Identification and transcriptional mapping of genes encoded at the IR/Us junction of equine herpesvirus type 1

Abstract

Two open reading frames (ORFs) encoded at the inverted repeat unique short (Us) junction of the Short (S) region of the equine herpesvirus type 1 genome were identified by DNA sequencing of a 2876 base pair (bp) genomic segment, and transcripts encoding these ORFs were characterized by Northern blot, S1 nuclease, and primer extension analyses. These studies also established the size of each inverted repeat to be 12,768 nucleotides (nts). The IR6 ORF (816 bp), mapping at nts 12,317-11,502 of the S region, is the last gene completely encoded within each inverted repeat and encodes a predicted 30.1-kDa protein of 272 amino acids, which does not exhibit homology to other alphaherpesvirus proteins. IR6 is expressed as an early transcript of 1.2 kb which is detected initially at 1.5 hr p.i. and up to 12 hr p.i. The transcription initiation and termination sites of IR6 were mapped by primer extension and S1 nuclease analyses to nts 12,465 and 11,408, respectively. The first ORF encoded within the Us segment (909 bp; EUS1), mapping at nts 13,397-12,489, encodes a predicted 33.5-kDa protein of 303 amino acids that exhibits 29% identity to the US2 protein of herpes simplex virus 1. EUS1 is expressed as a 2.3-kb mRNA of the gamma-1 class, as its synthesis begins prior to viral DNA replication at 4 hr p.i. but is retarded by phosphonoacetic acid, an inhibitor of viral DNA replication. The Tci and Tct sites of EUS1 were mapped by S1 nuclease analyses to nts 13,637 and 11,408, respectively. Interestingly, this termination site is also utilized by three late mRNAs of 5.8, 3.8, and 1.7 kb which originate within the Us and overlap the IR6 mRNA encoded in the terminal inverted repeat (TR) of the prototype genomic isomer. EUS1 is 3' coterminal with IR6 in the inverted repeat, whereas, the 5.8, 3.8, and 1.7 kb transcripts are 3' coterminal with IR6 of the TR.