This is likely due to different gp41- binding regions for the two drugs, as ENF aligns with its corresponding HR1 residues closer to the N-terminus of gp41 and RC-101 is known to bind to HR2. The V38A single mutant remaining susceptible to RC-101 provides evidence that RC-101 targets a region outside of the ENF binding site. This finding is particularly promising since RC-101, and similarly acting h-defensins, would likely remain active against HIV-1 harboring other ENF-resistance mutations such as those frequently observed in the ����GIV���� region of HR1. Irrespective of the specificity of either HR1 mutation, N126K provided some degree of resistance to both drugs despite the clear differences in not only the structure of both inhibitors tested, but also the different binding sites of these peptides on gp41. Moreover, we have shown that N126K has a different effect on HhAntag691 Hedgehog inhibitor fusion depending on the primary mutation present in HR1 responsible for providing drug resistance. The effect on fusion associated with Q66R and V38A may again be explained by their similar locations on the HR1 helix. Both Q66R and V38A occupy the same position of the helical turn directly interacting with the HR2 helix of the same molecule and could reasonably affect membrane fusion in a similar way. The comparable effect on fusion seen with HR1 mutations is MK-1775 contrasted by the difference in compensatory activity with the addition of N126K. While the V38A+N126K genotype appears to overcompensate for the loss of fusion associated with V38A, Q66R+N126K restores fusion to the level of the wild-type virus. This observation may explain differences in fitness in the absence of fusion inhibitors displayed by the two double-mutants. Previous studies have demonstrated that N126K leads to a rapidly fusing gp41 that is dependent on ENF for infection. However, with our Q66R+N126K mutant we observed that fitness was not affected and that fusion and entry kinetics were restored to the levels of the wild-type virus. This observation is perhaps due to the differences in how Q66R or V38A would affect the formation of the mature gp41 complex. The difference being, that while V38A merely exchanges one small hydrophobic residue for another, perhaps reducing HR1��s affinity for both HR2 and ENF, Q66R inserts a large cationic residue into the hydrophobic pocket of gp41 that could not only act to repel a cationic peptide such as RC-101, but would also sterically constrain two key tryptophan residues located in the corresponding region of HR2 that are believed to play a significant role in the activity of gp41. This key difference may explain why we see overcompensation in fusion observed with V38A+N126K, while Q66R+N126K only exhibits restored fusion when compared with the wild type.