In this study, we selected HIV-1 variants resistant to T1249 in vitro. Interestingly, gp41 amino acid position 38, implicated in T20 resistance, is also involved in T1249 resistance. Although the interface of the peptide inhibitor with HR2 is quite large, the resistance mutations primarily appear at or near position 38. Because of the large contact surface, the virus can perhaps easily compensate for point mutations, and therefore mutations at the docking site of the peptide inhibitor will have a more dramatic impact on peptide binding. The LLSGIV stretch has been shown to be a critical docking site for T20 (50
), and this may explain the critical role of position 38 in resistance development. Whereas T20 resistance is mediated by hydrophobic and noncharged amino acid substitutions at position 38 in HR1 (V38A, V38G, and V38W), T1249 resistance appears to require charged amino acids (V38E, V38R, and V38K). These results indicate that T20 and T1249 exhibit very similar inhibition modes that call for similar but not identical mechanisms of resistance. T1249 is a more potent inhibitor than T20, likely due to its higher HR1 binding affinity. A different gp41 amino acid substitution (involving charged residues) is apparently needed to prevent T1249 binding as opposed to T20 binding. A randomized mutagenesis study that focused on gp41 residues 37 and 38 previously showed the importance of residue 38 in T1249 resistance (3
). Our viral escape study, without an a priori bias for any specific residue, confirms the importance of residue 38 but also demonstrates that other changes can confer T1249 resistance. Besides mutations at position 38, substitutions at the C terminus of the HR1 domain (Q79E) and in the loop (K90E) were found to cause resistance to T1249 as well.
In single-cycle infection experiments, we measured up to 24-fold T1249 resistance for the V38E mutant and lower levels of resistance (3.0- to 6.7-fold) for the V38R, V38K, Q79E, and K90E variants. The previously described T20-resistant V38A, V38G, and V38W variants provided only a low level of T1249 resistance (2.1- to 2.7-fold) (Table ). Interestingly, none of the V38 variants provide cross-resistance to the third-generation fusion inhibitor T2635. In fact, some position 38 variants were found to be more susceptible to T2635 than the wild type was. In contrast, the Q79E and K90E mutants exhibited modest levels of resistance to all three spectra of peptide inhibitors. These observations suggest that resistance to T2635 differs mechanistically from T20 and T1249 resistance. While the position 38 substitutions directly affect the HR1-peptide interaction, this is probably not the case for the Q79E and K90E substitutions because they are located outside the actual peptide binding site. Possibly they accelerate the HR1-HR2 association and thereby restrict the time frame in which the peptides can act.
Similar to the HR1-T20/T1249 interaction, the HR1-HR2 interaction can be affected by the drug resistance mutations. Indeed, as for the V38A T20-resistant mutant (5
), a decrease in melting temperature of the six-helix bundle was seen for the V38E, V38R, and V38K variants. Consistent with these results, limited proteolytic experiments reveal not only a decrease in overall proteolysis resistance relative to that of the wild type but also a major change in the proteolytic pattern. This suggests that charged side chains at position 38 of gp41 perturb the six-helix bundle structure more dramatically than noncharged residues. Indeed, we measured a significantly destabilized six-helix bundle, reduced infectivity, and delayed replication for the resistant variants.
We analyzed the gp41 sequences after only one month of culturing under T1249 pressure. Upon prolonged culturing, we expect that further evolution will take place. It is likely that additional and/or compensatory mutations in gp41 or gp120 may provide further resistance to T1249 and/or improve viral fitness. This possibility is currently under investigation.
We initiated our in vitro T1249 escape studies with wild-type and T20-resistant virus variants. The input type of amino acid 38 appears to determine the outcome of evolution. Specifically, the V38R variant was generated exclusively from the T20-resistant V38G (three mutants) and V38W (four mutants) variants, whereas 38E was derived exclusively from the V38 wild type (one mutant) and the T20-resitant V38A variant (two mutants). Inspection of the underlying codon changes provides a likely explanation (Table ). Evolution of a 38E-encoding codon is relatively easy starting from V38-encoding and 38A-encoding codons (GTG→GAG and GCG→GAG, respectively), which require only a single transversion (T-to-A and C-to-A, respectively) (6
). However, both codons require double-hit mutations to make a 38R-encoding codon (GTG→CGG or AGG for V38; GCG→CGG or AGG for 38A). Interestingly, the situation is reversed for the G38- and W38-encoding codons, which prefer to evolve toward 38R. The G38E change requires only a single transition (GGG→GAG), but there are two simple routes toward R (GGG→CGG or AGG). The GGG→AGG change was in fact seen exclusively (three mutants), and it is linked to the most frequent G-to-A mutation that is needed (6
). Starting with a 38W-encoding codon also provides a route to a 38E-encoding codon (TGG→GAG, a double mutation) that is more difficult than that to 38R-encoding codon (TGG→CGG or AGG), two single mutation routes, of which the transition type (T to C; three mutants) is preferred over the transversion type (T to A; one mutant). Thus, the mutational bias of HIV-1 determines the precise evolution path toward drug resistance (28
The findings reported here are of potential clinical relevance as T20 therapy may trigger the selection of resistant viruses that influence resistance development under subsequent T1249 therapy. Although the further clinical development of T1249 has been halted (23
), the same selection phenomenon may occur with new entry inhibitors that use a similar mechanism of action. However, our observation that T2635 is not affected by T20 and T1249 resistance mutations at position 38 may disprove this argument. The results of this study also underscore the possibility that HIV-1 will lose fitness in the process of becoming resistant to potent fusion inhibitors, which may impact disease progression. Newer fusion inhibitors, including T2635, may reduce resistance development by a combination of improved potency and loss of Env function upon the acquisition of resistance. As such, the further development of this class of antivirals is warranted.