late promoters (32,44,55,57,64), and (iv) acting antagonistically with a third early major regulatory protein, EICP0, to selectively repress expression of certain promoters from all classes of EHV-1 promoters, including ␥2 late promoters (3,35).Sequence alignment of the EHV-1 IE protein and other homologs in the subfamily Alphaherpesvirinae defined five colinear regions that harbor specific functional domains. Region 1 contains an acidic transactivation domain (TAD; aa 3 to 89) (58) and a serine-rich tract (SRT; aa 181 to 220).…”

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

“…Our previous studies showed that the EICP0 protein interacts directly with basal transcription factors TFIIB and TBP as well as the IE protein (35) and contains a cysteine-rich zinc RING finger near the N terminus that is essential for activation of the E and L promoters (3). Of the many possibilities that may explain how NREBP abolishes the EICP0 protein's transactivation of its own promoter, one possible mechanism is that NREBP abolished the EICP0 protein's trans-activation by blocking or squelching its interaction with TFIIB and TBP.…”

“…Previous studies in our laboratory showed that the EICP0 protein is a powerful trans-activator that activates all classes of EHV-1 promoters (3,4,34). To determine whether the EICP0 protein trans-activates the EICP0 promoter, the pEICP0-CAT reporter plasmid was used for transient transfection assays.…”

“…more than 70% (3). The regulatory functions of the EICP0 protein are severely antagonized by the immediate-early (IE) protein (35), and thus the EHV-1 EICP0 protein differs from HSV-1 ICP0, which functions synergistically with ICP4 to activate expression of the HSV-1 E and L promoters (8,15).…”