Ty3 viruslike particle morphogenesis and host interactions

描述(申请人提供)：逆转录病毒是艾滋病和癌症的原因，逆转录病毒是真核基因组的重要组成部分。然而，在从组装到到达核孔的生命周期的细胞质阶段，逆转录病毒和逆转录元件与宿主细胞的相互作用仍然有相当大的未知数。我们正在使用酿酒酵母中的Ty3逆转录病毒样元件作为模型系统来研究生命周期的这一方面。Ty3由两个重叠的阅读框架GAG3和POL3组成，编码Gag3和Gag3-Pol3，它们被加工成成熟的衣壳、核衣壳、蛋白酶、逆转录酶和整合酶。这些蛋白质在结构和功能上与逆转录病毒相似。表达Ty3的细胞产生病毒样颗粒(VLP)，病毒样颗粒经历蛋白分解成熟、逆转录、脱壳和核进入。在过去的资助期间，我们已经开发了一些工具来促进拟议的工作，包括一组100多个在Ty3转座中存在缺陷的突变体、针对Ty3蛋白的抗体以及用荧光记者标记的Ty3元件。对产生Ty3的细胞的显微镜观察表明，Ty3的mRNA和蛋白首先出现在外周细胞质中，但被输送到核周团，在那里观察到VLP。拟议的工作有具体的目标A-E：。确定VLP组装和核传递所需的Ty3RNA和结构蛋白。蛋白质和RNA将分别以反式表达，以确定RNA是否必须编码Gag3才能被运输到核周区域。如果需要Gag3，则将确定是否可以以TRANS形式提供。将确定组装和运输Ty3蛋白和RNA所需的Gag3残基。已鉴定出130个影响Ty3转座的突变体。将对这些菌株的高优先级亚群中的Ty3表达以及蛋白质和cDNA中间体进行分析，以确定影响VLP的组装、加工、cDNA合成和核传递的突变。编码与Ty3结构蛋白相互作用的蛋白质的基因将通过用串联亲和标签标记Gag3和CA并对亲和纯化的复合体进行质谱分析来识别。突变谱和初步实验支持一种模型，在该模型中，细胞骨架和内体运输系统将Ty3蛋白质和RNA移动到与核有接触点的核周围簇，在那里很可能发生大量组装和cdna合成。对该模型进行了实验验证。

DESCRIPTION (provided by applicant):Retroviruses are the cause of AIDS and cancer and retroelements are a significant component of eukaryotic genomes. Nevertheless, there is a considerable amount which remains unknown about interactions of retroviruses and retroelements with host cells during the cytoplasmic phase of the lifecycle from assembly to arrival at the nuclear pore. We are using the Ty3 retroviruslike element in Saccharomyces cerevisiae as a model system to study this aspect of the lifecycle. Ty3 is comprised of two overlapping reading frames, GAG3 and POL3, encoding Gag3 and Gag3-Pol3 which are processed into mature capsid, nucleocapsid, protease, reverse transcriptase, and integrase. These proteins are analogous in structure and function to their retrovirus counterparts. Cells in which Ty3 is expressed produce virus like particles (VLPs) which undergo proteolytic maturation, reverse transcription, uncoating, and nuclear entry. Over past funding periods we have developed tools which will facilitate the proposed work including a set of over 100 mutants defective in Ty3 transposition, antibodies against the Ty3 proteins, and Ty3 elements tagged with fluorescent reporters. Microscopic observation of cells producing Ty3 has shown that Ty3 mRNA and proteins first appear in the peripheral cytoplasm, but are trafficked to perinuclear clusters where VLPs are observed. The proposed work has specific aims A-E:. A, B. Identification of Ty3 RNA and structural protein requirements for VLP assembly and nuclear delivery. Protein and RNA will be expressed separately and in trans to determine whether the RNA must encode Gag3 in order to be trafficked to the perinuclear region. If Gag3 is required, whether it can be supplied in trans will be determined. Gag3 residues required for assembly and trafficking of Ty3 protein and RNA will be determined. C. 130 mutants have been identified that affect Ty3 transposition. Ty3 expression and protein and cDNA intermediates will be analyzed in a high priority subset of these strains in order to identify mutations that affect assembly, processing, cDNA synthesis and nuclear delivery of the VLP. Genes that encode proteins that interact with Ty3 structural proteins will be identified by tagging Gag3 and CA with a tandem affinity tag and performing mass spectrometry on affinity-purified complexes. D, E. The spectrum of mutants and preliminary experiments support a model in which the cytoskeleton and endosomal trafficking systems move Ty3 proteins and RNAs toward a perinuclear cluster which has points of contact with the nucleus and where much of assembly and cDNA synthesis are likely to occur. Experiments are proposed to test this model.