Characterization of the SARSCoV frameshift signal

描述（由申请人提供）：病毒利用程序性核糖体移框（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。对Retro- virus和totivirus的研究表明，维持正确的PRF效率对病毒传播至关重要，因此确定了这一机制作为抗病毒治疗的潜在靶点。先前已证明PRF被冠状病毒（CoV）利用，对迅速出现的SARS-CoV（严重急性呼吸综合征（SARS）的病原）的初级序列分析显示，存在一种假定的PRF信号，预计该信号可将延长的核糖体向-1或5'方向移动一个碱基（-1 PRF）。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信号利用了一个复杂的、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-1 PRF的效率，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潜在的抗病毒特性。拟议研究的广泛目的是利用系统发育、分子、结构和病毒分析的组合来表征SARS-CoV -1 PRF信号的重要特征。具体来说，我们将1)利用基于人体内上皮细胞的检测系统表征SARS和小鼠肝炎冠状病毒mRNA伪结的变化如何影响移码效率，2)利用组织培养和小鼠模型系统中的感染性cDNA克隆确定移码效率变化对SARS-CoV传播的影响，以及3)利用核酸酶定位在结构上表征SARS-CoV -1 PRF信号。高分辨率核磁共振和量热法。

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.