Characterization of the SARSCoV frameshift signal

描述（由申请方提供）：病毒利用程序性核糖体移码（PRF）来转录后调节单顺反子病毒mRNA上编码的多个基因的表达。在逆转录病毒和全病毒中的研究表明，保持正确的PRF效率对于病毒繁殖至关重要，因此将这种机制确定为抗病毒治疗的潜在靶标。先前已显示PRF被冠状病毒（CoV）利用，并且对快速出现的SARS-CoV（严重急性呼吸综合征（SARS）的病原体）的初级序列分析揭示了推定的PRF信号的存在，该信号被预测为在-1或5 '方向上移动延长核糖体一个碱基（-1PRF）。我们实验室对该序列的初步比较、结构和功能分析表明，SARS-CoV（可能是整个冠状病毒家族）的-1 PRF信号利用了一种复杂的、迄今未知的mRNA假结结构，该结构包含三个茎环，而不是通常的两个茎环。此外，我们的研究结果表明，第三茎环的功能可能是调节-1PRF效率，从而调节病毒蛋白的表达。因此，与这种结构相互作用的小分子可能具有抗病毒特性。拟议研究的主要目的是使用系统发育、分子、结构和病毒检测的组合来表征SARS-CoV-1 PRF信号的重要特征。具体而言，我们将1）使用基于体内人上皮细胞的测定系统来表征SARS和小鼠肝炎CoV的mRNA假结的变化如何影响移码效率，2）使用组织培养物和小鼠模型系统中的感染性cDNA克隆来确定移码效率的变化对SARS-CoV繁殖的影响，以及3）使用核酸酶图谱、高分辨率NMR和量热方法对SARS-CoV-1 PRF信号进行结构表征。

DESCRIPTION (provided by applicant): Viruses utilize programmed ribosomal frameshifting (PRF) to post-transcriptionally regulate the expression of multiple genes that are encoded on monocistronic viral mRNAs. Studies in Retro- and Totiviruses have shown that maintaining correct PRF efficiencies is critical for virus propagation, thus identifying this mechanism as a potential target for antiviral therapeutics. PRF has previously been shown to be utilized by the Coronaviruses (CoV), and primary sequence analysis of the rapidly emerging SARS-CoV, the etiological agent of Severe Acute Respiratory Syndrome (SARS), reveals the presence of a putative PRF signal that is predicted to shift elongating ribosomes by one base in the -1 or 5' direction (-1 PRF). Initial comparative, structural and functional analysis of this sequence in our laboratory suggests that the -1 PRF signal of SARS-CoV (and possibly of the entire Coronavirus family) utilizes a complex, heretofore unknown mRNA pseudoknot structure that contains three stem loops as opposed to the usual two-stem loop variety. Further, our findings suggest that the function of the third stem-loop may be to regulate -1 PRF efficiency, and hence the expression of viral proteins. Thus, small molecules that interact with this structure may potentially have antiviral properties. The broad aim of the proposed research is to characterize important features of the SARS-CoV -1 PRF signal using a combination of phylogenetic, molecular, structural, and viral assays. Specifically, we will 1) characterize how changes in the mRNA pseudoknots of the SARS and Mouse Hepatitis CoV's affect frameshifting efficiency using an in vivo human epithelial cell based assay system, 2) determine the effects of changes in frameshift efficiency on propagation of the SARS-CoV using an infectious cDNA clone in tissue culture and in a mouse model system, and 3) structurally characterize the SARS-CoV -1 PRF signal using nuclease mapping, high-resolution NMR and calorimetric methodologies.