Restriction and ligation enzymes

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).

l-Cysteine hydrochloride monohydrate, l-cystine dihydrochloride, and other nonradioactive amino acids were purchased from Sigma-Aldrich. A 30% (wt/wt) solution of hydrogen peroxide (H₂O₂), bovine catalase, ovalbumin, O-nitrophenol-β-galactoside (ONPG), diethylenetriamine pentaacetic acid (DTPA), dithiothreitol (DTT), 2,2′-dipyridyl, β-mercaptoethanol, antibiotics, and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) were purchased from Sigma-Aldrich. Coomassie reagent was from Thermo Scientific. Medium reagents and buffer chemicals were from Fisher Chemical. Restriction and ligation enzymes were from New England BioLabs. Qiagen kits were used for genomic and plasmid DNA preparation. Colony PCR beads were from GE Healthcare, and PCR reagents were purchased from Invitrogen. DNA sequencing was performed at ACGT, Inc. Radiolabeled l-[1,2,1′,2′-¹⁴C]cystine and l-[¹⁴C(U)]leucine were purchased from Perkin Elmer; we note that putative radiolabeled cystine that was received from another supplier was found not to be authentic cystine. Transport measurement filters were purchased from Millipore Sigma (GSWP02500).