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 Med Virol. 1995 Dec;47(4):445-53. doi: 10.1002/jmv.1890470426.
Transformation of primary baby rat kidney cells by the human papillomavirus type 16 (HPV 16) E7 gene and efficient accumulation of E7 RNA have been shown by this laboratory to depend on the integrity of the nucleotide position (nt) 880 splice donor site. Here, the splice sites within the HPV 16 E6 open reading frame (ORF) and the sites of the SV40 splicing unit were examined for an ability to provide this requirement. Constructs containing the HPV 16 E6 sites and the SV40 splice site sequences were used for transformation and RNase protection assays. E6 splice sites supported a low level of transformation, in assays for complete HPV 16 early region constructs containing loss-of-function mutations of the nt 880 site. Using constructs with wild-type E6 or SV40 splice sites showed that both splice sites could substitute similarly for the requirement in cis of the nt 880 site for transformation. HPV 16 E6 mutated splice site and SV40 splice site in reverse, nonfunctional orientation relative to the promoter, were not transformation competent. The HPV 16 E7 RNA levels for the E6 splice site constructs correlated closely with the transformation frequency. The SV40 splice sites were required for E7 transcript accumulation. The results showed E6 splice site function and evidence for enhanced exon skipping from E6 splice donor site to acceptor sites 3' of the E7 ORF. This was shown with constructs containing loss-of-function mutations of the nt 880 site. These results confirmed the function of the splice sites by the transformation competent constructs and suggested lower transformation frequency than for wild type was due to skipping of the E7 exon. These patterns of transcripts may have a role in the regulation of gene expression during progression to malignancy. The combined results revealed that the general presence of a functional splice donor site was absolutely required for transformation by HPV 16 E7 and accumulation of E7 RNA.
本实验室已证明，人乳头瘤病毒16型（HPV 16）E7基因对原代幼鼠肾细胞的转化以及E7 RNA的有效积累取决于核苷酸位置（nt）880剪接供体位点的完整性。在此，对HPV 16 E6开放阅读框（ORF）内的剪接位点以及SV40剪接单元的位点进行了检测，以确定其是否能够满足这一要求。含有HPV 16 E6位点和SV40剪接位点序列的构建体用于转化和核糖核酸酶保护试验。在对含有nt 880位点功能缺失突变的完整HPV 16早期区域构建体进行的试验中，E6剪接位点支持低水平的转化。使用具有野生型E6或SV40剪接位点的构建体表明，这两个剪接位点在顺式条件下对nt 880位点转化的要求具有相似的替代作用。相对于启动子呈反向、无功能方向的HPV 16 E6突变剪接位点和SV40剪接位点不具备转化能力。E6剪接位点构建体的HPV 16 E7 RNA水平与转化频率密切相关。E7转录本积累需要SV40剪接位点。结果显示了E6剪接位点的功能以及从E6剪接供体位点到E7 ORF 3'端受体位点外显子跳跃增强的证据。这在含有nt 880位点功能缺失突变的构建体中得到了证实。这些结果通过具有转化能力的构建体证实了剪接位点的功能，并表明转化频率低于野生型是由于E7外显子的跳跃。这些转录本模式可能在恶性进展过程中的基因表达调控中发挥作用。综合结果表明，功能性剪接供体位点的普遍存在是HPV 16 E7转化和E7 RNA积累所绝对必需的。
