Mapping the sequences that mediate interaction of the equine herpesvirus 1 immediate-early protein and human TFIIB.

The sole immediate-early (IE) gene of equine herpesvirus 1 encodes a 1,487-amino-acid (aa) regulatory phosphoprotein that independently activates expression of early viral genes. Coimmunoprecipitation assays demonstrated that the IE protein physically interacts with the general transcription factor TFIIB. Using a variety of protein-binding assays that employed a panel of IE truncation and deletion mutants expressed as in vitro-synthesized or glutathione S-transferase fusion proteins, we mapped a TFIIB-binding domain to aa 407 to 757 of the IE protein. IE mutants carrying internal deletions of aa 426 to 578 and 621 to 757 were partially defective for TFIIB binding, indicating that aa 407 to 757 may harbor more than one TFIIB-binding domain. The interaction between the IE protein and TFIIB is of physiological importance, as evidenced by transient-cotransfection assays. Partial deletion of the TFIIB-binding domain within the IE protein inhibited its ability to activate expression of the viral thymidine kinase gene, a representative early promoter, and of the IR5 gene, a representative late promoter, by greater than 20 and 50%, respectively. These results indicate that the interaction of the IE protein with TFIIB is necessary for its full transactivation function and that the IE-TFIIB interaction may be part of the mechanism by which the IE protein activates transcription.

The equine herpesvirus 1 immediate-early protein interacts with EAP, a nucleolar-ribosomal protein.

The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The IE protein of 1487 amino acids contains a serine-rich tract (SRT) between residues 181 and 220. Deletion of the SRT decreased transactivation activity of the IE protein. Previous results from investigation of the ICP4 protein, the IE homolog of herpes simplex virus 1 (HSV-1), revealed that a domain containing a serine-rich tract interacts with EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein), a 15-kDa nucleolar-ribosomal protein (R. Leopardi, and B. Roizman, Proc. Natl. Acad. Sci. USA 93, 4572-4576, 1996). DNA binding assays revealed that (i) glutathione S-transferase (GST)-EAP disrupted the binding of HSV-1 ICP4 to its cognate DNA in a dose-dependent manner, (ii) GST-EAP interacted with the EHV-1 IE protein, but did not disrupt its binding to its cognate site in viral DNA. GST-pulldown assays indicated that the SRT of the IE protein is required for physical interaction with EAP. The IE protein and EAP colocalized in the cytoplasm of the infected equine ETCC cells at late times of the infection cycle. This latter finding may be important in EHV-1 gene regulation since late viral gene expression is greatly influenced by the EICP0 trans-activator protein whose function is antagonized by the IE protein.

Characterization of the transactivation domain of the equine herpesvirus type 1 immediate-early protein.

Equine herpesvirus type 1 (EHV-1) possesses a sole diploid immediate early gene (IE) that encodes a major regulatory protein of 1487 amino acids capable of modulating gene expression from both early and late promoters and also of trans-repressing its own promoter. Using a series of GAL-4-IE fusion constructs, we previously demonstrated that the minimal transactivation domain (TAD) of the IE protein maps within amino acids 3-89. Additional studies revealed that that the carboxyl terminus of the IE protein may be required for full transactivation activity in vitro. Analyses of the minimal TAD revealed the presence of 13 acidic amino acids and six basic residues giving the TAD region a net negative charge of -7. In addition, there are conserved hydrophobic residues (Leu(12) and Phe(15)) that may be critical for transactivation function. To identify residues essential for IE transactivation and to ascertain if the overall net negative charge of the TAD or the position of specific hydrophobic residues within the IE TAD are critical for the transactivation function, plasmids expressing mutant forms of the TAD were generated using specifically designed mutagenic oligonucleotides and PCR mutagenesis. Mutagenized TADs in which the acidic and hydrophobic amino acid residues were replaced, singly and in combination, with polar, uncharged amino acids were cloned into a GAL-4/CAT reporter expression system and assayed in transient transfection assays. To determine if the carboxyl terminus is necessary for full transactivation activity, a series of constructs that express forms of the IE protein-containing deletions within this region were generated and assayed for transactivation function in transient transfection assays. These assays demonstrated that mutation of any acidic residue, either singly or in combination, or deletion of the carboxyl terminus of the IE protein resulted in a severe impairment of transactivation activity. These results show that both acidic and hydrophobic residues within the IE TAD are critical for transactivation function and that the carboxyl terminus of the IE protein is required for full transactivation activity.

The defective interfering particles of equine herpesvirus 1 encode an ICP22/ICP27 hybrid protein that alters viral gene regulation.

The genomes of equine herpesvirus 1 (EHV-1) defective interfering (DI) particles that mediate persistent infection were shown to encode a unique hybrid open reading frame composed of sequences that encode the 196 N-terminal amino acids of ICP22 linked in-frame to the C-terminal 68 amino acids of ICP27. Previous studies demonstrated that this hybrid gene, designated as ICP22/ICP27. was expressed abundantly at both the mRNA and the protein levels in DI particle-enriched infections, but not in standard EHV-1 infection (Chen et al., 1996 J. Virol. 70, 313-320). Since the ICP22/ICP27 hybrid protein contains portions of two EHV-1 early regulatory proteins, its effect on EHV-1 gene regulation was investigated. In EHV-1-infected cells, the ICP22/ICP27 hybrid protein expressed from plasmid vectors significantly reduced expression of a reporter gene under the control of the EHV-1 immediate-early (IE) gene promoter and early gene promoter, such as the viral ICP27 gene. In uninfected cells, the ICP22/ICP27 hybrid protein moderately down-regulated the IE and ICP22 promoters, up-regulated late gene promoters such as IR5, and altered the regulatory function of the IE and 1CP22 proteins in co-transfected cells. These results demonstrated that DI particles might alter viral gene regulation by expression of a unique hybrid gene encoded on the DI particle genome.

Complementation of a replication-defective mutant of equine herpesvirus type 1 by a cell line expressing the immediate-early protein.

Equine herpesvirus type 1 (EHV-1) possesses a sole, diploid immediate-early (IE) gene that encodes a major regulatory protein of 1487 amino acids capable of modulating expression of both early and late EHV-1 promoters and capable of trans-repressing its own promoter. In this study, a rabbit kidney cell line (IE13.1) that constitutively expresses the EHV-1 IE protein was generated by cotransfection of rabbit kidney (RK-13) cells with the viral IE gene and a neomycin resistance marker. The IE protein expressed by this cell line was shown (1) to be expressed by and to localize to the nucleus of virtually all cells as demonstrated by indirect immunofluorescence, (2) to be the full-size IE polypeptide as judged by Western immunoblot analyses with an anti-IE protein-specific antibody, and (3) to be functional as shown by the transactivation of two representative EHV-1 early promoters linked to the chloramphenicol acetyltransferase reporter gene in transient transfection assays. The IE13.1 cell line was able to complement a recombinant virus in which both copies of the IE gene were replaced by insertion of the Escherichia coli lacZ gene. This IE deletion mutant, designated KyADeltaIE, was not able to replicate in equine, rabbit, or mouse cells but was capable of replication in the IE13.1 cells that provided the IE protein in trans. Rescue of the KyADeltaIE virus was achieved by recombination with a marker plasmid that harbors the wild-type IE gene, and the rescued virus (KyADeltaIER) was able to grow on noncomplementary cells. Overall, these results offer direct evidence that the IE gene is essential for EHV-1 replication and provide reagents useful for the analysis of IE protein function.