We investigated the evolution of resistance to the third-generation HIV-1 fusion inhibitor T2635 by culturing the virus under increasing pressure of T2635. It was previously shown that common T20 and T1249 resistance mutations do not cause high-level cross-resistance to T2635 (21
). We demonstrate that the accumulation of numerous mutations within multiple gp41 subdomains is needed to obtain high-level resistance to T2635 and that the number of mutations was strongly correlated with the level of resistance (). Furthermore, this accumulation of mutations was accompanied by a dramatic loss of Env function. We did not find evidence that any of the gp41 mutations were compensatory, i.e., served to improve Env function in the context of initial resistance mutations. In contrast, the data suggest that many of the observed changes within gp41, if not all, contribute to resistance and do not restore Env function. We did, however, find evidence that compensatory changes were selected in gp120. We have investigated the R298K substitution, which was selected in the majority of evolution cultures and which partially restored Env function without contributing to T2635 resistance. In the subsequent paragraphs, we discuss the mechanistic implications of the observations reported in this study.
Some T20 and T1249 resistance mutations present in the starting cultures contribute to T2635 resistance and were therefore preserved during T2635 escape. In particular, substitution N126K, which was present in 8 starting cultures, was always preserved and selected for in an additional 18 cultures, in addition to the selection of N126H in a single culture. In fact, at the end of the 6-month evolution experiment, only 3/30 cultures did not have a substitution at position 126. The IC50
measurements explain the selection of N126K: it causes modest T2635 resistance on its own, and in combination with other substitutions its effect can be additive (e.g., with Q79E) or synergistic (with Q66R). N126K does not cause significant resistance to T20 (), and its emergence during T20 escape compensates for loss of Env function (slower fusion kinetics) caused by resistance mutations in HR1 (6
Mutations involved in resistance to T20 and T1249 at position 38 rapidly reverted back to the wt valine. Apparently, they do not contribute to T2635 resistance and are lost because they negatively affect Env function (22
). In addition, they may disturb the RNA structure of the underlying Rev response element and only the wt codon creates an optimal RNA structure (48
). Mutations Q79E and K90E cause modest T2635 resistance () and have a large negative effect on virus infectivity. These mutations were therefore preserved only when no other resistance mutations were selected ().
Surprisingly few substitutions occurred in the assumed binding site of T2635 in HR1. Only substitutions L33S and Q66R were detected in multiple independent cultures. This is in sharp contrast to escape from first- and second-generation peptides, which is predominantly induced by one or multiple changes in the peptide binding site (particularly positions 36, 38, and 43 [19
]). The binding energy of T2635 for HR1 is high compared to that of T20 and T1249 (21
). We have previously shown that substitutions at position 38 had a considerable effect on the drug-HR1 binding energy. This was sufficient to cause resistance to T20 but not to T2635 because the relative contribution of residue 38 to peptide binding is much less (24
). Apparently, single amino acid substitutions in the binding site are not sufficient to prevent T2635 binding. Multiple mutations in the T2635 binding site may be necessary to sufficiently lower the T2635-HR1 binding energy to cause resistance, but this escape route has a high genetic barrier and therefore is more difficult and may be accompanied by an insurmountable loss of Env function and viral fitness. Therefore, other escape mechanisms are apparently preferred.
The virtually exclusive selection of HR1 substitutions at positions 33 and 66 on the outer edges of the T2635 binding site leads us to speculate that T2635 has another mechanism for initial binding other than T20 and T1249. The T20 and T1249 peptides lack secondary structure in solution, and they assume a secondary structure upon binding to HR1 (21
). T20 and probably T1249 are thought to dock onto HR1 around residues 36 to 43 (65
), which implies that they “zipper up” bidirectionally to acquire their helical structure, using the HR1 grooves as a template ( A). Our results suggest that T2635, which is stably helical in solution (21
), does not dock onto the center of HR1 but at either side, and then snaps into the HR1 grooves unidirectionally (B). T2635 binding may therefore depend more on residues near the ends of its binding site. What are the atomic interactions that underlie the interactions of residues 33 and 66 with T2635? The HR1-T2635 model shown in reveals that L33 engages in hydrophobic interactions with L35 of T2635 (corresponding to L149 of HR2). The L33S substitution will eliminate these interactions and probably decrease docking efficiency of T2635 at this end. Q66 can form a hydrogen bond with the nitrogen of the pyrrole ring of W3 of T2635 (corresponding to W117 in HR2) () (27
). W3 is the most N-terminal residue of T2635 that interacts with HR1 and packs in the hydrophobic groove formed by residues W60, I62, and L65 (30
). Between all these hydrophobic interactions, the polar interaction with Q66 may properly orient W3. The positively charged R side chain of Q66R will likely engage in a salt bridge with E4 on T2635 (). As mentioned before, establishing such a salt bridge would require a slight counterclockwise rotation of the T2635 helix, which could interfere with the proper packing of W3 into the HR1 groove. We have previously shown that adverse salt bridges can interfere with peptide packing onto HR1 and result in resistance to T20 and T1249 (24
). This theory is supported by the observation that a variant of T20, containing helix-stabilizing salt bridges similar to those of T2635, is no longer sensitive to resistance mutations at positions 38, 38, or 43 (50
). Furthermore, resistance to the stabilized SC29EK peptide occurs at position 37 (I37K), which is located at the end of its binding site (47
Fig. 6. Mechanism of T20 and T2635 docking onto HR1 and escape. The T20 and T1249 resistance “hotspots” at positions 38 and 43 are mapped onto the structures of the T20-HR1 complexes (A), as are positions 33 and 66 on the structure of the T2635-HR1 (more ...)
As we found many mutations in regions outside the HR1 binding site, T2635 resistance mechanisms other than direct effects on peptide binding should account for the resistance observed in most cultures. Several mutations were repeatedly selected in the central part of the HR2 domain (N126K, H132Q, E136G, E137G/V). Substitution N126K has been reported several times before (6
). It has been implicated in the appearance of a T20-dependent virus (7
) and is thought to speed up six-helix bundle formation, thereby limiting the window of opportunity for fusion inhibitors to act. Several studies have described an improved thermal stability and/or improved free energy of the six-helix bundle with substitutions at position 121, 126, 137, or 138 (7
). Furthermore, the removal of the glycan at position 126 enhances the thermal stability of the HR1-HR2 complex (67
). Ray et al. showed delayed fusion for resistance mutations N43D and Q66R in HR1, while the addition of mutations in HR2 can restore fusion kinetics (55
). Similarly, residue E154K is thought to improve fusogenicity (61
). Combined, these studies imply that substitutions in HR2 can favor the formation of a stable postfusion six-helix bundle, thereby accelerating fusion kinetics and decreasing the time frame for T2635 interference. We suggest a similar mechanism for the Q66R/N113D double mutant. An electrostatic interaction between N113D and Q66R may favor HR1 and HR2 association while disfavoring the binding of T2635 by an adverse salt bridge, resulting in a preferred binding of HR2 and accelerated fusion kinetics.
The kinetics of the six-helix bundle formation may also be affected at an earlier step. Premature interactions between HR1 and HR2 are prevented by the presence of gp120 domains, and before HR2 can fold onto HR1, the gp120 domains have to relax their grip, or even completely dissociate from gp41 (10
). This is accomplished only after engagement of the receptor and coreceptor, allowing the next steps in the fusion process (37
). Substitutions that weaken the gp120-gp41 interactions may accelerate six-helix bundle formation and cause T2635 resistance. Interestingly, several of the changes we observed (K77N/E, Q79E, K90E, T94N, N100D) are located in the gp41 loop domain that is known to interact with gp120 (11
). Possibly, these changes weaken the gp120-gp41 interactions, thus allowing faster six-helix bundle formation and leaving less time for T2635 to act. Premature six-helix bundle formation, i.e., in the absence of a target cell, will result in Env inactivation that may account for the loss of Env function.
This leaves us with a few less frequently observed non-HR1 substitutions. The A6V substitution, which as a single mutant provides the strongest T2635 resistance (7-fold), is located in the FP and may therefore alter the gp41 interactions with lipids. It may affect the speed and/or efficiency of the FP insertion in the target membrane which occurs prior to six-helix bundle formation (1
) or the transition from membrane hemifusion to full membrane merger. Alternatively, the FP substitution may affect the interaction with gp120. Interestingly, substitutions at positions 5, 7, and 8 in the FP were shown to confer resistance to the CCR5 inhibitor vicriviroc (3
), although the mechanism remains unclear. These findings reveal that the hydrophobic FP contributes to the entry process in ways we do not understand yet. Furthermore, we repeatedly observed the L210F change in the cytoplasmic tail of gp41. Since it does not cause resistance (), we guess that it may be a compensatory change. L210F was present in a virus isolate that showed enhanced Env incorporation into virions (8
). Possibly, the loss of Env function is compensated by incorporation of more Env molecules onto virions.
In summary, we present an escape from the third-generation peptide fusion inhibitor T2635 that requires multiple mutations in various gp41 subdomains and that comes at the expense of Env function. The mechanism of resistance is very distinct from escape routes observed for the first- and second-generation fusion inhibitors T20 and T1249. The emerging characteristics of third-generation fusion inhibitors like T2635, including enhanced stability, enhanced potency, and escape that requires multiple mutations and comes with a loss of function, warrant further studies on this class of inhibitors.