Ai W, Narahari J, Roman A
Department of Microbiology and Immunology, Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, Indiana 46202-5120, USA.
J Virol. 2000 Jun;74(11):5198-205. doi: 10.1128/jvi.74.11.5198-5205.2000.
Human papillomavirus type 6 (HPV-6) is a low-risk HPV whose replication cycle, like that of all HPVs, is differentiation dependent. We have previously shown that CCAAT displacement protein (CDP) binds the differentiation-induced HPV-6 E1 promoter and negatively regulates its activity in undifferentiated cells (W. Ai, E. Toussaint, and A. Roman, J. Virol. 73:4220-4229, 1999). Using electrophoretic mobility shift assays (EMSAs), we now report that Yin Yang 1 (YY1), a multifunctional protein that can act as a transcriptional activator or repressor and that can also inhibit HPV replication in vitro, binds the HPV-6 E1 promoter. EMSAs, using subfragments of the promoter as competitors, showed that the YY1 binding site is located at the 5' end of the E1 promoter. When a putative YY1 site was mutated, the ability of YY1 to bind was greatly decreased. The activity of the mutated E1 promoter, monitored with the reporter gene luciferase, was threefold greater than that of the wild-type promoter, suggesting that YY1 negatively regulates HPV-6 E1 promoter activity. Nuclear extracts from differentiated keratinocytes showed decreased binding of YY1 to the wild-type promoter. Consistent with this, in differentiated keratinocytes, the activity of the transfected luciferase gene transcribed from the mutated promoter was comparable to that of the wild-type promoter; both promoters were up-regulated in differentiated keratinocytes compared to undifferentiated cells. These data suggest that YY1 functions in undifferentiated keratinocytes but not in differentiated keratinocytes. Both the wild-type and mutated promoters could be negatively regulated by overexpression of a plasmid encoding CDP. Thus, both YY1 and CDP appear to be negative regulators of the differentiation-induced HPV-6 E1 promoter and thereby the HPV life cycle. In contrast, only binding of CDP was detected using the E1 promoter of the high-risk HPV-31.
人乳头瘤病毒6型（HPV-6）是一种低风险的人乳头瘤病毒，其复制周期与所有HPV一样，依赖于分化。我们之前已经表明，CCAAT置换蛋白（CDP）结合分化诱导的HPV-6 E1启动子，并在未分化细胞中负调控其活性（W.艾、E.图桑和A.罗曼，《病毒学杂志》73:4220-4229，1999年）。现在，我们通过电泳迁移率变动分析（EMSA）报告，阴阳1（YY1）这种多功能蛋白可作为转录激活因子或抑制因子，并且在体外也能抑制HPV复制，它能结合HPV-6 E1启动子。使用启动子的亚片段作为竞争物的EMSA表明，YY1结合位点位于E1启动子的5'端。当一个假定的YY1位点发生突变时，YY1的结合能力大大降低。用报告基因荧光素酶监测的突变E1启动子的活性比野生型启动子高3倍，这表明YY1负调控HPV-6 E1启动子活性。分化角质形成细胞的核提取物显示YY1与野生型启动子的结合减少。与此一致的是，在分化角质形成细胞中，从突变启动子转录的转染荧光素酶基因的活性与野生型启动子相当；与未分化细胞相比，两种启动子在分化角质形成细胞中均上调。这些数据表明，YY1在未分化角质形成细胞中起作用，而在分化角质形成细胞中不起作用。野生型和突变型启动子都可被编码CDP的质粒过表达负调控。因此，YY1和CDP似乎都是分化诱导的HPV-6 E1启动子以及HPV生命周期的负调控因子。相比之下，使用高危HPV-3l的E1启动子时，仅检测到CDP的结合。
