Cloning and Validation of HIV-1 Nef and Vpu Constructs

Nef and vpu amplicons were cloned into eukaryotic expression vectors as described (28 (link), 30 (link)). Briefly, second-round products were purified using the E.Z.N.A Cycle Pure kit (Omega Bio-tek). Each nef or vpu amplicon was then cloned into a modified version of pSELECT-GFPzeo (In vivoGen) that contained 5’ AscI and 3’ SacII sites, where expression is driven by a composite hEF1-HTLV promoter. pSELECT-GFPzeo also features an independent CMV/HTLV promoter driving the expression of GFP. The pSELECT-GFPzeo vector used for vpu cloning was further modified to include the HIV-1 Rev Responsive Element (RRE) sequence downstream of the multiple cloning site to enhance Vpu expression (pSELECT-RRE-GFP). Following ligation, DNA products were transformed into E. cloni 10G chemically competent cells (Lucigen) and plated on Luria-Bertani (LB) agar containing Zeocin. Colonies were isolated, grown in LB medium containing Zeocin, and plasmid DNA was purified using the E.Z.N.A Plasmid DNA Mini kit (Omega Bio-tek). Plasmid clones were validated by Sanger sequencing. Following phylogenetic authentication (see below), one intact nef and one intact vpu clone per participant was selected for in vitro functional analysis (Supplementary Figure 1).