| Equine herpesvirus type 1 (EHV-1) gene expression is regulated in an immediate-early (IE), early (E), and late (L) fashion by five regulatory proteins (IE, EICP27, EICP22, E-TIF, and EICP0). Molecular studies on the EICP0 gene and gene products revealed that: (i) the EICP0 open reading frame of 1,257 base pairs encodes a protein of 419 amino acids (aa); (ii) one early transcript of 1.4 kb predicted to encode the EICP0 protein is detected in Northern blot analyses; (iii) the EICP0 protein is expressed as multiple species of early proteins that are first detected at 3 hours post-infection; (iv) the EICP0 protein functions as a potent trans-activator of EHV-1 genes since it can independently trans-activate chloramphenicol acetyltransferase reporter constructs under the control of the IE, E, ;A panel of EICP0 mutant plasmids was generated and employed in transient trans-activation studies which revealed that: (i) carboxy-terminal truncation mutants exhibited a progressive loss of trans-activating ability as increasing portions of the carboxyl-terminus were removed; (ii) the amino-terminus, containing the RING finger (aa 8-46) and the acidic region (aa 71-84), is necessary but not sufficient for activation of EHV-1 promoters; (iii) the carboxy-terminus (aa 325-419) is dispensable for EICP0 trans-activation function; (iv) a motif resembling a nuclear localization signal (aa 289-293) was unnecessary for the EICP0 protein to trans-activate any promoter; and (v) the EICP0 protein is phosphorylated between 4 and 8 hours post-infection, and a serine-rich region (aa 210-217), a potential site for phosphorylation, contributes to its ability to activate the ;This early regulatory protein may be essential for EHV-1 replication since studies employing six EICP0-expressing cell lines and an EICP0-deletion mutant virus indicate that this mutant virus cannot be purified to homogeneity on non-complementing cells. |
