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Development of an anti-viral agent based on functional analysis of the human papillomavirus E2 protein

基于人乳头瘤病毒E2蛋白功能分析开发抗病毒剂

基本信息

批准号：
15591779
负责人：
FUJII Takuma
金额：
$ 2.18万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591779/
关键词：
peptide papillomavirus transcription replication nuclear localization signal

项目摘要

The E2 protein of HPV is a DNA-binding protein that regulates the expression of the E6 and E7 genes and is believed to be extremely important in the analysis of the mechanism underlying viral carcinogenesis. Accordingly, we attempted to isolate the peptide that binds to the E2 protein and inhibit its transcription. First we isolated a phage clone binding to the E2 protein of HPV type 16. As a result of clonal analysis, we identified a peptide sequence containing tryptophan binding to the E2 protein. When tryptophan was replaced with alanine and the binding activity to the E2 protein was analyzed, the modified peptide was found to exhibit reduced binding activity. Furthermore, we produced a synthetic peptide by adding a nuclear localization signal to the isolated peptide sequence, and examined its effect on the transcriptional activity of the E2 by means of the luciferase assay ; it was found in this experiment that the transcriptional activity was decreased. These findings showed that the isolated peptide binding to the E2 protein inhibited the transcriptional activity. Then, we incorporated a nucleotide sequence obtained on the basis of this peptide sequence in the expression vector, forcibly expressed the peptide into the cells and determined its effect on the transcriptional activity of E2. However, to date, we have not discovered any sequence that efficiently inhibits the transcription activity. A possible reason for this is that such a peptide is not effectively expressed in the cells. Since the E2 protein is also involved in virus replication, we established an in viiro system for studying viral gene replication using the E2 protein. We added the above-described peptide using this system to examine whether or not gene replication would be inhibited. However, we found no inhibitory effect of the peptide on the virus replication. These results indicate that the peptide identified by us specifically inhibits viral transcription.

HPV的E2蛋白是调节E6和E7基因表达的DNA结合蛋白，并且被认为在分析病毒致癌的潜在机制中极其重要。因此，我们试图分离与E2蛋白结合并抑制其转录的肽。首先，我们分离了与HPV 16型的E2蛋白结合的噬菌体克隆。作为克隆分析的结果，我们确定了含有色氨酸结合到E2蛋白的肽序列。当色氨酸被丙氨酸取代并分析与E2蛋白的结合活性时，发现修饰的肽表现出降低的结合活性。此外，我们通过将核定位信号添加到分离的肽序列来产生合成肽，并通过荧光素酶测定来检查其对E2的转录活性的影响;在该实验中发现转录活性降低。这些结果表明，分离的肽结合到E2蛋白抑制转录活性。然后，我们将基于该肽序列获得的核苷酸序列掺入表达载体中，将该肽强制表达到细胞中，并测定其对E2转录活性的影响。然而，迄今为止，我们还没有发现任何有效抑制转录活性的序列。一个可能的原因是这种肽在细胞中不能有效表达。由于E2蛋白也参与病毒复制，我们建立了一个体外系统，用于研究病毒基因复制使用E2蛋白。我们使用该系统添加上述肽以检查基因复制是否会被抑制。然而，我们发现肽对病毒复制没有抑制作用。这些结果表明我们鉴定的肽特异性抑制病毒转录。