A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike

Xia, Shuai; Yan, Lei; Xu, Wei; Agrawal, Anurodh Shankar; Algaissi, Abdullah; Tseng, Chien-Te K.; Wang, Qian; Du, Lanying; Tan, Wenjie; Wilson, Ian A.; Jiang, Shibo; Yang, Bei; Lu, Lu

The inhibitory activity of peptide against MERS-CoV replication was tested in Calu-3 cells using a modified standard microneutralization assay, as previously described (68) . Briefly, 60 l of a serially twofold diluted peptide was incubated with 60 l (120 TCID 50 ) of MERS-CoV in MEM medium supplemented with 2% FBS (M-2 medium) in duplicate wells of 96-well plates for ~60 min at room temperature. One hundred microliters of the peptide/MERS-CoV mixtures was then transferred into confluent Calu-3 cells grown in 96-well plates. Wells of Calu-3 cells cultured with M-2 medium with and without virus were included in these assays as positive and negative controls, respectively. While the advanced CPE of Calu-3 cells could develop within 24 to 36 hours in response to MERS-CoV infection at higher multiplicities of infection (MOIs) of 1 or 0.1 (69), we did not observe any prominent formation of CPE until ~ 60 to 72 hours after infection at an estimated MOI of ~ 0.001. Hence, to more accurately measure the efficacy of peptide inhibitors against MERS-CoV infection, we harvested the supernatants at 72 hours and quantified infectious virus titers by the standard Vero E6-based infectivity assays and expressed the titers as log 10 TCID 50 /ml.