Identification and characterization of Marek's disease virus genes homologous to ICP27 and glycoprotein K of herpes simplex virus-1

Abstract

We have identified two Marek's Disease Virus (MDV) genes within the EcoRI-B fragment of MDV-GA genomic DNA. EcoRI-B is 11.3-kb long and maps within the long unique region of MDV genomes. A 3.2-kb fragment of EcoRI-B has been sequenced and contains two open reading frames, ORF53 and ORF54. ORF53 (MDV gK), a homolog to HSV-1 glycoprotein K (gK), is 1062 nucleotides long and encodes 354 amino acids (39.5 kDa). ORF54, designated MDV ICP27, based on significant similarity to HSV-1 ICP27, is 1419 nucleotides long and encodes 473 amino acids (54.5 kDa). In Northern blot hybridization, two overlapping transcripts (2.9 and 1.6 kb) were detected in MDV-infected DEF cells treated with cycloheximide, suggesting that both transcripts belong to the immediate-early gene family. Amino acid sequence analysis of MDV gK shows some common glycoprotein features, including a putative N-terminal signal sequence, four N-linked glycosylation sites, and four potential transmembrane domains. Comparison of the predicted amino acid sequence of MDV ICP27 with that of HSV-1 ICP27 and VZV ORF4 shows a high degree of conservation within the C-terminus. The C-terminal region of HSV-1 ICP27 has been demonstrated to be critical to its function. A conserved zinc finger metal-binding motif C(442)-X4-C(447)-X13-H(461)-C(467) was also found in the C-terminus of MDV ICP27. Furthermore, MDV ICP27 upstream sequences contain four copies of consensus sequence elements similar to the tegument protein target sequence TAATGARAT. TrpE-ICP27 fusion protein was expressed in Escherichia coli, and rabbit antisera were generated using purified fusion protein. A 55-kDa protein has been detected in both MDV-GA- and Md11-infected cells using immunoblot analysis.