Harry J B, Wettstein F O
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles, 90095, USA.
J Virol. 1996 Jun;70(6):3355-62. doi: 10.1128/JVI.70.6.3355-3362.1996.
Cottontail rabbit papillomavirus induces on cottontail and domestic rabbits papillomas which progress at a high frequency to carcinoma. The virus encodes three transforming proteins; one is translated from open reading frame (ORF) E7 and binds the retinoblastoma protein, and two, LE6 and SE6, are translated from the first and second ATGs of ORF E6, respectively. Here we show that neither of the E6 proteins coprecipitated with p53 in vitro, nor did they bind to a recently identified E6-binding protein (J. J. Chen, C. E. Reid, V. Band, and E. Androphy, Science 269:529-531, 1995). This protein was shown to bind to the E6 proteins of the high-risk human papillomairus types 16 and 18 but not to the low-risk human papillomavirus types VI and II. In-frame deletions cloned into the pZipNeo vector were used to identify structural features of SE6 and LE6 important for transformation of NIH 3T3 cells. Three deletions covering the amino-terminal half of SE6 did not transform cells. In two of the three deletions, two Cys-X-X-Cys motifs were deleted, each deletion preventing the formation of one of the potential small Zn fingers of SE6. Among the LE6 deletions, only one had a reduced transformation efficiency, while seven transformed cells at least as efficiently as wild-type LE6. In each of three of these seven mutants, two Cys-X-X-Cys motifs were deleted. None of the three amino acid deletions which abolished transformation by SE6 reduced transformation by LE6. Furthermore, transformation did not correlate with the level of SE6 or LE6 proteins detectable. ORF E8 colinear with ORF E6, which could generate a 50-amino-acid protein with a hydrophobic segment, did not transform cells when cloned into the pZipNeo vector. However, mutation of the E8 ATG, which did not alter the amino acid sequence of LE6, increased transformation by LE6 without affecting the level of LE6 expression. The data suggest that transformation by the E6 proteins is not mediated by interfering with p53 function or through binding to the E6-binding protein. Furthermore, different structural features are important to maintain transformation functions and protein stability of LE6 and SE6. Finally, E8 seems not to be a transforming protein but rather appears to modulate transformation bv LE6.
棉尾兔乳头瘤病毒可在棉尾兔和家兔身上诱发乳头瘤，这些乳头瘤会以较高频率发展为癌。该病毒编码三种转化蛋白；一种由开放阅读框（ORF）E7翻译而来，可结合视网膜母细胞瘤蛋白，另外两种，即LE6和SE6，分别由ORF E6的第一个和第二个ATG翻译而来。在此我们表明，两种E6蛋白在体外均未与p53共沉淀，它们也不与最近鉴定出的一种E6结合蛋白结合（J. J. 陈、C. E. 里德、V. 班德和E. 安德鲁菲，《科学》269:529 - 531, 1995）。已证明该蛋白可与高危型人乳头瘤病毒16型和18型的E6蛋白结合，但不与低危型人乳头瘤病毒VI型和II型的E6蛋白结合。克隆到pZipNeo载体中的框内缺失用于鉴定SE6和LE6对NIH 3T3细胞转化重要性的结构特征。覆盖SE6氨基末端一半的三个缺失突变体不能转化细胞。在这三个缺失突变体中的两个中，两个Cys - X - X - Cys基序被删除，每次删除都阻止了SE6潜在的一个小锌指结构的形成。在LE6缺失突变体中，只有一个的转化效率降低，而七个突变体转化细胞的效率至少与野生型LE6一样高。在这七个突变体中的三个中，每个都删除了两个Cys - X - X - Cys基序。消除SE6转化作用的三个氨基酸缺失突变体均未降低LE6的转化作用。此外，转化作用与可检测到的SE6或LE6蛋白水平无关。与ORF E6共线性的ORF E8，可产生一个带有疏水片段的50个氨基酸的蛋白，当克隆到pZipNeo载体中时不能转化细胞。然而，E8 ATG的突变，并未改变LE6的氨基酸序列，却增加了LE6的转化作用，而不影响LE6的表达水平。这些数据表明，E^蛋白的转化作用不是通过干扰p53功能或通过与E6结合蛋白结合来介导的。此外，不同的结构特征对于维持LE6和SE6的转化功能和蛋白稳定性很重要。最后，E8似乎不是一种转化蛋白，而是似乎能调节LE6的转化作用。
