MECHANISM OF CORONAVIRUS RNA TRANSCRIPTION

冠状病毒是一种包膜的RNA病毒，会导致人类和动物的各种疾病。这项研究的长期目标是以小鼠肝炎病毒(MHV)为模型系统，阐明冠状病毒RNA合成的机制。冠状病毒RNA转录涉及前导RNA和基因间序列之间的相互作用。有人认为，这种相互作用可能是通过互补序列之间的直接RNA-RNA相互作用来调节的。利用突变的MHV(JHM2c)，我们最近发现MHV的RNA转录并不严格要求序列互补。一个特定的亚基因组mRNA物种在基因间隔区的不同位置启动，其中在前导和基因间模板之间没有序列互补性。这一发现导致我们假设，蛋白质-RNA和蛋白质-蛋白质相互作用而不是直接的RNA-RNA相互作用是启动mRNA转录的驱动力。我们还发现，异质性核核糖核蛋白(HnRNP)A1是调节MHV RNA转录的潜在细胞因子。我们建议使用JHM2c作为一种独特的手段，并使用生化和分子方法来验证这一假说，具体目的有两个：(I)定义hnRNP-A1与MHV RNA顺式作用序列之间的相互作用，并确定这种蛋白质-RNA相互作用在调节MHV RNA转录中的功能意义；(Ii)表征hnRNP-A1与MHV RNA聚合酶和核衣壳蛋白在体外和体内的相互作用，并确定这些蛋白质-蛋白质相互作用在调节MHV RNA转录方面的功能意义。这些研究将有助于从根本上理解冠状病毒和其他RNA病毒中RNA合成的调控机制。

Coronaviruses are enveloped RNA viruses that cause a variety of diseases in humans and animals. The long-term goal of the proposed research is to elucidate the mechanisms of coronavirus RNA synthesis by using mouse hepatitis virus (MHV), a prototype murine coronavirus, as a model system. Coronavirus RNA transcription involves the interaction between the leader RNA and intergenic sequence. It has been proposed that this interaction is probably mediated by direct RNA-RNA interactions between complementary sequences. Using a mutant MHV (JHM2c), we recently found that MHV RNA transcription does not strictly require sequence complementarity. A specific subgenomic mRNA species is initiated at various sites of the intergenic region where there is no sequence complementarity between the leader and the intergenic template. This finding leads us to hypothesize that protein-RNA and protein-protein interactions rather than direct RNA-RNA interactions are the driving force for the initiation of mRNA transcription. We also found that the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a potential cellular factor in the regulation of MHV RNA transcription. We propose to use JHM2c as a unique means and to employ biochemical and molecular approaches to test this hypothesis with the two Specific Aims: (I) To define the interactions between hnRNP-A1 and the cis-acting sequences of MHV RNA both in vitro and in vivo, and to determine the functional significance of this protein-RNA interactions in regulation of MHV RNA transcription; (II) To characterize the interactions between hnRNP-A1 and the MHV RNA polymerase and nucleocapsid proteins both in vitro and in vivo, and to determine the functional significance of these protein- protein interactions in regulation of MHV RNA transcription. These studies will contribute to the fundamental understanding of the mechanisms of regulation of RNA synthesis in coronaviruses and other RNA viruses as well.