Prives C, Covey L, Scheller A, Gluzman Y
Mol Cell Biol. 1983 Nov;3(11):1958-66. doi: 10.1128/mcb.3.11.1958-1966.1983.
Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.
对三种猿猴病毒40（SV40）转化的猴细胞系C2、C6和C11进行了分析，这些细胞系产生的T抗原变体无法启动病毒DNA复制，分析内容涉及它们对病毒复制起点调控序列的亲和力。C2和C11 T抗原均特异性结合病毒起源区域位点1和位点2的序列，而在所有测试条件下，C6 T抗原对任何病毒DNA序列均无特异性亲和力。最近已从C6细胞中克隆出编码C6 T抗原的病毒DNA序列，并用于转化已建立的大鼠细胞系。来自几个克隆的C6 - SV40转化大鼠系的T抗原未能特异性结合起点。C6 DNA包含三个突变：两个位于T抗原氨基末端附近的氨基酸位置30和51，第三个位于内部的氨基酸位置153。制备了两种重组SV40 DNA突变体，分别含有位置30和51的氨基末端突变（C6 - 1）或位置153的内部突变（C6 - 2），并用于转化大鼠2细胞。而来自C6 - 2转化细胞的T抗原对这些序列缺乏任何特异性亲和力。因此，氨基酸位置153处的单个突变（天冬酰胺突变为苏氨酸）足以消除T抗原的起点结合特性。一种T抗原特异性单克隆抗体PAb 100，先前已证明其能免疫沉淀T抗原的一种免疫上不同的起点结合亚类，可识别野生型或C6 - 1抗原，但不与C6或C6 - 2 T抗原反应。这些结果表明，病毒复制功能包含T抗原的特性，这些特性除了其特异性结合SV40调控序列的能力外还存在。此外，从这些数据得出结论，特异性病毒起点结合不是T抗原转化功能的必要特征。
