OPEN READING FRAMES IN A 4556-NUCLEOTIDE-SEQUENCE WITHIN MDV-1 BAMHI-D DNA FRAGMENT - EVIDENCE FOR SPLICING OF MESSENGER-RNA FROM A NEW VIRAL GLYCOPROTEIN GENE

A DNA segment of the MDV-1 BamHI-D fragment was sequenced, and the ope n reading frames (ORFs) present in the 4556 nucleotide fragment were a nalyzed by computer programs. Computer analysis identified 19 putative ORFs in the sequence ranging from a coding capacity of 37 amino acids (aa) (ORF-1a) to 684aa (ORF-1). The special properties of four ORFs ( 1a, 1, 2, and 3) were investigated. Two adjacent ORFs, ORF-1a and ORF- 1, were found by computer analysis to have the properties of two intro ns encoding a glycoprotein: ORF-la encodes an aa sequence with the pro perties of a signal peptide, and ORF-1 encodes a polypeptide with a me mbrane anchor domain and putative N-glycosylation sites in the aa sequ ence. ORF-1a and ORF-1 were found to be transcribed in MDV-1-infected cells. Two RNA transcripts were detected: a precursor RNA and its spli ced form. Both are transcribed from a promoter located 5' to ORF-1a, a nd splice donor and acceptor sites are used to splice the mRNA after c leavage of a 71-nucleotide sequence. This finding suggests that ORF-1a and ORF-1 are two introns of a new MDV-1 glycoprotein gene. The DNA s equence containing ORF-1 was transiently expressed in COS-1 cells, and the viral protein produced in these cells was found to react with ant i-MDV serotype-1 Antigen B-specific monoclonal antibodies. These studi es indicate that the protein encoded by ORF-1 has antigenic properties resembling Antigen B of MDV-1. A gene homologous to ORF-1 was detecte d in the genome of both MDV-2(SB1) and MDV-3(HVT), which serve as comm ercial vaccine strains. Two additional ORFs were noted in the 4556 nuc leotide sequence: ORF-2, which encodes a 333 aa polypeptide initiating in the U-L and terminating in the TR(L) prior to the putative origin of replication, and ORF-3, which encodes a 155 aa polypeptide that is partly homologous to the phosphoprotein pp38 encoded by the BamHI-H se quence. The 65 N-terminal aa of the two gene products are identical, b oth being derived from the nucleotide sequences in the TR(L) and IR(L) , respectively. Additional homologous aa sequences are the hydrophobic aa domain in the middle of both proteins. The functions of ORF-2, ORF -3, and additional ORFs are under study.