Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication

Abstract

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.

Human cytomegalovirus origin of DNA replication (oriLyt) resides within a highly complex repetitive region.

“…[3][4][5]. In this and other alphaherpesviruses, lytic replication origins are characterized by two conserved elements: a binding site for the viral origin-specific DNA binding protein and an adjacent A+Trich region (6)(7)(8). Although this type of origin is relatively simple, sequences flanking the core elements apparently contribute to the efficiency of replication (9).…”

Analysis of an origin of DNA replication located at the L terminus of the genome of pseudorabies virus

“…Domain A of the adenovirus core origin contains a similar A+T-rich region which is well conserved among otherwise divergent serotypes (reviewed by Kelly et al [17]). PrV L-ori also contains a stretch of A's followed by a G+C-rich region; a similar sequence is also present in HSV ori (1,7,8,9,18,19,25,33).…”

Nuclear localization and transcriptional activation activities of truncated versions of the immediate-early gene product of equine herpesvirus 1

“…Equine herpesvirus 1 (EHV-1), an alphaherpesvirus with worldwide distribution, is a causative agent of equine rhinopneumonitis, as well as epizootic abortion among pregnant mares (1,20). Within the laboratory, EHV-1 provides a model system with which to study lytic herpesvirus replication, persistent infection, and other important aspects of herpesvirus molecular biology (2,6,11,12,25,26). In particular, EHV-1 provides an excellent system for the study of alphaherpesvirus gene regulation (9,15,23,24,(29)(30)(31)35).…”