Several expression plasmids were generated from env genes of 4 cluster individuals (LTNP_1, LTNP_3, LTNP_RF_21, and AS7), 4 HIV-1 chronic progressors (I_10, IV_10, I_14, and RIS_06), and 3 laboratory-adapted viruses (SF162, NL4.3, and 89ES_061). The R5-tropic BaL.01-env (catalog no. 11445) glycoprotein plasmid was from the NIH AIDS Research and Reference Reagent Program. The env genes were amplified by nested PCR from proviral DNA (35 (link)). The products were cloned into the pcDNA3.1D/V5-His Topo expression vector (Invitrogen). In total, 9 clones derived from the 4 cluster patients (LTNP_1 clones 8, 10, 12, and 37; LTNP_3 clones 20, 22 and 3; LTNP_RF clone 21; and AS7), 6 clones from chronic progressors (I_10 clones 27 and 28, IV_10 clones 5 and 16, RIS_06 clone 2, and patient I_14 clone 9), and 4 clones from reference samples (SF162, 89ES_061, NL4.3, and BaL.01) were constructed. pcDNA3 plasmids coding for NL4.3-derived mutants D368R and 41.2, which abrogate CD4 binding and gp41-mediated membrane fusion, respectively, were generated by site-directed mutagenesis or by subcloning from original plasmids, as previously described (24 (link)). Expression plasmids were transformed in DH5α cells, and clones were sequenced to check the correct insertion of the env gene.
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