REGULATION OF PAPILLOMAVIRUS GENE EXPRESSION

乳头状病毒基因表达的调控

• 批准号: 6160912
• 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/6160912
• 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.

乳头瘤病毒是一种嗜上皮性病毒，可引起良性和 各种鳞状上皮的恶性病变，如皮肤和 宫颈。乳头瘤病毒的生命周期与 受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 可以用来研究整个病毒生命周期的系统。艾莉森 麦克布莱德与这个实验室合作，成功地获得了 克隆牛乳头瘤病毒1型(BPV-1)的生产性感染 DNA导入牛角质形成细胞的培养和鉴定 器官分型联合培养对这些细胞的分化 和裸鼠异种移植。我们还克隆了人乳头瘤病毒16型基因组DNA 一种携带染色体外HPV-16的细胞系(W12)。初步 实验表明，我们能够将原代人类 用这种DNA包皮角质形成细胞(HfK)并建立细胞系 保持染色体外的HPV-16DNA。这些系统应该允许我们 利用突变型乳头瘤病毒研究病毒顺式元件和 病毒生命周期中的反式因子。我们还开发了一种裸体 小鼠异种移植系统研究依赖分化的加工 乳头瘤病毒的前mRNAs。本系统采用集成表达方式 载体，并允许检测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，老鼠被移植到 HaCat细胞表达BPV-1晚期Pre-mRNA(包含E4和 E5 ORF，而不是E6或E7 ORF)发展为大肿瘤 乳头瘤病毒所致乳头状瘤的特点。这个系统将会 也被用来研究E5通过的信号通路 E4和E5的功能及其在乳头状瘤发生发展中的作用 BPV-1晚期前mRNAs在分化中交替剪接 依赖的态度。这种可选的剪接是一个必不可少的组件 病毒基因表达的早到晚的切换。几个顺位- 已经确定了调控剪接位点选择的因素 体外培养。紧接在两个备选3‘拼接中的第一个的下游 SITS是一个由嘌呤组成的两部分剪接调控元件- 富含外显子剪接增强子(ESE)和富含嘧啶的外显子 剪接抑制因子(ESS)。第二个类似ESE的元素位于一个短的 第二个备选3‘拼接点上游的距离。此元素 既可以作为上游3‘剪接位点的增强子，也可以作为 位于下游3‘剪接部位的抑制子。我们现在已经证明了 体内ESE开关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 一组SR剪接因子。这种安排潜在地允许 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刻画 ESS的成员。几种异源前体的体外剪接研究 MRNA表明，抑制剪接需要次优的3‘ Splice站点，而不是ESE，这表明ESS直接在 3‘拼接位点。天然凝胶电泳法已被用于 证明ESS抑制剪接体组装的早期步骤。 ESS的突变分析表明，尽管整个ESS是 最大限度地抑制体外剪接所必需的中央C- 富裕地区是最重要的。紫外光交联和 免疫沉淀研究表明，U2AF65与富含U/C的5‘结合 一半，富含C的中心区与30和55 kDa的SR蛋白结合，而 富含银的3‘端与SR蛋白ASF/SF2结合。