Evolution of the T20-dependent HIV-1 variant. (A) Replication of wild-type (GIV-SNY), T20-dependent (GIA-SKY) and HIV-1 variants obtained in evolution cultures in the absence of T20. Bars represent CA-p24 values at day 4 post-infection, which represent the relative differences in replication capacity. Infection of fresh SupT1 cells was performed with an equal amount of the viral stocks obtained at day 42 of evolution. (B) Summary of observed mutations, replication capacity and syncytia formation of the six evolution cultures after 42 days of evolution. Replication without T20 and syncytia formation represent relative differences observed in infection experiment displayed in 1A (-, no replication or syncytia; ++++, high replication or all cells involved in syncytia). (C) Schematic of mutations in the Env protein. The complete Env gene is shown (not to scale) with the location of the original GIA-SKY mutations and the second site changes selected in the evolution cultures. The G431R change that was selected in multiple cultures is marked in bold. Both gp160 and gp41 HxB2 numbering references are included.