State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changbai Rd 155, Beijing 102206, People's Republic of China.
BMC Mol Biol. 2012 Feb 15;13:5. doi: 10.1186/1471-2199-13-5.
The human papillomavirus (HPV) E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa) substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells.
CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA) showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2.
These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.
人乳头瘤病毒（HPV）E2 蛋白是一种多功能 DNA 结合蛋白。HPV E2 的转录活性通过与 HPV 基因组上游调控区的特定结合位点结合来介导。先前我们报道了一位寻常疣患者的 HPV-2 变体，其 E2 ORF 中有五个点突变，即 L118S、S235P、Y287H、S293R 和 A338V。在 HPV-2 LCR 的控制下，突变 HPV E2 的共表达诱导病毒早期启动子的活性增加。在本研究中，构建了一系列编码 E2 蛋白的哺乳动物表达质粒，这些质粒的 E2 蛋白带有一个至五个氨基酸（aa）取代这些突变。将这些构建体转染到 HeLa、C33A 和 SiHa 细胞中。
CAT 表达分析表明，增强的启动子活性是由于在 DNA 结合域内包含 A338V 突变的 E2 构建体的共表达所致。Western blot 分析表明，瞬时转染的 E2 表达质粒，无论是原型还是 A338V 突变体，在细胞中均持续表达。为了研究 E2 突变对其 DNA 结合活性的影响，表达并纯化了一系列具有不同长度的重组 E2 蛋白。电泳迁移率变动分析（EMSA）表明，A338V 突变的 E2 蛋白与含有两个 E2 结合位点的人工探针或 HPV-2 和 HPV-16 启动子近端 LCR 序列的结合亲和力明显强于 HPV-2 原型 E2。此外，含有 A338V 突变的构建体的共表达显示对异源 HPV-16 早期启动子 P97 的活性高于原型 E2。
这些结果表明，aa338 从丙氨酸突变为缬氨酸对 E2 的 DNA 结合及其转录调控至关重要。
