Cloning and expression of an equine herpesvirus 1 origin-binding protein

• Published in: Journal of Virology 1994-06, Vol.68 (6), p.3674-3681
• Main Authors: Martin, D.W, Deb, S
• Format: Article
• Language: English
• Subjects: ADN; Animals; Base Sequence; Binding Sites; Biological and medical sciences; CLONACION; CLONAGE; Cloning, Molecular; DNA Primers - genetics; DNA Replication; DNA, Viral - genetics; DNA, Viral - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; EXPRESION GENICA; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; GENE; Gene Expression; GENES; Genes, Viral; Genetics; Herpesvirus 1, Equid - genetics; Herpesvirus 1, Equid - metabolism; HERPESVIRUS EQUIN; HERPESVIRUS EQUINO; Microbiology; Molecular Sequence Data; Mutagenesis; PROTEINAS; PROTEINE; SECUENCIA NUCLEICA; Sequence Deletion; SEQUENCE NUCLEIQUE; Viral Proteins - genetics; Viral Proteins - metabolism; Virology
• ISSN: 0022-538X; 1098-5514
• DOI: 10.1128/jvi.68.6.3674-3681.1994

Equine herpesvirus 1 (EHV-1) is an important pathogen of horses and is closely related to several important human pathogens, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus. The EHV-1 genome contains open reading frames similar in sequence to the HSV-1 replication genes. PCR was used to clone EHV-1 gene 53, which is similar in sequence to the HSV-1 UL9 gene. The gene 53 product has regions of striking similarity to the HSV-1 UL9 and VZV gene 51 products. In vitro transcription and translation of this gene generated a protein of 87 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further characterization of this protein was accomplished through the use of gel shift analysis. The in vitro-synthesized protein bound sequence specifically to EHV-1 Oris as well as HSV-1 Oris. A series of triple-base-pair substitutions across the previously defined HSV-1 origin-binding protein consensus site was used in gel shift analysis to show that the EHV-1 origin-binding protein bound to the same consensus site as the HSV-1 origin-binding protein, 5'-CGTTCGCACTT-3'. Using a nuclear extract of EHV-1-infected RK13 cells, we have identified an activity that interacts similarly with this consensus site. In gel shift assays, the retarded band arising from the nuclear extract migrated similarly to the retarded band arising from in vitro-translated EHV-1 gene 53. An N-terminal deletion of EHV-1 gene 53 was also created, expressed in vitro, and used in gel shift assays to localize the DNA-binding domain. Results of these experiments indicated that amino acids 1 to 499 were dispensable for binding and that the C-terminal fragment (amino acids 500 to 888) recognized the same consensus site as did the wild-type protein. Thus, the product of EHV-1 gene 53 is an origin-binding protein with a high degree of similarity to the HSV-1 and varicella-zoster virus origin-binding proteins