REGULATION OF ALPHAVIRUS TRANSCRIPTION

甲型病毒是蚊媒脑炎的重要原因。我们的 总体目标是确定甲型病毒转录和转录的机制 为了了解病毒正链和负链RNA的合成是如何 受监管的。甲型病毒的病毒编码非结构蛋白 引发病毒的辛德比斯病毒(SIN)和塞姆利基森林病毒(SFV) 通过将感染病毒的正链基因组RNA转录成 互补的负链rna将根据它们的功能进行表征。 在病毒复制中。SIN和SFV的温度敏感突变体和 我们之前描述过的它们的返回体将被用于 确定病毒蛋白的功能，这些蛋白是 并确定负链RNA的合成是否调节 负链RNA合成涉及顺式或反式作用病毒 功能。我们将研究SIN HR的缺陷干扰粒子(DI) 以确定无缺陷病毒是否能够关闭 DI RNA负链模板的合成。我们将进行排序， 通过逆转录酶扩增引物的方法， 以纯化的病毒基因组为模板的双脱氧核苷酸， 编码SIN HR、TS6、TS11第三和第四非结构蛋白 和TS24，以及这些突变体的回复突变体。我们之前的研究让我们 怀疑第三或第四种非结构蛋白 甲型病毒基因组由B或F顺反子编码。我们将继续 为了开发一种体外实验来表征甲型病毒的转录， 特别是，使用体外产生的病毒RNA进行负链合成 克隆的病毒核糖核酸。我们计划克隆非结构基因，并要求 SIN HR和选定的温度敏感基因的侧翼序列 Sin HR突变体导入核探针Gemini载体的分析 甲型病毒合成和调控的序列要求 正链和负链RNA合成。最后，我们将确定和 确定处理多蛋白的病毒编码的蛋白酶的特征 病毒非结构蛋白的前体，这似乎是 SIN HR ts17的温度敏感性。

Alphaviruses are an important cause of mosquito-borne encephalitis. Our overall aim is to determine the mechanism of alphavirus transcription and to understand how the synthesis of viral minus and plus strand RNA is regulated. The viral encoded nonstructural proteins of the alphaviruses Sindbis virus (SIN) and Semliki Forest virus (SFV) which initiate viral replication by transcribing the infecting viral plus-strand genome RNA into a complementary minus-strand RNA will be characterized as to their function in viral replication. Temperature sensitive mutants of SIN and SFV and their revertants that we have characterized previously will be used to identify the functions of the viral proteins that are required for minus-strand RNA synthesis and to determine if the regulation of minus-strand RNA synthesis involves cis-acting or trans-acting viral functions. We will study defective interferring particles (DIs) of SIN HR to determine whether nondefective virus is capable of shutting off the synthesis of the minus-strand template for the DI RNA. We will sequence, by the method of primer extension using reverse transcriptase and dideoxynucleotides with purified viral genomes as templates, the genes encoding the third and fourth nonstructural proteins of SIN HR, ts6, ts11 and ts24 and revertants of these mutants. Our previous research leads us to suspect that the third or the fourth nonstructural protein of the alphavirus genome is encoded by the B or the F cistron. We will continue to develop an in vitro assay to characterize alphavirus transcription, particularly, minus strand synthesis using viral RNA produced in vitro from cloned viral RNA. We plan to clone the nonstructural genes and required flanking sequences of both SIN HR and selected temperature sensitive mutants of SIN HR into Riboprobe Gemini plasmid vectors in order to analyze the sequence requirements for synthesis and regulation of alphavirus plus-strand and minus-strand RNA synthesis. Lastly, we will identify and characterize the viral encoded protease that processes the polyprotein precursor to the viral nonstructural proteins and that appears to be temperature sensitive in SIN HR ts17.