A novel cellular tool for screening human pan-coronavirus antivirals

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a pressing global threat, having emerged in December 2019. Alongside it, Middle East respiratory syndrome coronavirus (MERS-CoV), a highly pathogenic human coronavirus, was identified in 2012 and continues to cause seasonal outbreaks in the Middle East. The persistence of these deadly human coronaviruses underscores the need for ongoing research on broad-spectrum antivirals. Human alveolar A549 cells have been widely used to study respiratory virus infections; however, there is a lack of standardized cell models that are permissive to these diverse lethal coronaviruses. To facilitate the assessment and validation of antiviral treatments, a robust human cell model that is susceptible to SARS-CoV-2, MERS-CoV, and other human coronavirus family members is indispensable. SARS-CoV-2 uses the angiotensin converting enzyme 2 (Ace2) receptor for entry and transmembrane serine protease 2 (Tmprss2) to prime its spike protein, while MERS-CoV relies on the dipeptidyl peptidase 4 receptor (Dpp4) for cellular entry, both of which are minimally expressed in A549 cells. In this study, we fine-tuned the expression levels of each receptor for optimal viral entry and infectivity using lentiviral transduction, cell sorting and clone selection. We successfully developed a robust human cell model expressing multiple viral receptors and demonstrated its susceptibility to both lethal coronaviruses and seasonal human coronaviruses, OC43 and 229E. We also compared two known 3C-like protease inhibitors and found that Nirmatrelvir is superior to Pomotrelvir in terms of pan-coronavirus antiviral activity. Furthermore, we tested 13 known antimalarial drugs and identified Halofantrine as having antiviral activity against SARS-CoV-2. Our findings suggest that this novel human cell model is a valuable and versatile tool for the screening and identification of pan-CoV antiviral drugs.