Project Summary We aim to determine the molecular basis for SARS-CoV-2 viral packaging and to develop a screening strategy to identify inhibitors of this key step in the coronavirus infection cycle. Selective packaging of the viral genome, over more abundant transcripts, involves specific interactions between an RNA packaging signal, viral structural proteins and possibly other factors. Inhibition of this process would block formation of infectious virions and thereby contribute to a therapeutic regimen that would prevent or treat infection. Building on our laboratory’s extensive expertise in RNA biochemistry and virus-like particle research, we propose to determine the functional SARS-CoV-2 packaging signal and to develop a robust small molecule-based assay for SARS-CoV-2 packaging inhibition. To determine the components of the SARS-CoV-2 packaging mechanism, we will generate virus-like particles (VLPs) that contain the structural proteins of the virus but not the viral genome. The absence of the genome renders these VLPs non-infectious and therefore safe to work with. Methods for generating these VLPs derive from published research with other coronaviruses as well as our own lab’s experience working with influenza and HIV VLPs. SARS-CoV-2 VLPs will be produced by co-expressing the viral spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. RNA molecules containing the packaging signal can be packaged into these VLPs and delivered into receiver cells, providing an assay for packaging signal detection. In parallel, this approach will be used to establish a screening assay to identify viral packaging inhibitors. These two aims are independent, yet the results of each workstream will inform both the fundamental and applied aspects of the project. Our long-term objective is to develop a small molecule inhibitor of SARS-CoV-2 viral packaging. This approach has the following advantages: 1) we will naturally detect nucleocapsid inhibitors, which can be potent antiviral drugs as shown for HIV and other viruses due to strict constraints on nucleocapsid function; 2) our approach targets a step in the viral infection cycle that is not currently the focus of major therapeutic discovery efforts, enhancing the opportunity to find a new and/or complementary antiviral strategy; and 3) our screening approach does not require live virus and can be executed safely in most high-throughput screening facilities. The research proposed here will enable the development of new antiviral strategies for treating coronaviruses. SARS-CoV-2 is the third betacoronavirus to trigger a zoonotic outbreak in the last 18 years and estimates suggest that ~5000 related viruses are circulating within bat populations around the world. Our proposal targets a critical yet relatively understudied step of the coronavirus life cycle that is a promising target of selective small- molecule inhibition. The results of this work will inform and enable other viral inhibition efforts...