Petersen R, Kempler G, Barklis E
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
Mol Cell Biol. 1991 Mar;11(3):1214-21. doi: 10.1128/mcb.11.3.1214-1221.1991.
Retrovirus expression in embryonal carcinoma (EC) cells is blocked at a postintegration stage of the viral life cycle, in part because of the inadequate function of the viral long terminal repeat promoter in this cell type. However, selection for retrovirus expression in EC cells has identified mutations in Moloney murine leukemia virus (M-MuLV) located in the tRNA primer-binding site (PBS) region which relieve the EC cell-specific repression. We have found that exchanging the M-MuLV proline PBS for a glutamine one in a recombinant virus permits expression in EC cells. By using the recombinant virus as a backbone, the EC cell-specific repressor-binding site (RBS) element has been mapped to M-MuLV nucleotides 147 to 174. The RBS does not require precise positioning downstream of the M-MuLV promoter and can function in either orientation and in an intron, indicating that the regulatory effect is probably at the DNA, rather than RNA, level. We also show that the RBS element can repress heterologous promoters from an upstream position. Our results indicate that the RBS acts as a silencer that its inhibitory effect is mediated by a trans-acting factor, and that the mechanism of action is probably at the level of transcription. Through in vitro binding assays we have identified a binding factor which specifically recognizes the wild-type RBS sequence (binding factor A). The binding characteristics of factor A suggest that it is a stem cell repressor which acts at the M-MuLV RBS. Our DNA-binding assays also have identified a unique binding factor (binding factor Hp) which specifically recognizes a hemimethylated form of the wild-type RBS. This factor may play a role in methylation mediated control of retrovirus expression in EC cells.
逆转录病毒在胚胎癌细胞（EC）中的表达在病毒生命周期的整合后阶段被阻断，部分原因是这种细胞类型中病毒长末端重复启动子的功能不足。然而，对EC细胞中逆转录病毒表达的筛选已经鉴定出莫洛尼鼠白血病病毒（M-MuLV）位于tRNA引物结合位点（PBS）区域的突变，这些突变可缓解EC细胞特异性抑制。我们发现，在重组病毒中将M-MuLV脯氨酸PBS替换为谷氨酰胺PBS可使病毒在EC细胞中表达。以重组病毒为骨架，已将EC细胞特异性阻遏物结合位点（RBS）元件定位到M-MuLV核苷酸147至174。RBS不需要精确位于M-MuLV启动子下游，并且可以在任一方向和内含子中发挥作用，这表明调节作用可能在DNA水平而非RNA水平。我们还表明，RBS元件可以从上游位置抑制异源启动子。我们的结果表明，RBS作为一种沉默子，其抑制作用由反式作用因子介导，作用机制可能在转录水平。通过体外结合试验，我们鉴定出一种特异性识别野生型RBS序列的结合因子（结合因子A）。因子A的结合特性表明它是一种在M-MuLV RBS处起作用的干细胞阻遏物。我们的DNA结合试验还鉴定出一种独特的结合因子（结合因子Hp），它特异性识别野生型RBS的半甲基化形式。该因子可能在EC细胞中逆转录病毒表达的甲基化介导控制中发挥作用。
