IDENTIFICATION OF PERSISTENT RNA-DNA HYBRID STRUCTURES WITHIN THE ORIGIN OF REPLICATION OF HUMAN CYTOMEGALOVIRUS

Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor bi nding sites. We identified two site-specific alkali-labile regions wit hin oriLyt which flank an alkali-resistant DNA segment. These alkali-s ensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus- associated RNA, vRNA, was identified by DNase I treatment of HCMV DNA obtained from sucrose gradient purified virus. This heterogeneous popu lation of vRNA was end labeled and used as a hybridization probe to ma p the exact location of vRNAs within oriLyt. vRNA-1 is localized betwe en restriction endonuclease sites XhoI at nucleotide (nt) 93799 and Sa cI at nt 94631 and is approximately 500 bases long. The second vRNA, v RNA-2, lies within a region which exhibits a heterogeneous restriction pattern located between the SphI (nt 92636) and BamHI (nt 93513) and is approximately 300 bases long. This region was previously shown to b e required for oriLyt replication (D. G. Anders, M. A. Kacica, G. S. P ari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). RNase H anal ysis determined that vRNA-2 forms a persistent RNA-DNA hybrid structur e in the context of the viral genome and in an oriLyt-containing plasm id used in the transient-replication assay.