We performed mutagenesis on the HR2 domain and tested the activity of the lead compounds against the EBOV mutants to identify which HR2 residues interacted with the lead compounds. Briefly, each HR2 residue (residues 610 to 636) was separately replaced with alanine (A) by site-directed mutagenesis (Agilent Technologies) and confirmed by sequencing (BGI, Beijing, China). The resulting plasmids were used to package the EBOV mutants. The infectivity of the EBOV mutants and the antiviral activity against them were evaluated as described above. The HR2 domain from influenza virus was mapped in the same manner by using amino acid substitutions.

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