Huang P S, Patrick D R, Edwards G, Goodhart P J, Huber H E, Miles L, Garsky V M, Oliff A, Heimbrook D C
Department of Cancer Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
Mol Cell Biol. 1993 Feb;13(2):953-60. doi: 10.1128/mcb.13.2.953-960.1993.
Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.
人乳头瘤病毒（HPV）是引起尖锐湿疣的病原体，并在人类宫颈癌的发生中起作用。HPV E7基因产物在这些疾病中表达，HPV 16型和18型的E7基因可促使哺乳动物细胞发生转化。对该基因的突变和缺失分析表明，该蛋白质产物的转化活性位于与视网膜母细胞瘤基因产物（pRB）直接形成复合物的同一结构域中。该结构域是E7和其他与pRB结合的病毒癌蛋白（包括腺病毒E1A蛋白）共有的两个保守区域（分别命名为CRI和CRII）之一。先前已证明HPV 16型E7蛋白与pRB的结合会影响pRB结合DNA以及与其他细胞蛋白形成复合物的能力。在本研究中，我们通过监测60 kDa版本的pRB（pRB60）与细胞转录因子E2F之间的结合，来确定E7蛋白与pRB之间的功能相互作用。我们观察到，完全阻断全长E7蛋白结合的E7的CRII（氨基酸20至29）对于抑制E2F/pRB60复合物的形成是必要的，但并不充分。虽然E1A的CRI（氨基酸37至55）似乎足以与E2F竞争结合pRB60，但E7的等效区域既不是必需的，也不充分。只有同时包含CRII和至少一部分锌结合结构域（氨基酸60至98）的E7片段才能抑制E2F/pRB60复合物的形成。这些结果表明，pRB60通过重叠但不同的结构域与E7和E2F结合。
