GENE EXPRESSION OF NEGATIVE STRAND RNA VIRUSES

这项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）家族病毒致病性的分子基础。nsRNA病毒由大量的病毒组成，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严重的损害和破坏。狂犬病、麻疹、腮腺炎、副流感、呼吸道合胞病毒等病毒都属于这类nsRNA病毒。近年来，新型致病性汉塔病毒和埃博拉病毒的出现，为研究nsRNA病毒致病性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对抗这些致命病原体的试剂的基础。我们的主要重点是建立VSV的关键病毒蛋白的功能，如RNA聚合酶L，转录因子P和基因组RNA结合核衣壳蛋白N。这些蛋白在感染细胞内构成转录核糖核蛋白（RNP）复合体感染。我们已经成功地利用重组表达载体在原核细胞和真核细胞中以生物活性形式表达了这些多肽。在当前的授权期内，我们取得了几个重要的发现，特别是关于L蛋白的亚基组成，包括细胞翻译延伸因子和假定的复制酶亚基复合物。在目前的提案中，我们打算使用生化和反向遗传学方法详细研究VSV转录酶和复制酶复合物的结构和功能。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-1 alphabetagamma在L活性中的作用；(2)推测的复制酶复合体L-(N-P)的结构和功能以及N-P复合体和细胞功能在复制酶功能中的作用；(3) P蛋白结构域II磷酸化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蛋白各结构域的精确功能，并表征狂犬病病毒P蛋白磷酸化所涉及的独特蛋白激酶。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，这对我们了解VSV和其他nsRNA病毒发病机制的分子基础至关重要。

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.