Equine herpesvirus 1 (EHV-1) has been shown to synthesize a 6.0-kilobase (kb) species of immediate-early (IE) mRNA in productively infected cells. This IE gene region maps within the outer portion (map units 0.79 to 0.83 and 0.96 to 1.00) of the two inverted repeat segments of the short genomic region, and elucidation of its DNA sequence has revealed multiple potential open reading frames (ORFs), including a major ORF of 4,461 nucleotides (F. J. Grundy, R. P. Baumann, and D. J. O'Callaghan, Virology 172:223-236, 1989). Analyses of IE polypeptides synthesized in EHV-1-infected cells (in vivo) and in vitro translation of hybrid-selected IE mRNA indicated that multiple species of IE proteins are encoded by this IE mRNA species. To address the nature of the 6.0-kb IE RNA species, Northern (RNA) blot hybridization, S1 nuclease mapping, and primer extension analyses have been employed. These data revealed that no major introns were detected within the body of the IE transcript. However, the IE mRNA was shown to be spliced at the 5' terminus, such that a 372-base intron containing two small ORFs (19 and 51 amino acids) was removed from the leader region of the transcript. This splicing event reduced the leader region from 625 to 253 bases. S1 and primer extension analyses of the 5' terminus of this transcript revealed that the transcription initiation site is located 24 to 26 bases downstream of the consensus TATAAA motif. The 3' transcription termination site was mapped by S1 nuclease analysis to approximately 10 to 20 bases downstream of the polyadenylation signal, AATAAA. The distance from the stop codon of the major ORF to the polyadenylation site is approximately 300 bases. Results from S1 nuclease experiments indicated that splicing does not occur at the 3' terminus. These studies indicated that the EHV-1 6.0-kb IE mRNA is spliced at the 5' terminus and that alternative splicing of this transcript may function in regulating translation of the IE mRNA species
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