Gene Expression of Negative strand RNA Viruses

描述(申请人提供)：水泡性口炎病毒(VSV)是一种动物病原体，是研究非节段负链(NNS)RNA病毒在这类病毒生命周期中复杂的转录和复制过程的分子机制的典范。NNS RNA病毒构成了大量的病原体，对人类、哺乳动物、鸟类、鱼类和植物造成了巨大的损害和破坏。属于这一组的主要人类病原体是狂犬病、麻疹、腮腺炎、人类副流感、流感和许多其他疾病，它们仍然通过致命的感染困扰着人类。最近出现的汉坦病毒和埃博拉病毒和马尔堡病毒，也属于这个家族，重新刺激了对NNS RNA病毒的研究。我们实验室一直在积极研究VSV的两个关键病毒多肽：L(大蛋白)和磷蛋白P(转录因子)的结构和功能，它们共同构成了RNA聚合酶复合体。在最后一次授予期间，该领域的一个主要贡献是坚定地建立了细胞蛋白与昆虫细胞中表达的L蛋白的联系，并提出了转录酶和复制酶是具有不同亚基组成的两个不同实体的新概念。在这一更新应用中，我们主要集中在详细检测从感染的哺乳动物细胞中纯化的VSV转录酶和复制酶的结构和功能。我们受到最近首次成功纯化转录酶复合体和来自感染细胞的复制酶的极大鼓舞，并证明细胞热休克蛋白HSP60、翻译延伸因子EF-1α和鸟苷转移酶GT是转录酶全酶的一部分，而复制酶缺乏宿主蛋白。基于这些发现，我们提出了三个具体的目标：(1)详细研究转录酶相关蛋白的功能；(2)表征复制酶和复制反应；(3)探讨L蛋白中宿主蛋白的结合域以及其他NNS病毒中宿主蛋白的相互作用。从这些研究中收集到的知识将有助于提供关于VSV基因表达机制的有价值的信息，并推广到其他NNS RNA病毒。

DESCRIPTION (provided by applicant): Vesicular stomatitis virus (VSV), an animal pathogen, remains a paradigm of the non-segmented negative strand (NNS) RNA viruses to study the intricate molecular mechanisms of transcription and replication processes during the life-cycle of this class of viruses. The NNS RNA viruses constitute a vast multitude of pathogens, which inflict immense damage and destruction to humans, mammals, birds, fish and plants. The major human pathogens belonging to this group are rabies, measles, mumps, human parainfluenza, influenza, and many others, which still scourge the human race by their deadly infection. Recent emergence of hantaviruses and Ebola and Marburg viruses, also belonging to this family, has restimulated intense investigation to study NNS RNA viruses. Our laboratory has been actively engaged in examining the structure and function of the two key viral polypeptides of VSV; L (the large protein) and the phosphoprotein P (transcription factors), which together constitute the RNA polymerase complex. A major contribution in the last granting period to the field has been the firm establishment of the association of cellular proteins with the L protein expressed in insect cells, and the proposition of a new concept that the transcriptase and replicase are two distinct entities with different subunit compositions. In this renewal application, we have primarily focused on examining in detail the structure and function of both transcriptase and replicase of VSV purified from the infected mammalian cells. We are greatly inspired by our recent success in purifying for the first time the transcriptase complex to homogeneity and also the putative replicase from the infected cells and demonstrate that cellular heat shock protein, Hsp60, translation elongation factor, EF-1alpha and guanylytransferase, GT, are part of the transcriptase holoenzyme, whereas the replicase lacks the host proteins. Based on these findings, we have proposed three specific aims (1) to study in detail the functions of associated proteins of the transcriptase (2) to characterize the replicase and the replicative reactions and (3) to probe into the binding domain of the host protein in the L protein and association of host proteins in other NNS viruses. The knowledge gleaned from these studies will help provide valuable information with regard to the mechanism of gene expression of VSV and by extension to other NNS RNA viruses.