Characterization and expression of the pseudorabies virus early gene UL54

Abstract

Pseudorabies virus (PRV) is an alphaherpesvirus, and its gene organization and regulation are similar to the well-characterized human simplex virus (HSV). Sequence analysis of the complete coding region of PRV UL54 gene revealed that the UL54 gene consisted of 1092 nucleotides encoding a protein of 363 amino acids and the gene showed homology to HSV immediate-early protein ICP27. Detection of the UL54 transcript in infected cells by reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that the UL54 gene belonged to the early kinetic class based on sensitivity to cycloheximide and insensitivity to phosphonoacetic acid (PAA). To study the structure and function of UL54 protein, this gene was subcloned on Escherichia coli expression vector pET28b for overexpression, and the expressed product was applied to generate specific antibody against UL54 protein. The specificity of the mouse immuneserum was confirmed by its ability to react with a 40 kDa viral protein present in the PRV infected cells in Western immunblotting assay, detected as early as 4 h after infection. In addition, immunoperoxidasing staining of PRV infected cells undertaken with this antibody demonstrated mainly nuclear staining pattern. Furthermore, the RNA binding potential of UL54 protein was demonstrated by its binding activity to poly(G) RNA homopolymer in Northwestern blotting assay.