DNA Sequence and Transcriptional Analyses of the Region of the Equine Herpesvirus Type 1 Kentucky A Strain Genome Encoding Glycoprotein C

Abstract

DNA sequence and transcriptional analyses were performed on the region of the equine herpesvirus type 1 (EHV-1) genome (KyA strain) (map units 0.129 to 0.152) encoding open reading frames (ORFs) 15 and 16. ORF 16 encodes a homolog of glycoprotein C of HSV-1 (herpes simplex virus type 1), while ORF 15 corresponds in position to HSV-1 UL45 but exhibits no significant homology at the amine acid level. Sequence analyses revealed that the EHV-1 gC ORF of 468 amino acids and ORF-15 of 227 amino acids mapped at nucleotides (nt) 716 to 2119 and 2397 to 3077, respectively (relative to the 5′ end of the Bam HI recognition site at map unit 0.152). ORF 15 exhibited significant homology (69% identity) at the amino acid level to the EHV-4 gone located 3′ of the EHV-4 gC homolog. Sequence analyses identified potential CAAT and TATA boxes for the EHV- 1 gC ORF and a TATA box for ORF 15. While no consensus polyadenylation signal was detected between ORFs 15 and 16, two polyadenylation signals were detected 3′ of ORF 15. Northern blot and S1 nuclease analyses were used to map and characterize the gC and ORF 15 mRNAs, and metabolic inhibitors were used to identify the kinetic class of these two genes. The results revealed that gC is a γ-1 gene which encodes a 2.8-kb mRNA, while ORF 15 is γ-2 gene encoding a 0.9-kb mRNA which is 3′ coterminal with the gC transcript. The gC and ORF 15 mRNAs were shown by S1 nuclease analyses to initiate approximately 34 and 26 nucleotides downstream of their respective TATA boxes and to have a common termination site 18 to 20 nucleotides downstream of a consensus polyadenylation signal. Comparative sequence analysis revealed that the KyA strain gC protein differs in only three amino acid residues from the gC protein of the EHV-1 Ab4 and T431 strains, and one of the three amino acid differences occurred within a segment of six contiguous amino acids showing high degree of hydrophilicity in the gC molecule. Further comparative sequence analysis revealed that KyA strain genome has a major deletion in the region of ORF I7 which lies 5′ of gC in the Ab4 strain. This finding that 1038 base pairs (bp) of the 1203-bp ORF 17 is deleted indicates that ORF 17 is nonessential for EHV-1 replication in cell culture. To examine the regulation of the EHV-1 gC gene, transient transfection assays using CAT (chloramphenicol acetyltransferase) reporter gene constructs of gC were performed. The results showed that expression from the EHV-1 gC promoter required the presence of both the EHV-1 IE protein and the EHV-1 UL3 protein (an ICP27 homolog) for significant activation as has been shown with other EHV-1 late gene promoters.