REGULATION OF ALPHAVIRUS TRANSCRIPTION

甲型病毒之所以引起广泛关注，是因为它们在 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一样。它们广阔的复制潜力 对它们的传播至关重要，从而对它们的传播至关重要 相关疾病。多个感染性后代的成功合成 首先是将感染病毒的基因组翻译和复制到 互补的或负链的RNA模板。了解 甲型病毒RNA合成和翻译机制及甲型肝炎病毒 负链转录的调节将有助于我们的 了解细胞如何发挥作用并调节其表达 遗传信息。此外，甲型病毒会导致持续 蚊子中的感染，一定是进化出了一种机制，可以让 病毒复制，但防止感染EMG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 甲型病毒和风疹病毒的分子机制 病毒，创造一个病毒复制复合体，并调节它们的速度 抄写。我们在下一个授权期的三个目标是：1. 纯化和鉴定甲型病毒复制复合体，这将是 从感染了亲本病毒的细胞中分离出来， 条件致死突变体，或缺陷干扰粒子 含有比基因组RNA短得多的DI RNA，并且 允许外源模板体外复制的条件。2. 鉴定与甲型病毒有关的宿主细胞成分 复制复合体，并确定它们在病毒复制中的作用。3. 将病毒非结构蛋白的功能定位为阴性 链合成与负链的暂时终止 综合。使用具有感染性的辛德比斯克隆，我们将绘制突变图谱 在防止负链合成TS4中以及在TS17和TS133中 它还具有在后期合成负链的能力 感染，即24R表型，并检验假设nsP2，in 除NSP4外，NSP4还在负性反应的暂时停止中发挥作用 链合成。使用定点突变，我们将确定 除了TS24和24R外，NSP4的哪些氨基酸发生了变化， 允许在感染后期继续负链合成， 即，给出24r表型，我们将定义氨基酸的变化 Nspi，不同于ts11中的nspi，它产生负链合成。 NSP-1对温度的敏感性及其在负性反应中的作用 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.