GRL-216 fails to block protease dimerization with V82I and I84V substitutions. To examine whether an amino acid substitution(s) emerged upon selection of HIV-1NL4-3 with GRL-216, recombinant clones containing the L24I, V82I, I84V, V82I/I84V, or L10I/L24I/M46L/L63P/V82I/I84V mutation(s) were generated in the setting of the FRET-based HIV-1 expression assay (23). With L24I, V82I, or I84V alone, the protease dimerization-blocking activity of 0.1 μM GRL-216 was not affected; however, with either set of the V82I/I84V or L10I/L24I/M46L/L63P/V82I/I84V substitutions, GRL-216 failed to block the dimerization.