Identification of an infectious laryngotracheitis virus gene encoding an immunogenic protein with a predicted M r of 32 kilodaltons

Abstract

The nucleotide sequence of an infectious laryngotracheitis virus (ILTV) gene which maps immediately upstream from the glycoprotein 60 (gp60) gene was determined. The gene, designated p32, encodes a predicted polypeptide of 298 amino acids with an estimated M r of 32000 daltons. The predicted protein sequence has four potential N-glycosylation sites and a signal sequence at the N-terminal region. Amino acid residues in the NH 2-terminal region of the p32 protein exhibit similarity to glycoprotein X (gX) of pseudorabies virus (PRV) and its homolog in equine herpesvirus type 1 (EHV-1). Within the conserved (N-terminus) region, one putative N-linked glycosylation site and four cysteine residues are aligned in these proteins. These common structural features of the gX-like proteins were also found in glycoprotein G (gG) of human herpes simplex virus type 2 (HSV-2) and equine herpesvirus type 4 (EHV-4). High level bacterial production of the p32 protein was achieved by cloning the p32 open reading frame into a pGEX-2T expression vector. Western blot analysis of the fusion protein produced in E. coli using immune chicken sera confirms that p32 protein is of viral origin and is an immunogen in birds with infectious laryngotracheitis (ILT). An antiserum from chicken immunized with the fusion protein detected a substantial amount of p32 protein in the medium of ILTV-infected cells in Western blotting. Moreover tunicamycin treatment of cells infected with the virus indicated that p32 was glycosylated. This allows us to conclude that p32 is a glycoprotein and like gX of PRV accumulates in the medium of infected cells.