CHARACTERIZATION OF THE PAPILLOMAVIRUSES

乳头瘤病毒的特征

• 批准号: 2463587
• 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/2463587
• 关键词: Papillomavirus; athymic mouse; binding proteins; crosslink; gene expression; genetic enhancer element; genetic regulatory element; host organism interaction; human tissue; immunoprecipitation; keratinocyte; posttranscriptional RNA processing; transcription factor; virus genetics; virus protein; virus replication

The papillomavirus life cycle is intimately linked with the differentiation state of the squamous epithelium as skin and the cervix. which it infects. One goal of this project is to set up an animal model that can be used to study the full viral life cycle. We The papillomaviruses are epitheliotropic viruses which induce benign and malignant lesions in a variety of squamous epithelia such are now ready to begin grafting human keratinocytes onto the backs of nude mice. In this environment these cells form a fully differentiated squamous epithelium. Wild type and mutant human papillomaviruses will be introduced into these cells to study the roles of cis elements and trans factors in the viral life cycle. Previously we have used in situ hybridization to demonstrate that alternative splicing of BPV-1 late pre-mRNAs is regulated in a differentiation dependent manner. Several cis-elements have been identified which regulate splice site choice. Immediately downstream of the first of two alternative 3' splice sites is a purine-rich positive element known as an exonic splicing enhancer (ESE) which is required for efficient utilization of that splice site. Immediately downstream of this element is a pyrimidine-rich negative element known as an exonic splicing suppressor (ESS). Together these two elements form a bipartite splicing regulatory element which modulates utilization of the upstream splice site. Only two other bipartite splicing regulatory elements are known. Further studies on the ESS indicate that it can suppress splicing of HIV-1 and RSV pre-mRNAs which contain suboptimal splice sites but has no effect on a -globin pre-mRNA containing strong splice sites. A third element similar to the exonic splicing enhancer has been identified a short distance upstream of the second alternative 3' splice site. Although this element can function as a splicing enhancer when located near a 3' splice site in an exonic position, we speculate that it functions in its normal location as an intronic splicing suppressor. This arrangement potentially allows the coordinated regulation of two alternative splice sites by the same trans-acting factors. Combined UV crosslinking and immunoprecipitation experiments indicated that both splicing enhancers bind a subset of the SR family of splicing factors (ASF/SF2, SRp55, and SRp75). Mutational analysis indicated that this binding is functionally relevant. The ESS binds a 65 kDa protein which may be the splicing factor U2AF65. We are currently investigating whether the activity of these factors is regulated by differentiation of the epithelial cells. In other studies we have screened an expression library for proteins which bind to the BPV-1 early 3' untranslated region. Only one protein, a bovine Y-box protein, was identified. The functional significance of this binding is still unknown.

乳头状瘤病毒的生命周期与 鳞状上皮细胞的分化状态，如皮肤和 子宫颈 它感染的病毒。 该项目的一个目标是建立一个 动物模型，可用于研究病毒的整个生命周期。 我们 乳头状瘤病毒是嗜上皮病毒， 和各种鳞状上皮的恶性病变， 准备开始将人类角质形成细胞移植到 小鼠 在这种环境中，这些细胞形成完全分化的 鳞状上皮 野生型和突变型人乳头瘤病毒将 导入这些细胞，研究顺式元件的作用， 病毒生命周期中的反式因子 以前，我们已经使用原位杂交来证明， BPV-1晚期前体mRNA的选择性剪接在一种 分化依赖方式。一些顺式元件已经被 确定了调节剪接位点选择的基因。 紧下游 两个可变3'剪接位点中的第一个是富含嘌呤的 一个被称为外显子剪接增强子（ESE）的阳性元件， 这是有效利用剪接位点所必需的。 立即 该元件的下游是富含嘧啶的负元件， 外显子剪接抑制子（ESS）。 这两个元素结合在一起 形成二分剪接调节元件， 上游剪接位点的利用。 只有另外两个两党 剪接调节元件是已知的。 关于ESS的进一步研究 表明它可以抑制HIV-1和RSV前体mRNA剪接， 含有次优剪接位点，但对 - 珠蛋白 含有强剪接位点的前mRNA。 第三个元素类似于 外显子剪接增强子已被鉴定为短距离 在第二选择性3 ′剪接位点的上游。 虽然这 当位于3'端附近时，元件可以起到剪接增强子的作用 剪接位点，我们推测它在 它作为内含子剪接抑制子的正常位置。 这 这种安排可能允许协调调节两个 通过相同的反式作用因子选择剪接位点。 组合 紫外交联和免疫沉淀实验表明， 剪接增强子结合剪接因子SR家族的一个子集 (ASF/SF 2、SRp 55和SRp 75）。突变分析表明， 绑定功能相关。 ESS结合一种65 kDa的蛋白质， 可以是剪接因子U2 AF 65。 我们目前正在调查 这些因子的活性是否受到分化的调节 的上皮细胞。 在其他研究中，我们筛选了一个表达文库， 其与BPV-1早期3'非翻译区结合。 只有一 蛋白，牛Y-盒蛋白，被鉴定。 功能 这种结合的意义仍然未知。