MECHANISM OF TRANSCRIPTION OF CORONAVIRUS

冠状病毒是一组感染冠状病毒的囊膜RNA病毒。 不同种类的动物引起呼吸道、胃肠道和 神经系统疾病。小鼠肝炎病毒作为模型系统 病毒诱导脱髓鞘的研究。我们实验室的目标是 了解冠状病毒复制的独特机制。一本小说 LEADER-RNA启动转录的机制被提出来描述 冠状病毒mRNAs的合成在这个模型中，领导者RNA是 合成，从基因组模板RNA解离并重新结合到 基因间隔区的负链RNA模板 前导RNA的3‘端与RNA的互补序列 模板。转录从前导RNA引物开始并继续 到模板的末尾。我已经开发了一个体外系统来 确定这需要哪些蛋白质和前导RNA序列 独特的转录机制。这个系统利用了外生型领导。 在序列上与病毒前导RNA略有不同的RNA。这个 将合成的前导RNA添加到制备的细胞质提取物中 冠状病毒感染细胞。我修改了溶血磷脂的治疗方案 治疗感染的细胞以建立体外转录系统和 开发了一种非常灵敏的聚合酶链式反应方法来检测转录产物。 使用这个系统，我能够演示病毒的前导RNA启动 MRNAs。该系统代表了第一个直接的生化演示 自由前导RNA在冠状病毒mRNA合成中的作用。我们建议 利用该系统分析冠状病毒转录的机制。 冠状病毒的另一个不同寻常的特征是它的基因组比率很高 重组。高频重组被认为是由于 冠状病毒RNA复制的不连续性质导致 核糖核酸中间体的合成。利用外源RNA进行体外培养 转录系统，我已经能够演示复制选择 重组。我们建议调查以下方面的序列要求 利用体外系统进行冠状病毒RNA重组。 其独特的转录和重组机制 冠状病毒牵涉到一种具有有趣特性的聚合酶，例如 可以识别碱基错配的假定的内切核活性。 我们对冠状病毒聚合酶的功能的理解是 由于该基因及其基因产物的大小(22kb)而受阻 (&gt；800 kDa)。我们已经证明了蛋白质产物是一种多蛋白质，它 被切割成亚基，但对其他功能知之甚少 域名。我们建议研究细胞的功能域。 通过鉴定温度敏感型突变体来鉴定多蛋白 这个基因。并通过产生针对假定的功能结构域的抗体 这种多蛋白。抗体将被用来识别病毒中的蛋白质 感染细胞，并在体外转录系统中进行测试 抑制RNA合成。

Coronaviruses are a group of enveloped RNA viruses which infect diverse species of animals causing respiratory, gastrointestinal and neurological diseases. Mouse hepatitis virus serves as a model system for the study of viral induced demyelination. The goal of our laboratory is to understand the unique mechanisms of coronavirus replication. A novel mechanism of leader-RNA primed transcription has been proposed to describe the synthesis of coronavirus mRNAs. In this model, leader RNA is synthesized, dissociates from the genomic template RNA and rebinds to the negative-strand RNA template at intergenic regions where there is a complementary sequence between the 3'-end of the leader RNA and the RNA template. Transcription initiates off the leader RNA primer and continues to the end of the template. I have developed an in vitro system to determine what proteins and leader RNA sequences are required for this unique mechanism of transcription. This system utilizes exogenous leader RNA which differs slightly in sequence from the viral leader RNA. The synthetic leader RNA is added to cytoplasmic extracts prepared from coronavirus infected cells. I adapted the protocol of lysolecithin treatment of infected cells to set up the in vitro transcription system and developed a very sensitive PCR method to detect transcription products. Using this system, I was able to demonstrate leader RNA priming of viral mRNAs. This system represents the first direct biochemical demonstration of the role of free leader RNA in coronavirus mRNA synthesis. We propose to use this system to analyze the mechanism of coronavirus transcription. Another unusual feature of coronavirus is its high rate of genome recombination. The high frequency recombination is proposed to be due to the discontinuous nature of coronavirus RNA replication which results in the synthesis of RNA intermediates. Using exogenous RNA in the in vitro transcription system, I have been able to demonstrate copy-choice recombination. We propose to investigate the sequence requirements for coronavirus RNA recombination using the in vitro system. The unique transcription and recombination mechanisms of coronavirus implicate a polymerase with interesting -properties such as a putative endonucleolytic activity which would recognize base mismatches. Our understanding of the functions of the coronavirus polymerase have been hampered because of the large size of the gene (22 kb) and its gene product (> 800 kDa). We have shown that the protein product is a polyprotein which is cleaved into subunits, but little is known about other functional domains. We proposed to investigate the functional domains of the polyprotein by characterizing temperature sensitive mutants which map to this gene. and by generating antibodies to putative functional domains of the polyprotein. The antibodies will be used to identify proteins in virus infected cells and tested in the in vitro transcription system for inhibition of RNA synthesis.