MATRIX PROTEINS OF ENVELOPED VIRUSES

负链病毒的外周基质(M)蛋白，如 水疱性口炎病毒(VSV)在病毒粒子组装和转录过程中起关键作用。 转录调控。我们建议对分子基础进行表征 对于膜结合、核衣壳结合，以及 VSV M蛋白通过下列途径发挥转录抑制功能 化学、免疫学、遗传学和生物物理方法： 1.用特定的化学物质和蛋白酶裂解产生的多肽 将与合成的寡肽一起进行测序、测绘和测试， 它们与磷脂小泡和核衣壳结合的能力，以及 来抑制病毒转录。 2.制备了M蛋白的单抗，并对其进行了检测 它们与特定多肽的反应性和它们的能力 抑制M蛋白及其多肽的各种生物学功能。 3.互补III组温度敏感突变M蛋白， 受到转录抑制的限制，突变的M蛋白将被 测试它们与脂泡、核衣壳和 特异性的单抗。 4.突变型、突变型和野生型M蛋白的重组DNA克隆 将准备基因并对它们的cDNA进行测序，以确定 M蛋白与囊泡和核衣壳结合缺陷的病变和 在转录抑制中。我们最终希望能制备出人的cdna克隆 在表达载体中插入M基因，形成定点缺陷的M蛋白。 5.野生型和突变型M蛋白的生物物理研究 多肽将被用来分析膜动力学的扰动 通过与膜反应的荧光去偏振来测量 由圆二色光谱测定。 6.VSV-New Jersey和A/WSN/33流感病毒M蛋白及其相互作用 单抗也将通过这些方法进行检测以进行比较。 与M蛋白在功能和进化上的相关性 VSV-印第安纳州。

The peripheral matrix (M) protein of negative-strand viruses, such as vesicular stomatitis virus (VSV), plays a key role in virion assembly and transcription regulation. We propose to characterize the molecular basis for the membrane binding, nucleocapsid binding, and transcription-inhibition functions of the VSV M protein by the following chemical, immunological, genetic, and biophysical methods: 1. Peptides generated by cleavage with specific chemicals and proteases will be sequenced, mapped, and tested, along with synthetic oligopeptides, for their capacity to bind to phospholipid vesicles and nucleocapsids, and to inhibit viral transcription. 2. Monoclonal antibodies to M protein have been prepared and will be tested for their reactivity with specific peptides and for their capacity to inhibit the various biological functions of M protein and its peptides. 3. Complementation group III temperature-sensitive mutant M proteins, restricted in transcription-inhibition, and revertant M proteins will be tested for their capacity to bind to lipid vesicles, nucleocapsids, and specific monoclonal antibodies. 4. Recombinant DNA clones of mutant, revertant, and wild-type M-protein genes will be prepared and their cDNAs sequenced to identify the sites of lesions in M proteins defective in binding to vesicles and nucleocapsid and in transcription-inhibition. We eventually hope to prepare cDNA clones of M genes in expression vectors to make site-directed defective M proteins. 5. Biophysical studies with wild-type and mutant M proteins and their peptides will be performed to assay perturbations of membrane dynamics measured by fluorescence depolarization reacting with membranes as determined by circular dichroism spectroscopy. 6. M proteins of VSV-New Jersey and A/WSN/33 influenza virus and their monoclonal antibodies will also be examined by these methods for comparison with the functional and evolutionary relatedness of the M protein of VSV-Indiana.