CHARACTERIZATION OF THE PAPILLOMAVIRUSES

乳头瘤病毒的生命周期与 鳞状上皮作为皮肤的分化状态 宫颈。它会感染这种病毒。这个项目的一个目标是建立一个 可用于研究病毒全生命周期的动物模型。我们 乳头瘤病毒是一种嗜上皮性病毒，可引起良性病变。 以及各种鳞状上皮中的恶性病变 准备开始将人类角质形成细胞移植到裸鼠的背部 老鼠。在这种环境中，这些细胞形成了完全分化的 鳞状上皮。野生型和突变型人乳头瘤病毒将 被引入到这些细胞中来研究顺式元件和 病毒生命周期中的反式因子。 此前，我们已经使用原位杂交来证明 BPV-1晚期前mRNAs的选择性剪接在 分化依赖方式。几个顺式元素已经被 确定了哪些规范剪接位点的选择。紧随其后 在两个可供选择的3‘剪接位点中，第一个是富含嘌呤的 被称为外显子剪接增强子(ESE)的正性元件 有效利用该剪接部位所需的。立马 在这个元素的下游是一种已知的富含嘧啶的负元素 作为外显子剪接抑制因子(ESS)。把这两个元素加在一起 形成调控的两部分剪接调控元件 上游剪接位点的利用率。只有另外两个二人组 剪接调控元件是已知的。关于ESS的进一步研究 表明它可以抑制HIV-1和RSV前mRNAs的剪接， 含有次优剪接位点，但对α-珠蛋白没有影响 含有强剪接位点的前信使核糖核酸。类似于的第三个元素 外显子剪接增强子已经在短距离内被发现 位于第二个备选3‘拼接位点的上游。虽然这件事 当元件位于3‘端附近时，可以起到剪接增强剂的作用 剪接位点处于外显子位置，我们推测它在 它作为内含子剪接抑制因子的正常位置。这 安排可能允许协调监管两个 由相同的反式作用因子产生的选择性剪接位点。组合在一起 紫外光交联和免疫沉淀实验表明 剪接增强剂结合SR家族剪接因子的一个子集 (ASF/SF2、SRp55和SRp75)。突变分析表明，这是 绑定在功能上是相关的。ESS结合一个65 kDa的蛋白， 可能是剪接因子U2AF65。我们目前正在调查 这些因子的活性是否受分化的调节 上皮细胞。 在其他研究中，我们已经筛选了蛋白质的表达文库 它与BPV-1早期的3‘非翻译区结合。只有一个 鉴定了一种牛Y-box蛋白。功能界别 这种结合的意义尚不清楚。

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.