Abstract
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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have