REGULATION OF PAPILLOMAVIRUS GENE EXPRESSION

乳头状病毒基因表达的调控

• 批准号: 6100812
• 负责人: C C BAKER
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6100812
• 关键词: RNA splicing; bovine papillomavirus; cell differentiation; gene expression; genetic mapping; genetic regulation; genetic regulatory element; human immunodeficiency virus 1; human papillomavirus; human tissue; keratinocyte; messenger RNA; microorganism culture; nucleic acid sequence; polyadenylate; posttranscriptional RNA processing; tissue /cell culture; transcription factor; virus RNA; virus genetics; virus protein; yeast two hybrid system

The papillomaviruses are epitheliotropic viruses which induce benign and malignant lesions in a variety of squamous epithelia such as skin and the cervix. The papillomavirus life cycle is intimately linked with the differentiation state of the squamous epithelium which it infects. One goal of this project is to establish both tissue culture and animal systems that can be used to study the full viral life cycle. Alison McBride, in collaboration with this lab, has successfully obtained productive infection with cloned bovine papillomavirus type 1 (BPV-1) DNA by transfection into bovine keratinocytes in culture and differentiation of these cells with a combination of organotypic culture and nude mouse xenografts. We have also cloned HPV-16 genomic DNA from a cell line (W12) which harbors extrachromosomal HPV-16. Preliminary experiments indicate that we are able to transfect primary human foreskin keratinocytes (HFK) with this DNA and establish cell lines that maintain extrachromosomal HPV-16 DNA. These systems should allow us to use mutant papillomaviruses to study the roles of viral cis elements and trans factors in the viral life cycle. We have also developed a nude mouse xenograft system to study the differentiation-dependent processing of papillomavirus pre-mRNAs. This system uses integrated expression vectors and allows the assay of cis-processing elements that overlap viral genes essential for viral replication. Surprisingly, mice grafted with HaCat cells expressing a BPV-1 late pre-mRNA (containing the E4 and E5 ORFs but not the E6 or E7 ORFs) developed large tumors with characteristics of papillomavirus-induced papillomas. This system will also be used to investigate the signaling pathways through which E5 functions and the roles of E4 and E5 in the development of papillomas. BPV-1 late pre-mRNAs are alternatively spliced in a differentiation dependent manner. This alternative splicing is an essential component of the early to late switch in viral gene expression. Several cis- elements have been identified which regulate splice site choice in vitro. Immediately downstream of the first of two alternative 3' splice sites is a bipartite splicing regulatory element consisting of a purine- rich exonic splicing enhancer (ESE) and a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE-like element is located a short distance upstream of the second alternative 3' splice site. This element could act either as an enhancer on the upstream 3' splice site or as a repressor on the downstream 3' splice site. We have now shown that mutation of either ESE switches splice site usage in vivo, highlighting the importance of these two elements. Both ESE elements bind the same set of SR splicing factors. This arrangement potentially allows the coordinated regulation of two alternative splice sites by the same transacting factors. We have now carried out a detailed characterization of the ESS. In vitro splicing studies using several heterologous pre- mRNAs indicate that suppression of splicing requires a suboptimal 3' splice site but not an ESE, suggesting that the ESS works directly on the 3' splice site. Native gel electrophoresis has been used to demonstrate that the ESS inhibits early steps in spliceosomal assembly. Mutational analysis of the ESS showed that although the entire ESS is necessary for maximal suppression of splicing in vitro, the central C- rich region is the most important. UV cross-linking and immunoprecipitation studies showed that U2AF65 binds the U/C-rich 5' half, the C-rich central region binds 30 and 55 kDa SR proteins, and the AG-rich 3' end binds the SR protein ASF/SF2.

乳头瘤病毒是亲上皮病毒，可诱导良性和 皮肤、皮肤等多种鳞状上皮的恶性病变 子宫颈。乳头瘤病毒的生命周期与 其感染的鳞状上皮的分化状态。一 该项目的目标是建立组织培养和动物 可用于研究完整病毒生命周期的系统。艾莉森 McBride 与该实验室合作，成功获得 克隆牛乳头瘤病毒 1 型 (BPV-1) 的生产性感染 将 DNA 转染至培养物中的牛角质形成细胞中并 结合器官型培养对这些细胞进行分化 和裸鼠异种移植物。我们还克隆了 HPV-16 基因组 DNA 携带染色体外 HPV-16 的细胞系 (W12)。初步的 实验表明我们能够转染原代人类 包皮角质形成细胞 (HFK) 具有该 DNA 并建立细胞系 维持染色体外 HPV-16 DNA。这些系统应该让我们能够 使用突变乳头瘤病毒研究病毒顺式元件的作用和 病毒生命周期中的反式因子。我们还开发了裸体 小鼠异种移植系统研究分化依赖性加工 乳头瘤病毒前体 mRNA。本系统采用集成表达 载体并允许分析重叠的顺式加工元件 病毒复制所必需的病毒基因。令人惊讶的是，小鼠被移植了 HaCat 细胞表达 BPV-1 晚期前 mRNA（包含 E4 和 E5 ORF（但不是 E6 或 E7 ORF）产生了大肿瘤 乳头瘤病毒诱发的乳头状瘤的特征。该系统将 也可用于研究 E5 所通过的信号通路 E4 和 E5 在乳头状瘤发展中的功能和作用。 BPV-1 晚期前 mRNA 在分化过程中选择性剪接 依赖方式。这种选择性剪接是一个重要组成部分 病毒基因表达的早期到晚期的转变。几个顺式 已鉴定出调节剪接位点选择的元件 体外。紧邻两个替代 3' 剪接中第一个的下游 位点是一个二分剪接调节元件，由嘌呤组成 丰富的外显子剪接增强子（ESE）和富含嘧啶的外显子 剪接抑制器（ESS）。第二个类似 ESE 的元件位于短路处 第二个替代 3' 剪接位点上游的距离。这个元素 可以作为上游 3' 剪接位点的增强子或作为 下游 3' 剪接位点上的阻遏物。我们现在已经证明 任一 ESE 开关体内剪接位点使用的突变，突出显示 这两个要素的重要性。两个 ESE 元素绑定相同 SR 拼接因子集。这种安排可能允许 同一基因对两个选择性剪​​接位点的协调调控 交易因素。我们现在已经进行了详细的表征 ESS 的。使用几种异源预剪接的体外剪接研究 mRNA 表明剪接抑制需要次优的 3' 剪接位点但不是 ESE，表明 ESS 直接在 3'剪接位点。天然凝胶电泳已被用于 证明 ESS 抑制剪接体组装的早期步骤。 ESS 的突变分析表明，虽然整个 ESS 是 体外最大抑制剪接所必需的，中央C- 富裕地区是最重要的。紫外线交联和 免疫沉淀研究表明 U2AF65 结合富含 U/C 的 5' 一半，富含 C 的中心区域结合 30 和 55 kDa SR 蛋白，并且 富含 AG 的 3' 末端结合 SR 蛋白 ASF/SF2。