Manfredi J J, Prives C
Department of Biological Sciences, Columbia University, New York, New York 10027.
J Virol. 1990 Nov;64(11):5250-9. doi: 10.1128/JVI.64.11.5250-5259.1990.
To identify regions on the large T antigens of simian virus 40 (SV40) and polyomavirus which are involved in oncogenic transformation, we constructed plasmids encoding hybrid polyomavirus-SV40 large T antigens. The hybrid T antigens were expressed in G418 sulfate-resistant pools of rat F2408 cells, and extracts of such pools were immunoprecipitated with an antibody against p53. Two hybrid T antigens containing SV40 amino acids 337 to 708 bound to p53, whereas another hybrid T antigen containing SV40 amino acids 412 to 708 did not. This suggests that a binding domain on SV40 large T antigen for p53 is contained within amino acids 337 to 708, with amino acids 337 to 411 playing an important role. One of the two hybrids that bound to p53 was chosen for further study. This T antigen contained SV40 large T antigen amino acids 336 to 708 joined to polyomavirus large T antigen amino acids 1 to 521 (PyT1-521-SVT336-708). Immunoprecipitation with antibodies directed against the product of the retinoblastoma susceptibility gene, p105-RB, showed that this hybrid bound p105-RB as well as p53. Pools expressing the hybrid PyT1-521-SVT336-708 did not grow in soft agar, nor did they form foci on confluent monolayers of nontransformed F2408 cells. The hybrid T antigen was expressed at levels comparable to those seen in retrovirus-infected F2408 cells expressing only SV40 large T antigen, which do show a transformed phenotype. Thus, this level of expression was sufficient for transformation by SV40 large T antigen but not for the hybrid large T antigen. These data, combined with genetic studies from other laboratories, suggest that complex formation with p53 and p105-RB is necessary but not sufficient for the oncogenic potential of papovavirus large T antigens.
为了确定猿猴病毒40（SV40）和多瘤病毒大T抗原中参与致癌转化的区域，我们构建了编码杂交多瘤病毒 - SV40大T抗原的质粒。杂交T抗原在对硫酸G418有抗性的大鼠F2408细胞群体中表达，并且用针对p53的抗体对这些细胞群体的提取物进行免疫沉淀。两种含有SV40氨基酸337至708的杂交T抗原与p53结合，而另一种含有SV40氨基酸412至708的杂交T抗原则不与p53结合。这表明SV40大T抗原上与p53结合的结构域位于氨基酸337至708内，其中氨基酸337至411起着重要作用。选择与p53结合的两种杂交体之一进行进一步研究。这种T抗原包含与多瘤病毒大T抗原氨基酸1至521连接的SV40大T抗原氨基酸336至708（PyT1 - 521 - SVT336 - 708）。用针对视网膜母细胞瘤易感基因产物p105 - RB的抗体进行免疫沉淀表明，这种杂交体与p105 - RB以及p53都结合。表达杂交体PyT1 - 521 - SVT336 - 708的细胞群体在软琼脂中不生长，在未转化的F2408细胞的汇合单层上也不形成集落。杂交T抗原的表达水平与仅表达SV40大T抗原的逆转录病毒感染的F2408细胞中所见的水平相当，而后者确实表现出转化表型。因此，这种表达水平对于SV40大T抗原的转化是足够的，但对于杂交大T抗原则不足够。这些数据，结合其他实验室的遗传学研究，表明与p53和p105 - RB形成复合物对于乳头瘤病毒大T抗原的致癌潜力是必要的，但不是充分的。
