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

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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.