Identification of host dependency factors for MERS-CoV infection and discovery of repurposing drugs for therapeutic uses

Abstract

Human infection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was first appeared in Saudi Arabian in 2012. Since then more than 2000 laboratory confirmed cases and more than 800 deaths with comorbidities including diabetes and chronic renal or heart diseases have been reported. However, the underlying pathophysiology of MERS-CoV infection is still not fully understood and no antivirals or vaccine are commercially available. In this study we applied two strategies, 1) genome-wide CRISPR-Cas9 knockout screen and 2) virus-host protein-protein interactome, to identify the host dependency factors or their corresponding pathways for the MERS-CoV infection. For the CRISPR-Cas9 survival screen we identified 13 candidates that are essential for MERS-CoV infection in cultured cells. One of the candidates is the host receptor DPP4 for MERS-CoV entry. For the virus-host protein-protein interactome study, we applied immunoprecipitation followed by liquid chromatography - mass spectrometry (LC-MS/MS) and successfully identified more than 300 host factors associated with MERS-CoV proteins including nonstructural proteins (NSP 1-16), five accessary proteins (orf3, orf4a, orf4b, orf5 and orf8) and four structural proteins (Spike, Envelope, Membrane, Nucleoprotein). Through the pathway analysis we have chosen nine candidates playing the key roles on known signaling pathways for further investigation. It includes prostaglandin synthesis pathway, GSK3 kinase pathway, deacetylation pathway, MHC class I antigen presentation, deubiquitination, spindle assembly checkpoint and interferon signaling pathway. Small molecule inhibitors targeting these host factors and/or pathways were tested and the treatment of two FDA-approved drugs potently inhibited MERS-CoV infection in the cultured cells. Taken together, both CRISPR-Cas9 screen and virus-host protein-protein interaction studies not only provide fundamental knowledge on virus-host interaction, the host factors identified in this study may be used as the host druggable targets. Further in vivo experiment using DPP4 knockin mice will provide important insights into the development of these repurposing drugs for therapeutic uses. This study was supported by Research Grant Council – Theme-based Research Scheme (T11-707/15-R).