INTERACTION OF HIV-1 REV WITH NUCLEOLAR PROTEIN B23

这项研究的总体目标是了解核仁的作用， 蛋白B23作为HIV-1 Rev的可能受体和/或转运蛋白 蛋白 蛋白质B23（Mr 38，000）是一种RNA相关的非核糖体蛋白， 磷蛋白和假定的核糖体装配因子。 蛋白质B23形式 与HIV-1 Rev蛋白的特异性复合物，并建议， B23是Rev.The Rev的核仁受体和/或转运蛋白 蛋白质调节病毒mRNA的剪接，使HIV-1的结构 蛋白质在感染后期产生。 我们描述了两个 B23的形式（B23.1和B23.2），我们能够通过 在细菌中表达。 我们现在建议进一步描述 Rev-B23相互作用，并研究B23在 Rev蛋白的运输。 具体目标是：（a）确定 B23亚型结构如何影响Rev结合， 翻译后修饰 自然和自然的相对亲和力 将确定细菌产生的B23.1和B23.2版本。 类似 将用酪蛋白体外磷酸化的B23.1进行比较 激酶II和CDC2型蛋白激酶。 B23的影响 将通过电子显微镜研究亚型对Rev纤维形成的影响 显微镜(b)来确定B23序列的哪一部分 Rev蛋白。 这些实验将涉及竞争， B23的合成肽、部分蛋白水解和缺失突变 在细菌中表达。(c)为了检验B23运输Rev 从细胞质到细胞核或核仁。 为此，我们将使用 为核输入研究设计的毛地黄皂苷透化细胞系统。 荧光标记的Rev和B23在细胞核中的积累将 可以通过荧光显微镜和图像分析来测量。 的影响 将测量添加的B23对Rev蓄积动力学的影响。 研究 还将对共价交联的RevB23复合物进行， 确定B23是否真的携带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， 互动，而且还有助于开发针对目标的手段 可能干扰病毒的药物对特定HIV蛋白的影响 复制的

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.