Luciw P A, Oppermann H, Bishop J M, Varmus H E
Mol Cell Biol. 1984 Jul;4(7):1260-9. doi: 10.1128/mcb.4.7.1260-1269.1984.
To assess the factors required for integration and expression of retroviral DNA, we have examined viral DNA, RNA, and protein in NIH/3T3 mouse cells transformed by transfection with various forms of cloned Rous sarcoma virus (RSV) DNA. Linear RSV DNA molecules, derived from circular DNA containing two long terminal repeats (LTRs) and permuted by cleavage at the SacI restriction endonuclease site in the leader sequence, were integrated near the ends of the linear molecule, with the LTRs on the 3' side of the src gene. Integration of a subgenomic RSV DNA fragment containing the viral src gene without intact LTRs also occurred near the ends of the linear molecule. Head-to-tail tandem arrays of RSV DNA species were observed in some transformed cell lines that received fully digested DNA and in all cell lines that received DNA ligated to produce oligomers before transfection. Closed circular RSV DNA, with one or two LTRs, integrated without apparent specificity within several regions of the viral genome. After transfection with SacI-permuted RSV DNA still linked to arms of the lambda bacteriophage vector DNA, bacteriophage sequences were joined to host DNA. Transformed cell lines produced by transfection with the various forms of RSV DNA produced similar levels of viral src protein, although the efficiency of successful transformation varied by at least two orders of magnitude. Analyses of viral polyadenylated RNA, together with the patterns of viral DNA in transformed cells, indicated that viral DNA can be integrated and expressed without regard to LTR sequences, with adjacent host DNA presumably supplying signals required for the promotion and processing of functional src mRNA.
为了评估逆转录病毒DNA整合和表达所需的因素，我们检测了用各种形式的克隆劳氏肉瘤病毒（RSV）DNA转染转化的NIH/3T3小鼠细胞中的病毒DNA、RNA和蛋白质。线性RSV DNA分子来源于含有两个长末端重复序列（LTRs）的环状DNA，并通过在前导序列中的SacI限制性内切酶位点切割而重排，它整合在线性分子的末端附近，LTRs位于src基因的3'侧。含有病毒src基因但没有完整LTRs的亚基因组RSV DNA片段也在线性分子的末端附近整合。在一些接受完全消化DNA的转化细胞系以及所有接受在转染前连接产生寡聚物的DNA的细胞系中，观察到了RSV DNA种类的头对头串联阵列。带有一个或两个LTRs的闭环RSV DNA在病毒基因组的几个区域内无明显特异性地整合。在用仍与λ噬菌体载体DNA臂相连的经SacI重排的RSV DNA转染后，噬菌体序列与宿主DNA连接。用各种形式的RSV DNA转染产生的转化细胞系产生了相似水平的病毒src蛋白，尽管成功转化的效率相差至少两个数量级。对病毒多聚腺苷酸化RNA的分析以及转化细胞中病毒DNA的模式表明，病毒DNA可以不考虑LTR序列而进行整合和表达，相邻的宿主DNA大概提供了促进和加工功能性src mRNA所需的信号。
