CONTROL OF PAPILLOMAVIRUS LATE GENE EXPRESSION

乳头状病毒晚期基因表达的控制

• 批准号: 2463637
• 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/2463637
• 关键词: RNA splicing; bovine papillomavirus; cell differentiation; gene expression; genetic mapping; genetic regulatory element; human immunodeficiency virus 1; keratinocyte; messenger RNA; microorganism culture; nucleic acid sequence; polyadenylate; posttranscriptional RNA processing; transcription factor; virus RNA; virus genetics; virus protein; yeast two hybrid system

The papillomaviruses are small DNA tumor viruses which induce benign and malignant lesions in squamous epithelia of higher vertebrates. The complete life cycle of these viruses occurs only in the differentiated keratinocytes of a squamous epithelium. This project currently focuses on posttranscriptional regulatory mechanisms which may be important in regulating papillomavirus gene expression during keratinocyte differentiation, with particular emphasis on the regulation of bovine papillomavirus type 1 (BPV-1) translation and polyadenylation. Four short open reading frames (uORFs) in the 5' untranslated region (UTR) of the major capsid protein (L1) mRNA have been shown to block translation of this mRNA. Mutation of each AUG singly and in combination showed that mutation of the second and third AUGs gave the greatest increase in translation in transfection assays. It is not known whether the mechanism of translational inhibition is a block to ribosome scanning or a direct effect of one of the peptides. However, the second uORF encodes a short arginine-rich peptide with similarities to the RNA-binding domain of the human immunodeficiency virus (HIV-1) tat protein, suggesting that it may have an RNA target. We have also carried out a yeast two hybrid screen with this peptide to identify potential protein targets. This screen gave two unknown proteins, one of which also interacts with HIV-1 tat. The 3'UTR of the L1 mRNA also contains a negative posttranscriptional regulatory element. This element has been mapped to a 5' splice site which binds at least one splicing factor, the U1 snRNP, but is not used for splicing. In vivo studies from the LTVB as well as in vitro studies from other labs have shown that this element inhibits polyadenylation. We have also shown that the HIV-1 Rev protein can block the effect of this element. We are currently using mutant Rev proteins to investigate whether this function of Rev is due to its ability to facilitate nucleocytoplasmic transport or to interact with the splicing machinery. We are also testing other elements, such as retroviral constitutive transport elements and viral elements which confer splicing independent expression, for their ability to counteract the effect of a regulatory 5' splice site. Preliminary data indicates that the hepatitis B Virus (HBV) PRE element does not have this ability, indicating that it does not function in the same way as the HIV Rev protein.

乳头状瘤病毒是小的DNA肿瘤病毒， 以及高等脊椎动物鳞状上皮的恶性病变。 的 这些病毒的完整生命周期只发生在分化的 鳞状上皮的角质细胞。 该项目目前重点 转录后调节机制，这可能是重要的， 角质形成细胞中乳头瘤病毒基因表达的调控 分化，特别强调牛的调节 乳头瘤病毒1型（BPV-1）翻译和多聚腺苷酸化。 四 5'非翻译区（UTR）中的短开放阅读框（uORF） 的主要衣壳蛋白（L1）mRNA已被证明可以阻断 翻译这个mRNA。 每个AUG的突变单独和在 组合显示，第二和第三AUG的突变给出了 转染测定中翻译的最大增加。 不 已知翻译抑制的机制是否是阻断 核糖体扫描或其中一种肽的直接作用。 然而，在这方面， 第二个uORF编码一个短的富含精氨酸的肽， 与人类免疫缺陷病毒（HIV-1）的RNA结合结构域 达特蛋白，这表明它可能有一个RNA靶点。 我们还 用该肽进行酵母双杂交筛选， 潜在的蛋白质靶点。该筛选得到两种未知蛋白质，一种 其中还与HIV-1达特相互作用。 L1 mRNA的3 'UTR也含有负转录后调控区。 调节元件。 该元件已被映射到5'剪接位点 其结合至少一种剪接因子U1 snRNP，但不被使用 用于拼接。 LTVB的体内研究以及体外研究 其他实验室的研究表明， 聚腺苷酸化 我们还表明，HIV-1 Rev蛋白可以 阻止这个元素的影响。 我们现在用的是变种人Rev 研究Rev的这种功能是否是由于其 促进核质转运或与 拼接机器 我们还在测试其他元素，例如 逆转录病毒组成型转运元件和病毒元件， 赋予剪接独立表达，因为它们能够抵消 调节性5'剪接位点的作用。 初步数据表明 B型肝炎病毒（HBV）的PRE元件不具有这种 能力，表明它的功能与 HIV Rev蛋白。