Fitness loss caused by the T242N mutation was partially restored by the preexisting compensatory amino acids. The same amount (2.5 ng p24) of the T/F and its mutant was mixed to infect freshly purified CD4+ T cells, and the proportion of each virus in the culture was determined by the PASS fitness assay. The relative fitness of the T242N mutants was determined for CH58 (a), CH470 (b), and CH40 (c) by comparing each T242N mutant to its corresponding T/F virus. The increased fitness loss of the T242N mutants after removing the compensatory amino acids was determined for CH58 (d), CH470 (e), and CH40 (f) by comparing each double-mutation virus to its corresponding T/F virus. The impact on fitness in the T/F viruses by the compensatory amino acids alone was determined by introducing those amino acids in the corresponding T/F viral genomes for all three viruses: CH58I247V(g), CH470I247V(h) and CH40Q219H(i).