Prichard M N, Jairath S, Penfold M E, St Jeor S, Bohlman M C, Pari G S
Iconix Pharmaceuticals, Inc., Mountain View, California 94043, USA.
J Virol. 1998 Sep;72(9):6997-7004. doi: 10.1128/JVI.72.9.6997-7004.1998.
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 binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive 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 population of vRNA was end labeled and used as a hybridization probe to map the exact location of vRNAs within oriLyt. vRNA-1 is localized between restriction endonuclease sites XhoI at nucleotide (nt) 93799 and SacI at nt 94631 and is approximately 500 bases long. The second vRNA, vRNA-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 be required for oriLyt replication (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). RNase H analysis determined that vRNA-2 forms a persistent RNA-DNA hybrid structure in the context of the viral genome and in an oriLyt-containing plasmid used in the transient-replication assay.
人巨细胞病毒（HCMV）裂解期DNA复制起始于顺式作用复制起点oriLyt。oriLyt是一个结构复杂的区域，包含重复元件和转录因子结合位点。我们在oriLyt内鉴定出两个位点特异性碱不稳定区域，它们位于一个耐碱DNA片段两侧。这些碱敏感区域是嵌入oriLyt并与病毒DNA共价连接的两种RNA分子降解的结果。通过对从蔗糖梯度纯化病毒中获得的HCMV DNA进行DNase I处理，鉴定出病毒相关RNA（vRNA）。对这种异质的vRNA群体进行末端标记，并用作杂交探针来确定oriLyt内vRNA的确切位置。vRNA - 1定位于核苷酸（nt）93799处的XhoI和nt 94631处的SacI限制性内切酶位点之间，长度约为500个碱基。第二种vRNA，即vRNA - 2，位于一个区域内，该区域在SphI（nt 92636）和BamHI（nt 93513）之间呈现异质限制性图谱，长度约为300个碱基。先前已证明该区域是oriLyt复制所必需的（D. G. Anders、M. A. Kacica、G. S. Pari和S. M. Punturieri，《病毒学杂志》66:3373 - 3384，1992年）。RNase H分析确定，在病毒基因组和瞬时复制分析中使用的含oriLyt的质粒背景下，vRNA - 2形成了一种持久的RNA-DNA杂交结构。
