Constructing and Characterizing Env Mutants

All env genes were amplified by PCR from their parental plasmids and cloned into the Xho1 and Eag1 restriction sites of the pEBB expression plasmid [107 (link)] (oligos from Eurofins, PCR enzyme Pfu Turbo from Agilent, restriction and ligation enzymes from New England Biolabs). V3-loop swaps were performed as previously described [63 (link)]. Briefly, PCR amplification of the Env_HXB2 or Env_SF162 V3 loop using overhang primers produced short dsDNA segments that were subsequently used as primers in QuikChange PCR (Agilent) to introduce the desired changes. Point mutations in the Env sequences were achieved through QuikChange PCR using overlapping oligonucleotides (Eurofins and IDT). All Env constructs were confirmed through Sanger sequencing of the entire open-reading frame prior to use (Genewiz).