REGULATION OF ALPHAVIRUS TRANSCRIPTION

甲病毒是普遍感兴趣的，因为它们在一个特定的环境中产生疾病。 包括人类在内的各种动物，因为它们在 无脊椎动物和脊椎动物。 它们广泛的复制潜力 对于它们的传播至关重要，因此对于它们的传播至关重要。 相关疾病。 成功合成许多感染性子代 首先是将感染的基因组翻译和复制成 互补或负链RNA模板。 了解 甲病毒RNA合成和翻译的机制以及 负链转录的调节将有助于我们 了解细胞如何运作和调节其表达 遗传信息。 此外，甲病毒引起持续的 蚊子的感染，并且必须进化出一种机制， 病毒复制，但防止Emg感染的蚊子，这是他们的 自然界中的水库 因此，我们的长远目标是发展 实验方法，使我们能够调查的性质， 甲病毒，很可能还有风疹 病毒，创造一个病毒复制复合体，并调节它们的速度， 转录。 我们下一个资助期的三个目标是：1。到 纯化和表征甲病毒复制复合物， 分离自被任一亲本病毒感染的细胞， 条件致死突变体或缺陷干扰颗粒 含有比基因组RNA短得多的DI RNA， 允许体外复制外源模板的条件。2. 鉴定与甲病毒相关的宿主细胞成分 复制复合物，并确定其在病毒复制中的作用。3. 为了明确病毒非结构蛋白的功能， 链合成和负链的暂时停止 合成. 使用感染性辛德毕斯克隆，我们将绘制突变 在防止负链合成TS 4中以及在TS 17和TS 133中 它们也具有在细胞生长后期合成负链的能力， 感染，即，24R表型，并测试假设，nsP2， 除了nsP4，在负性的时间停止中起作用。 链合成 使用定点突变，我们将确定 nsp4中的氨基酸改变，除了ts24和24R中的氨基酸改变， 允许负链合成在感染后期继续， 也就是说，给出24R表型，我们将定义氨基酸变化， nsPI，而不是ts11中的nsPI，其进行负链合成 温度敏感，并进一步探讨nsP 1在负性 链合成

Alphaviruses are of general interest because they produce disease in a variety of animals, including humans, and because they replicate in invertebrates as well as vertebrates. Their broad replicative potential is essential for their transmission and thus for the spread of their associated diseases. Successful synthesis of many infectious progeny starts with the translation and copying of the infecting genomic into a complementary, or negative-strand, RNA template. Understanding the mechanism of alphavirus RNA synthesis and translation and of the regulation of negative strand transcription will contribute to our understanding of how cells function and regulate the expression of their genetic information. Furthermore, alphaviruses cause persistent infection in mosquitos and must have evolved a mechanism that allows viral replication but prevents Emg infected mosquitos which are their reservoir in nature. Therefore, our long range goal is to develop experimental methods that allow us to investigate the nature of the molecular mechanism by which alphaviruses, and most probably also rubella virus, create a viral replication complex and regulate their rate of transcription. Our three aims for the next granting period are: 1. To purify and characterize alphavirus replication complexes which will be isolated from cells infected with either parental virus, conditionally-lethal mutants, or defective-interfering particles containing DI RNA that is much shorter than genome RNA and to develop conditions that permit in vitro replication of exogeneous templates. 2. To identify host cell components that are associated with alphavirus replication complexes and determine their role in viral replication. 3. To define the functions of viral nonstructural proteins in negative strand synthesis and in the temporal cessation of negative strand synthesis. Using the infectious Sindbis clone, we will map the mutations in ts4 which prevent negative strand synthesis and in ts17 and ts133 which also possess the ability to synthesize negative strands late in infection, i.e., the 24R phenotype, and test the hypothesis that nsP2, in addition to nsP4, plays a role in the temporal cessation of negative strand synthesis. Using site-directed mutagenesis, we will determine which amino acid alterations in nsp4, other than the one in ts24 and 24R, allow the continuation of negative strand synthesis late in infection, i.e., give the 24R phenotype and we will define amino acids changes in nsPI, other than the one in ts11 , that makes negative strand synthesis temperature-sensitive and probe further the role of nsP 1 in negative strand synthesis.