The γ2 Late Glycoprotein K Promoter of Equine Herpesvirus 1 Is Differentially Regulated by the IE and EICP0 Proteins

Abstract

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.

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

“…All of the in vitro expression plasmids used in this study were transcribed with SP6 RNA polymerase and translated in rabbit reticulocyte lysate (Promega) in the presence of [ 35 …”

“…Experiments involving the in vitro-synthesized proteins were performed in parallel in the presence or absence (competition assays) of 35 S-labeled proteins. Radioactive products were analyzed by SDS-PAGE followed by autoradiography and either stored at Ϫ70°C or used immediately for in vitro protein-binding assays.…”

“…Protein-protein interactions of the EHV-1 IE proteins (in vitro synthesized, bacterially expressed, or nuclear extracted) with TFIIB were carried out by a modification of previously described procedures (13). Aliquots of proteins were thawed on ice, and 2 g of the appropriate GST fusion protein was incubated with the 35 S-labeled proteins (see Fig. 3 to 5, lanes INPUT) in a final volume of 600 l of NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl [pH 8.0], 0.5% NP-40).…”

“…The gels were dried, and the bands were quantitated by PhosphorImager analysis (Molecular Dynamics). The competition assays were performed essentially as before except that unlabeled in vitro-synthesized proteins were employed to compete for the binding of the 35 S-labeled IE protein. To assess interactions with native IE proteins, 20 l of nuclear extracts from mockinfected or EHV-1-infected cells were allowed to bind with 2 g of GST or GST-hIIB protein in a final volume of 600 l of NETN buffer as described above.…”

“…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).…”