2.2. Mutagenesis

Site-directed mutagenesis was carried out using the standard QuikChange^TM protocol (Stratagene). Plasmids p66RTB containing the p66-coding sequence of HIV-1_BH10 and the codon-optimized p68-coding sequence of HIV-2_EHO were used as templates. Complementary mutagenic primers (Supplementary Table S1) were used to amplify entire plasmids in a thermocycling reaction carried out with high-fidelity Pfu DNA polymerase. Double mutants E138K/M184I and E138K/M184V were prepared from the single-mutant E138K HIV-1 RT, previously described [17]. Quadruple mutants E138K/M184I/N348I/T369I and E138K/M184V/N348I/T369I were obtained after introducing mutations E138K and M184I/V in sequential order, using as template the plasmid p66RTB containing the p66-coding sequence of mutant N348I/T369I HIV-1_BH10 RT. Mutant G344E/D345P/ins346F of HIV-2_EHO RT was prepared with mutagenic oligonucleotides containing the nucleotide changes required to introduce G344E and D345P substitutions in the presence of the insertion of Phe346, and the template RT-coding region encoding the HIV-2_EHO RT with the 346F insertion alone. The mutant H342Y/G344E/D345P/ins346F/V351T was derived from mutant G344E/D345P/ins346F, after introducing mutations H342Y and V351T in sequential order with the mutagenic primers indicated in the Supplementary Table S1. After mutagenesis, RT-coding regions were entirely sequenced and, if correct, used for RT expression and purification.