GENE EXPRESSION OF NEGATIVE STRAND RNA VIRUSES

这一更新应用的长期目标是以水疱性口炎病毒(VSV)为原型病毒，了解负链RNA(NsRNA)家族病毒致病的分子基础。NsRNA病毒由大量的病毒组成，这些病毒对包括脊椎动物、无脊椎动物和植物界在内的所有生物造成深刻的破坏和破坏。狂犬病、麻疹、流行性腮腺炎、副流感、呼吸道合并症等病毒都属于nsRNA病毒类别。近年来，新型致病汉坦病毒和埃博拉病毒的出现为阐明nsRNA病毒致病的分子基础提供了新的动力。彻底了解这些病毒的转录和复制模式是开发抗击这些致命病原体的试剂的基础。我们朝着这个目标的主要重点是建立VSV的关键病毒蛋白的功能，如L，RNA聚合酶，P，转录因子和N，基因组RNA结合的核衣壳蛋白。这些蛋白质构成了感染细胞内的转录核糖核蛋白(RNP)复合体感染。我们已经成功地利用重组表达载体在原核和真核细胞中以生物活性的形式表达了这些多肽。在目前的授权期内，我们在L蛋白的亚基组成方面有了一些重要的发现，涉及细胞翻译延长因子和可能的复制酶亚单位复合体。在目前的方案中，我们打算用生化和反向遗传学的方法详细研究VSV转录酶和复制酶复合体的结构和功能。我们将详细研究(1)L蛋白的结构和功能，特别是翻译延伸因子EF-1α在L活性中的作用；(2)可能的复制酶复合体L-(N-P)的结构和功能，以及N-P复合体和细胞功能在复制功能中的作用；(3)P蛋白的结构和功能，涉及结构域II在体外复制和反向遗传学中的作用。我们将进行详细的突变研究，以确定P蛋白中各个结构域的确切功能，并表征参与狂犬病病毒P蛋白磷酸化的独特蛋白激酶。这些研究有可能深入了解VSV基因组RNA转录和复制的生物合成途径，这是我们理解VSV和其他nsRNA病毒发病的分子基础的基础。

The long-term goal of this renewal application is to understand the molecular basis of pathogenicity of ,viruses belonging to the negative strand RNA (nsRNA) family using vesicular stomatitis virus (VSV) as the prototype virus. The nsRNA viruses comprise a vast multitude of viruses that inflict profound damage and destruction to all living organisms including vertebrates, invertebrates, and plant kingdom. The viruses, such as rabies, measles, mumps, parainfluenza, respiratory syncitial, and many more fall in this nsRNA virus category. Recently, the emergence of novel pathogenic hanta and Ebola viruses has provided renewed impetus to delineate the molecular basis of pathogenicity of the nsRNA viruses. A thorough understanding of the mode of transcription and replication of these viruses is fundamental to develop reagents to combat these deadly pathogens. Our major emphasis toward this goal has been to establish the functions of the key viral proteins of VSV, such as L, the RNA polymerase, P, the transcription factor and N, the genome RNA-binding nucleocapsid protein. These proteins constitute the transcribing ribonucleoprotein (RNP) complex infection within the infected cells. We have been successful in expressing, in biologically active form, these polypeptides in procaryotic and eucaryotic cells using recombinant expression vectors. During the current granting period, we made several important discoveries specially with respect to the subunit composition of the L protein involving cellular translation elongation factors and the putative replicase subunit complex. In the current proposal, we intend to study in detail the structure and function of the transcriptase and replicase complexes of VSV using biochemical and reverse genetics approaches. We will investigate in detail (1) the structure and function of the L protein, specifically the role of translation elongation factor EF-1 alphabetagamma in L activity; (2) the structure and function of the putative replicase complex L-(N-P) and the roles of N-P complex and cellular functions in the replicase function; (3) the structure and function of the P protein with regard to the role of domain II phosphorylation in replication in vitro and using reverse genetics. We will undertake detail mutational studies to determine the precise functions of various domains in the P protein and characterize the unique protein kinase involved in rabies es virus P protein phosphorylation. These studies have the potential to gain insight into the biosynthetic pathways leading to transcription and replication of VSV genome RNA, which is fundamental to our understanding of the molecular basis of pathogenesis of VSV and other nsRNA viruses.