REGULATION OF ALPHAVIRUS TRANSCRIPTION

甲病毒是蚊媒脑炎的重要病因。 我们 总体目标是确定甲病毒转录的机制， 了解病毒正负链RNA的合成 监管. 甲病毒编码的非结构蛋白 辛德毕斯病毒（SIN）和塞姆利基森林病毒（SFV），它们启动病毒性 通过将感染的病毒正链基因组RNA转录成 互补负链RNA的功能将被表征 在病毒复制中。 SIN和SFV的温度敏感突变体， 他们的回复突变体，我们已经确定了以前将被用来 确定病毒蛋白质的功能， 负链RNA的合成，并确定是否调节 负链RNA合成涉及顺式或反式作用的病毒 功能协调发展的 我们将研究SIN HR的缺陷干扰粒子（DI 以确定非缺陷病毒是否能够关闭 合成DI RNA的负链模板。 我们会测序， 通过使用逆转录酶的引物延伸方法， 以纯化的病毒基因组为模板， 编码SIN HR、ts6、ts11的第三和第四非结构蛋白 和TS 24以及这些突变体的回复突变体。 我们之前的研究让我们 怀疑第三个或第四个非结构蛋白的 甲病毒基因组由B或F顺反子编码。 我们将继续 为了开发表征甲病毒转录的体外测定， 特别地，使用从体外产生的病毒RNA进行负链合成， 克隆病毒RNA。 我们计划克隆非结构基因， SIN HR和选择的温度敏感性基因的侧翼序列 将SIN HR突变体插入Riboprobe Gemini质粒载体中，以分析 甲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 表征加工多蛋白的病毒编码蛋白酶 病毒非结构蛋白的前体， SIN HR ts17中的温度敏感性。

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.