Generation of A3H Haplotype Constructs

Plasmids containing A3H haplotypes I through IV were previously described [16 (link)], and used as templates to generate C-terminally HA-tagged constructs. All constructs used in this study were cloned into pcDNA3.1 (Thermo Fisher, Waltham, MA, USA) using EcoRI/XhoI restriction sites. Mutations were introduced using standard PCR or using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent, 210515, Santa Clara, CA, USA). Double A3H fusion constructs I–I, II–II, I–II, and II–I were previously described [20 (link)]. The double A3H fusion constructs unique to this study, III–III, III–II, and II–III were made through amplification of haplotypes II and III with a 5’ or 3’ primer containing the linker sequence GGT GGT GGT GGT GGC GCC (Gly-Gly-Gly-Gly-Gly-Ala). The 5’ and 3’ haplotype domains were digested using the KasI restriction site in the linker, and EcoRI (for 5’ domains) or XhoI (for 3’ domains). The 5’ domains, 3’ domains, and the EcoRI/XhoI double digested pcDNA3.1 vector were joined using T4 DNA Ligase (New England BioLabs, M0202, Ipswich, MA, USA). Infectivity experiments were performed using the HIV-1 molecular clone pLAIΔenvLuc2Δvif, which was previously described [28 (link)].