Ty3 viruslike particle morphogenesis and host interactions

Ty3病毒样颗粒形态发生和宿主相互作用

基本信息

批准号：
7884990
负责人：
SUZANNE SANDMEYER
金额：
$ 19.19万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-20 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7884990
关键词：
Abbreviations Acids Acquired Immunodeficiency Syndrome Affect Affinity Affinity Chromatography Alanine Antibodies Applied Genetics Atomic Force Microscopy Biochemical Biological Assay Biological Models Capsid Cell Fractionation Cell Nucleus Cells Collection Complementary DNA Complex Cytoplasm Cytoskeleton DNA Polymerase III Data Set Databases Docking Electron Microscopy Electrons Elements Endoplasmic Reticulum Eukaryota Fluorescence Resonance Energy Transfer Funding Gagging Gene Deletion Genes Genetic Genome HIV High Pressure Liquid Chromatography Human Indium Integrase Integration Host Factors Isocyanates Learning Life Long Terminal Repeats Malignant Neoplasms Mason-Pfizer monkey virus Mass Spectrum Analysis Mediating Membrane Messenger RNA Microscopic Modeling Morphogenesis Multivesicular Body Murine leukemia virus Mutation Nuclear Nuclear Envelope Nuclear Pore Nuclear Pore Complex Nuclear Pore Complex Proteins Nucleocapsid Ontology Open Reading Frames Pattern Peptide Hydrolases Peripheral Pharmaceutical Preparations Phase Phenotype Process Proteins RNA RNA Binding RNA Polymerase III RNA-Directed DNA Polymerase Reading Frames Reporter Research Personnel Retroelements Retrotransposon Retroviridae Reverse Transcription Rhodamine Rhodamines Role Saccharomyces Saccharomyces cerevisiae Scanning Site Site-Directed Mutagenesis Sorting - Cell Movement Structural Protein Structure System TATA-Box Binding Protein Technology Testing Type D Retrovirus Viral Virus-like particle Work Yeasts genome database genome-wide in vitro testing mutant particle programs protein expression protein function repository research study tetramethylrhodamine isothiocyanate tomography tool trafficking

项目摘要

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.

描述（由申请人提供）：逆转录病毒是艾滋病的原因，癌症和追溯元素是真核基因组的重要组成部分。然而，在生命周期的细胞质相中，从组装到到达核孔中，逆转录病毒和与宿主细胞的相互作用与宿主细胞的相互作用尚不清楚。我们将酿酒酵母中的TY3逆转录病毒样元素用作研究生命周期这一方面的模型系统。 TY3由两个重叠的读取帧GAG3和POL3组成，编码GAG3和GAG3-POL3，它们被处理成成熟的capsid，nucleocapsid，蛋白酶，蛋白酶，逆转录酶和集成酶。这些蛋白质的结构和功能与逆转录病毒对应物相似。 TY3表达的细胞产生了像颗粒（VLP）一样产生蛋白水解成熟，逆转录，脱落和核进入的细胞。在过去的资金期间，我们开发了工具，这些工具将有助于拟议的工作，包括一组TY3转置有缺陷的100多个突变体，针对TY3蛋白的抗体以及用荧光记者标记的TY3元素。产生TY3的细胞的显微镜观察表明，TY3 mRNA和蛋白质首先出现在外周细胞质中，但被运输到观察到VLP的核周簇。拟议的工作具有特定的目标A-E：。 A，B。鉴定TY3 RNA和VLP组装和核输送的结构蛋白质需求。蛋白质和RNA将分别表达并在反式中以确定RNA是否必须编码GAG3才能被贩运到核周区。如果需要GAG3，是否可以在Trans中提供它。将确定组装和运输TY3蛋白和RNA所需的GAG3残基。 C.已鉴定出影响TY3换位的130个突变体。 TY3表达，蛋白质和cDNA中间体将在这些菌株的高优先级子集中进行分析，以鉴定影响组装，加工，cDNA合成和VLP的核输送的突变。编码与TY3结构蛋白相互作用的蛋白质的基因将通过标记具有串联亲和力标签的GAG3和CA来识别，并在亲和纯化的复合物上执行质谱。 D，E。突变体和初步实验的光谱支持了一个模型，其中细胞骨架和内体运输系统将TY3蛋白和RNA移向核周簇，该核心群与核与核的接触点以及可能发生的许多组装和cDNA合成。提出了实验来测试此模型。