SIMIAN VIRUS-40 LARGE T-ANTIGEN POINT MUTANTS THAT ARE DEFECTIVE IN VIRAL-DNA REPLICATION BUT COMPETENT IN ONCOGENIC TRANSFORMATION
SIMIAN VIRUS-40 LARGE T-ANTIGEN POINT MUTANTS THAT ARE DEFECTIVE IN VIRAL-DNA REPLICATION BUT COMPETENT IN ONCOGENIC TRANSFORMATION
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DOI：
10.1128/mcb.4.6.1125
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发表时间：
1984-01-01
影响因子：
5.3
通讯作者：
GLUZMAN, Y
中科院分区：
文献类型：
--
作者：
MANOS, MM;GLUZMAN, Y
SV 40的大T抗原是一种多功能蛋白，在病毒裂解周期和SV 40引起的细胞致癌转化中都是必需的。为了研究T抗原在裂解和转化过程中的许多生化和生理活性的作用，研究了DNA复制缺陷型、转化能力型大T抗原突变体。本文还对两个这样的突变体C2/SV 40和C11/SV 40进行了遗传和生化分析。通过从C2和C11细胞（用UV照射的SV 40转化的非洲绿色猴肾CV-1细胞系）中回收整合的SV 40 DNA来分离突变体。将突变的病毒早期区克隆到质粒载体pK 1中以产生pC 2和pC 11。通过标记拯救定位导致病毒DNA复制缺陷的突变。随后的DNA测序显示了点突变，预测了两种突变体中蛋白质三分之一的羧基中的氨基酸取代。pC 2突变预测了Lys →的变化。在氨基酸516处的Arg。pC 11有2个突变，一个预测Pro →的变化。在残基522处预测Ser，另一个预测Pro →。氨基酸549处的Arg变化。2个C11突变彼此分离，在pC 11 A和pC 11B中形成2个不同的病毒基因组。pC 2、pC 11、pC 11 A和pC 11B能够转化原代和已建立的啮齿动物细胞培养物。C11和C11 A T抗原在ATP酶活性方面是缺陷的，这表明野生型水平的ATP酶活性对于T抗原引起的细胞致癌转化不是必需的。
The large T antigen of SV40 is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, DNA replication-deficient, transformation-competent large T-antigen mutants were studied. The genetic and biochemical analyses of 2 such mutants, C2/SV40 and C11/SV40 is described. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (African green monkey kidney CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys .fwdarw. Arg at amino acid 516. pC11 has 2 mutations, one predicting a change of Pro .fwdarw. Ser at residue 522, and another predicting a Pro .fwdarw. Arg change at amino acid 549. The 2 C11 mutations were separated from each other to form 2 distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.