Regulation of HPV16 late gene expression during keratinocyte differentiation

描述（由申请人提供）：项目摘要。乳头瘤病毒的晚期基因在其生命周期的所有阶段都被转录。特别地，在mRNA和蛋白质水平上的晚期基因表达被限制在角质形成细胞分化的晚期阶段，此时正在产生感染性病毒体。虽然这种限制的分子基础尚不清楚，但一个合理的贡献因素是在病毒晚期基因的3'UTR中发现的负调控元件（NRE）。牛乳头瘤病毒1型（BPV1）和人乳头瘤病毒16型（HVP 16）的NRE在原代角质形成细胞（天然宿主细胞）或各种细胞系中抑制基于荧光素酶的测定中的晚期基因表达。BPV1 NRE通过与U1 snRNP剪接因子结合而抑制，导致polyA尾添加的抑制。如果没有polyA尾，病毒晚期基因mRNA永远不会成熟，并且可能被核外泌体降解。与BPV1相反，HPV 16 NRE远未被理解，并且更复杂，因为它由4个弱的重叠的U1 snRNP结合位点组成，所述结合位点连接到功能未知的下游富含GU的区域。该提案有两个总体目标，首先是表征HPV16 NRE的机制，其次是确定其对分化角质形成细胞的自然环境中的病毒生命周期的重要性。值得注意的是，与转录水平相比，HPV16基因在RNA加工水平的调控明显不足。这项工作的成功完成将严格测试NRE对病毒生命周期的重要性，并将导致对NRE功能的机械理解。本案无关据估计，全世界每年约有40万例宫颈癌新发病例，是仅次于乳腺癌的女性癌症相关死亡的第二大原因。人乳头瘤病毒（HPV）DNA在超过90%的宫颈癌中发现，在肛门生殖器癌中也很普遍。流行病学和实验室研究表明，HPV和HPV 16在这些癌症中起着致病作用。出于这个原因，HPV 16株的分子生物学已经被深入研究。如果这一提议成功地鉴定出调节病毒晚期基因表达的新蛋白质，那么可以追求通过破坏这些调节蛋白质来破坏病毒生命周期的新治疗方法。还可以开发基于这些调节蛋白的差异表达的诊断测试。

DESCRIPTION (provided by applicant): Project Summary. The late genes of the papillomaviruses are transcribed throughout all stages of its life cycle. Paradoxically, late gene expression at the mRNA and protein level is restricted to late stages of keratinocyte differentiation when infectious virions are being produced. Although the molecular basis of this restriction is not known, a plausible contributory factor is the Negative Regulatory Element (NRE) found in the viral late gene's 3'UTR. The NREs of Bovine Papillomavirus 1 (BPV1) and human papillomavirus 16 (HVP16) inhibit late gene expression in reporter-based assays in both primary keratinocytes (the natural host cell) or in various cell lines. The BPV1 NRE inhibits by binding to the Ul snRNP splicing factor resulting in inhibition of polyA tail addition. Without a polyA tail, the viral late gene mRNA never matures and is presumably degraded by the nuclear exosome. In contrast to BPV1, the HPV16 NRE is far less understood and is more complicated because it consists of 4 weak, overlapping Ul snRNP binding sites linked to a downstream GU rich region of unknown function. This proposal has two overall goals, first to characterize the HPV16 NRE mechanistically and second to determine its importance to the viral life cycle within the natural environment of differentiating keratinocytes. It is important to note that the regulation of HPV16 genes at the RNA processing level is strikingly understudied when compared to the transcriptional level. The successful completion of this work will rigorously test the importance of the NRE to the viral life cycle and will lead to a mechanistic understanding of NRE function. Relevance. It is estimated that about 400,000 new cases of cervical carcinoma occur worldwide every year, and it is the second leading cause of cancer-related deaths among women after breast cancer. Human papillomavirus (HPV) DNA is found in over 90% of cervical cancers and is also prevalent in anogenital cancers. Epidemiologic and laboratory studies have shown a causative role for HPV and HPV 16 in these cancers. For this reason the molecular biology of the HPV 16 strain has been intensely studied. If this proposal successfully identifies new proteins that regulate viral late gene expression then new therapeutic approaches that disrupt the viral life cycle by disrupting these regulatory proteins can be pursued. Diagnostic tests, based on differential expression of these regulatory proteins, can also be developed.