Figure 3
Evaluation of the effect of the different resistance-associated mutations on HIV-1 entry efficiency and viral growth kinetics. (A) Mutations H105Y, V255M, S375R/N, G471R and D474N were introduced in the SF162 envelope by site-directed mutagenesis and subsequently cloned into a full –length pBRNL4.3 plasmid, generating replication competent virus. TZM-bl cells were infected with the different viruses using equal amounts of RT activity (40 pg RT). The entry efficiency is expressed as a percentage relative to the pBRNL4.3 bearing the wild type envelope sequence of SF162. The H105Y mutation is related to the rM48BaL virus; however for these experiments this mutation was also introduced in the SF162 envelope. (B) Viral replication of the original resistant SF162 and BaL viruses and their respective control wild type viruses in monocyte-derived macrophages (MDM). Results of two independent experiments are shown. Viral titers were first determined on PHA/IL-2 stimulated PBMCs from each of the two donors. Next, monocytes from the same donor were differentiated into MDM and infected at a multiplicity of infection of 10-3. Cultures were incubated for 15 days, and viral growth was determined on different time points by measuring the p24 amount in the supernatants.