Belaguli N S, Pater M M, Pater A
Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada.
J Virol. 1992 May;66(5):2724-30. doi: 10.1128/JVI.66.5.2724-2730.1992.
Mutations within coding sequences of the various human papillomavirus type 16 (HPV-16) genes have been used to demonstrate that the HPV-16 E7 gene is necessary and sufficient for transformation of rodent cells. We now provide evidence that, in addition to E7 coding sequences, a small cis-acting region immediately flanking the 3' end of E7 coding sequences is also required for transformation. This was shown by translation termination linker insertion, progressive deletion analysis, and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within the 3'-flanking region by deletion of as few as 4 nt or substitution of 3 nt totally abolished transformation. Regeneration of the wild-type sequence in a previously transformation-incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in a stronger transformation phenotype, while mutating the +2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the amount of E7 mRNA in transformation-deficient splice site mutants was much lower. Nuclear runoff experiments revealed that there was no change in the rate of synthesis of E7 message in the nt 880 splice site mutant. Furthermore, mutations of HPV-16 sequences indicated that the two other early region splice donor sites have no more than minor roles in transformation and efficient RNA accumulation. These results indicate that the specific integrity of the nt 880 splice donor site is essential for both accumulation of E7 RNA and efficient E7-mediated transformation.
多种16型人乳头瘤病毒（HPV - 16）基因编码序列内的突变已被用于证明HPV - 16 E7基因对于啮齿动物细胞的转化是必要且充分的。我们现在提供证据表明，除了E7编码序列外，紧邻E7编码序列3'端的一个小的顺式作用区域对于转化也是必需的。这通过翻译终止接头插入、渐进式缺失分析和定点诱变得以证明。通过删除少至4个核苷酸或替换3个核苷酸破坏3'侧翼区域内的核苷酸（nt）880剪接供体位点，完全消除了转化。在先前无转化能力的剪接位点突变体中恢复野生型序列可恢复转化。将野生型剪接供体位点突变为共有剪接位点会导致更强的转化表型，而将共有序列的 +2位置突变则显著降低转化频率。核糖核酸酶保护试验表明，转化缺陷型剪接位点突变体中E7 mRNA的量要低得多。核转录延伸实验表明，nt 880剪接位点突变体中E7信息的合成速率没有变化。此外，HPV - 16序列的突变表明，其他两个早期区域剪接供体位点在转化和有效的RNA积累中作用不大。这些结果表明，nt 880剪接供体位点的特定完整性对于E7 RNA的积累和有效的E7介导的转化都是必不可少的。
