INTERACTION OF HIV-1 REV WITH NUCLEOLAR PROTEIN B23

HIV-1 REV 与核仁蛋白 B23 的相互作用

• 批准号: 2069380
• 负责人: Mark O Olson
• 金额: $ 10.61万
• 依托单位: UNIVERSITY OF MISSISSIPPI MED CTR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2069380
• 关键词: digitonin; electron microscopy; fluorescence microscopy; gene deletion mutation; human immunodeficiency virus 1; intermolecular interaction; laboratory rat; molecular cloning; nucleoproteins; posttranslational modifications; protein isoforms; protein kinase; protein structure; protein transport; proteolysis; synthetic protein; virus protein

The general goal of this research is to understand the role of nucleolar protein B23 as a possible receptor and/or transporter of the HIV-1 Rev protein. Protein B23 (Mr 38,000), is a RNA-associated nonribosomal phosphoprotein and putative ribosome assembly factor. Protein B23 forms a specific complex with the HIV-1 Rev protein and it is suggested that B23 is the nucleolar receptor and/or transporter for Rev. The Rev protein regulates the splicing of viral mRNA so that HIV-1 structural proteins are produced late in the infection. We have characterized two forms of B23 (B23.1 and B23.2) and we are able to produce these by expression in bacteria. We now propose to further characterize the Rev-B23 interactions and to investigate the possible role of B23 in the transport of the Rev protein. The specific aims are: (a) to determine how Rev binding is affected by B23 isoform structure and posttranslational modification. Relative affinities of natural and bacterially produced B23.1 and B23.2 for Rev will be determined. Similar comparisons will be done with B23.1 phosphorylated in vitro with casein kinase II and with a CDC2 type protein kinase. The effects of B23 isoforms on Rev fiber formation will be investigated by electron microscopy. (b) to determine what part of the B23 sequence interacts with the Rev protein. These experiments will involve competition with synthetic peptides, partial proteolysis and deletion mutations of B23 expressed in bacteria. (c) to test the hypothesis that B23 transports Rev from the cytoplasm to the nucleus or nucleolus. For this we will use a digitonin-permeabilized cell system designed for nuclear import studies. The accumulation of fluorescently labeled Rev and B23 in the nucleus will be measured by fluorescence microscopy and image analyses. The effect of added B23 on the kinetics of Rev accumulation will be measured. Studies will also be performed on covalently cross-linked RevB23 complex to determine whether B23 actually carries Rev into the nucleus or nucleolus. These studies should not only answer questions about the Rev-B23 interactions but also aid in the development of a means of targeting a specific HIV protein by drugs which might interfere with viral replication.

这项研究的总体目标是了解核仁的作用。 B23蛋白可能作为HIV-1 Rev的受体和/或转运体 蛋白。蛋白质B23(Mr 38,000)，是一种与RNA相关的非核糖体 磷酸蛋白和可能的核糖体组装因子。蛋白质B23形式 与HIV-1 Rev蛋白的特异性复合体，提示 B23是Rev.的核仁受体和/或转运体 蛋白质调节病毒mRNA的剪接，使HIV-1的结构 蛋白质是在感染后期产生的。我们给出了两个特征 B23(B23.1和B23.2)的表格，我们能够通过以下方式生产这些表格 在细菌中表达。我们现在建议进一步描述 REV-B23的相互作用，并研究B23在 REV蛋白的运输。具体目标是：(A)确定 B23亚型结构对REV结合的影响 翻译后修饰。自然与自然的相对亲和力 将对REV细菌产生的B23.1和B23.2进行检测。类似 将与酪蛋白体外磷酸化的B23.1进行比较 蛋白激酶II和CDC2类型的蛋白激酶。B23的影响 REV纤维形成的异构体将用电子研究 显微镜。(B)确定B23序列的哪个部分与之相互作用 Rev蛋白。这些实验将涉及与 B23的合成肽、部分蛋白分解和缺失突变 在细菌中表达。(C)检验B23运输REV的假设 从细胞质到核或核仁。为此，我们将使用 专为核进口研究设计的洋地黄渗透细胞系统。 荧光标记的REV和B23在细胞核内的聚集 通过荧光显微镜和图像分析进行测量。的影响 测定了添加B23对REV累积动力学的影响。研究 还将对共价交联的RevB23复合体执行 确定B23实际上是否携带REV进入细胞核或核仁。 这些研究不仅应该回答关于REV-B23的问题 交互，而且还有助于开发一种针对 可能干扰病毒的药物产生的特异性HIV蛋白 复制。