Development of a virus-free sensor system to repurpose approved drugs for blocking Coronavirus replication

COVID19大流行突显了及时发现抗病毒治疗药物的重要性，特别是在几乎没有关于已开发(和等待批准)的疫苗是否能够实现较长免疫期的信息的情况下。因此，开发高性价比、高通量的传感器系统以快速将批准的药物重新用作抗病毒药物是非常必要的。我们已经开发了一种无病毒传感器系统-pShiftSensor，它可以通过抑制核糖体移码来特异性地筛选阻止SARS-CoV2复制的药物，核糖体移码是产生病毒复制酶所必需的。传感器系统是基于荧光的，因此允许经济高效的高通量定量读数。该系统基于程序化核糖体移码(-1\f25 PRF)所需的顺式元件，这是一种在所有冠状病毒中普遍存在的非规范翻译机制，用于合成病毒蛋白质，包括病毒复制所必需的RNA依赖的RNA聚合酶(RDRP)。我们已经开发了pShiftSensor-v1，这是一个包含SARS-CoV-2-1PRF盒式磁带(即光滑序列UUUAAAC和3‘伪结)的双报告系统。该系统是双顺反子的，从而避免了由于对照和测试报告表达盒之间的差异递送而产生的表达伪影。通过这项建议，我们将把我们的系统升级到pShiftSensor-V2，它针对的是主要细胞类型，如小气道肺细胞，SARS-CoV2感染的关键进入细胞类型，以及对全身感染至关重要的初级白细胞，特别是单核/巨噬细胞和T细胞。PShiftSensor-V2将允许在生理环境中对病毒复制抑制化合物进行具有成本效益的、高通量的细胞类型特异性筛选。

The COVID19 pandemic has highlighted the importance of timely discovery of antiviral therapeutics, especially when there is little information as to whether vaccines developed (and pending approval) enable a significant period of immunity. Therefore the development of cost effective, high-throughput sensor systems to rapidly repurpose approved drugs as antiviral agents is extremely desirable. We have developed a virus-free sensor system - pShiftSensor that can specifically screen for drugs to block SARS-CoV2 replication by inhibiting the ribosomal frameshift that is essential for production of the viral replicase. The sensor system is fluorescent-based, therefore permitting cost-effective high-throughput quantitate readout.The system is based on the cis-element required for -1 programmed ribosomal frameshifting (-1 PRF), a non-canonical translation mechanism that is ubiquitous throughout all coronaviruses and used to synthesis viral proteins including the RNA-dependent RNA polymerase (RdRP), essential for viral replication. We have developed pShiftSensor-v1, which is a dual-reporter system that contains the SARS-CoV-2 -1PRF cassette (i.e. the slippery sequence UUUAAAC and the 3' pseudoknot). The system is bi-cistronic thereby avoiding expression artifacts due to differential delivery between the control and test reporter expression cassette. Through this proposal, we will upgrade our system to pShiftSensor-V2, which targets primary cell types such as small airway lung cells, the key entry cell type for SARS-CoV2 infection, and primary leukocytes, especially monocytes/macrophages and T-cells, essential for systemic infection. pShiftSensor-V2 will permit cost-effective, high-throughput cell-type specific screening of viral replication inhibitory compounds in a physiological environment.