Cloning and characterization of a Marek's disease virus (MDV) gene homologous to herpes simplex virus type 1 (HSV-1) UL9 gene

Marek's disease virus (MDV), a highly cell-associated avian herpesvirus, induces Marek's disease (MD) which is characterized by malignant lymphoma of T cells. Viral replication represents a point at which pharmacological control of herpesvirus infection may be most successful. Studies in HSV-1 DNA replication implicate the UL9 protein as a key initiator of replication. In this study, a protein with apparent molecular size similar to HSV-1 UL9, was identified in infected cell extracts by western blot analysis with anti-HSV-1 UL9 antibody. A putative MDV UL9 gene was subsequently identified through sequencing of MDV genome fragments (BamHI G and C). Extended DNA sequence analysis revealed an open reading frame (ORF) which could encode a protein homologous to HSV-1 UL9. The MDV UL9 ORF encodes an 841 amino acid polypeptide which is highly similar to HSV-1 UL9 and VZV gene 51 (VZV UL9). MDV UL9 shares numerous structural motifs with HSV-1 and VZV gene 51, including six conserved N-terminal helicase motifs, an N-terminal leucine zipper motif, a C-terminal pseudo-leucine zipper sequence, and a putative helix-turn-helix structure. The above results suggest that MDV UL9 gene may have the similar biochemical activities to HSV-1 UL9, specifically the origin-binding activity.;A MDV UL9 protein of 95 kd was detected in nuclear extracts of MDV infected cells by western blot analysis with anti-MDV UL9 antibody. PCR was used to clone the MDV UL9 gene. In vitro transcription-translation of this gene generated a protein with apparent molecular size of 95kd. Electrophoretic mobility shift assays (EMSAs) with in-vitro expressed MDV UL9 protein or the infected nuclear extracts showed that MDV UL9 protein could bind to the HSV-1 UL9 binding site I and MDV UL9 binding site II. Competitive EMSAs with a mutant MDV UL9 site II DNA indicated that the last nucleotide (T) within MDV UL9 binding site II was essential for the binding of MDV UL9 protein to MDV UL9 binding site II. Competitive EMSAs with a series of mutant MDV UL9 site I DNAs demonstrated that the last two nucleotides (TT) within HSV-1 UL9 binding site I were essential for the binding of MDV UL9 protein in vitro.