The PCR products were cloned into the pcDNA 3.1D/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA). Subsequently, HA alanine mutants were generated using Gene Tailor Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA). A total of 20 A/PR/8/34-HA mutants were generated (His25Ala, His45Ala, Thr315Ala, Asn336Ala, Ile337Ala, Pro338Ala on the HA1 subunit and Trp357, Thr358, Gly359, Met360, Ile361, Asp362, Gly363, Trp364, Thr384, Ile388, Thr392, Val395, Asn396, Glu400 on the HA2 subunit. Sequence numbering refers to A/PR/8/34, GenBank accession number ABO21709).