Identification of latency-associated transcripts that map antisense to the ICP4 homolog gene of Marek's disease virus

Two small RNAs (0.9 and 0.75 kb), named Marek's disease virus (MDV) small RNAs (MSRs) and a 10-kb RNA, all of which map antisense to the MDV ICP4 homolog gene, have been readily detected in MDCC-MSB1 MDV-transformed T-lymphoblastoid cells. These RNAs were not detectable in reticuloendotheliosis virus-transformed T cells. When MDV was reactivated by treatment of lymphoblastoid cells with 25 micrograms of iododeoxyuridine per ml, the relative levels of the transcripts decreased. These RNAs were not detected by Northern (RNA) hybridization in productively infected chicken embryo fibroblasts 48 h postinfection; however, they were apparent 140 h postinfection. By using Northern hybridization, RNase protection assays, and primer extension analysis, the MSRs were determined to map antisense to the predicted translational start site of the ICP4 homolog gene. The conclusion most consistent with the data is that the two MSRs are overlapping, spliced RNAs. Both small RNAs contain a latency promoter binding factor consensus recognition sequence located toward their 5' ends as well as two potential ICP4 recognition consensus sequences, one in each orientation. The region contains a number of small open reading frames on each side and within the MSRs. Although the exact endpoints are unknown, the large 10-kb species spans the entire ICP4 homolog region. We believe that this group of RNAs, which map antisense to the ICP4 homolog gene, are latency-associated transcripts of MDV