The pathway identified here for early resistance to the N-peptide fusion inhibitor N44* highlights important structure-function relationships in Env that inform treatment strategies and provide insights into Env-mediated fusion. We show that two mutations, one each in the N- and C-HRs, confer a two- to fivefold increase in resistance to several N-peptide fusion inhibitors, as well as cross-resistance to C-peptide fusion inhibitors. The N mutation also altered viral sensitivity to sCD4. Thus, the resistance mutations reveal residues that broadly influence Env activation and fusion, as demonstrated in two Env proteins that use different coreceptors.
What do these findings tell us about mechanisms of resistance and Env-mediated fusion? In theory, several mechanisms of resistance to peptide fusion inhibitors are possible. In one case, resistance could occur directly by mutation of contact residues that impair inhibitor binding, as has been suggested for some T20-resistant viruses (31
). Alternatively, resistance could arise indirectly, by reducing inhibitor access or competitiveness to the binding site. Reduced access could reflect (i) steric issues or (ii) decreased time that the binding site is available to the inhibitor due to kinetics of conformational changes and fusion. Reduced inhibitor competitiveness could occur by making contacts in the endogenous 6HB more favorable than those involving the inhibitor. In the case of our escape mutant, our results appear to rule out the direct mechanism. The cross-resistance against the N- and C-peptide inhibitors argues against resistance being due to mutation of contact residues, because the N and C peptides make distinct contacts with gp41. Molecular modeling also does not support a model in which the resistance mutations impair helical interactions. Most convincing, the thermal denaturation studies indicate that the resistance mutations could actually stabilize 6HB interactions rather than destabilize them.
The biophysical experiments introduce thermodynamic considerations into the discussion of resistance. In our peptide models of the 6HB (Fig. ), the resistance mutations increased the structural stability of the resistant 6HB (NmCm) relative to the wild-type (sensitive) 6HB (NwCw). Interestingly, the mutation in the (noncognate) N-HR benefits the endogenous 6HB (NmCm), but it cannot benefit the 6HB formed by N44* binding to the C-HR (N44*Cm). It thus appears that the virus evolved a way to improve endogenous 6HB stability without improving inhibitor binding. The relevance of this pathway as a mechanism of resistance is supported by a recent study of a patient who developed resistance to T20 (Enfuvirtide) by generating point mutations in the N- and C-HRs (2
). Although the mutations reported by Baldwin et al. (2
) differ from those described here, it is striking that in vivo resistance to T20 (a C-peptide inhibitor) involved a mutation in the noncognate C-HR that resulted in a 3°C improvement in stability of the resistant 6HB, analogous to our in vitro findings involving an N-peptide fusion inhibitor.
Thermodynamic arguments relating to peptide inhibition suggest a model in which peptide is in direct competition with the endogenous HR at some step of the fusion process. With respect to the increased stability of the resistant 6HB, our data are consistent with an equilibrium model of inhibition by peptide proposed by Caffrey et al. (4
). Alternatively, it is thought that the free energy released during formation of 6HB helps overcome the free energy barrier for membrane fusion (27
). Stabilization of the endogenous 6HB could simply release more energy to promote fusion pore stabilization and expansion that are needed for complete membrane merge (26
), resulting in faster fusion kinetics and less time for the inhibitor to bind. Further experiments are under way to dissect the thermodynamic parameters contributing to enhanced stability of the resistant 6HB and its consequences for resistance.
Our data also support kinetic parameters in the development of resistance. In fact, the biophysical experiments showing that the 6HB involving the N44* peptide inhibitor is less stable than the sensitive (WT) 6HB (NwCw Tm
> N44*Cw Tm
) indicate that free energy considerations alone do not control inhibition, because N44* remains a potent inhibitor against WT virus. Rather, the data support a kinetic mechanism in which the inhibitor has preferential access to the gp41 target sites during refolding steps in virus entry. Experiments involving sCD4 further suggest kinetic parameters in resistance. In two different strains of HIV, the N mutation altered sensitivity to sCD4, indicating changes in the activation energy for the transition from native to fusion intermediate conformations. For the JR-CSF Env, which is much more resistant to sCD4 than the HXB2 Env, the N mutation increased sensitivity to sCD4, perhaps by destabilizing the native conformation. However, the opposite pattern was observed for the HXB2 Env, which is already easy to activate. This apparent discrepancy is difficult to interpret, but it is tempting to hypothesize that there is an optimal range of Env triggering that reduces the kinetic window for the inhibitor to bind Env. For the JR-CSF Env, easier receptor activation might allow more Env proteins to trigger and organize into fusion pores, speeding up fusion and acquisition of the peptide-resistant 6HB. For the HXB2 Env, reduced hair-trigger activation and consequent premature inactivation of Env might also lead to more efficient fusion kinetics. In this way the N mutation could influence the kinetics of fusion and the time available for the inhibitor to bind gp41. A kinetic model of fusion that is modulated by receptor density and affinity has been previously proposed to explain sensitivity to peptide fusion inhibitors (30
Indirect models of resistance offer an appealing strategy of escape for dominant-negative inhibitors. Dominant-negative inhibitors mimic interactions in the target protein and would be expected to impose constraints on the development of resistance; mutations that impair inhibitor binding would also likely impair function of the target protein. It is likely that C-HR mutations that reduce binding to N peptides would also destabilize the 6HB comprised of endogenous HR (37
). Such destabilizing mutations would also likely reduce infectivity of the mutant virus. A correlation between decreased 6HB stability and reduced infectivity has been reported for other mutations in gp41 (16
). Yet T20-resistant viruses can acquire resistance by mutating residues that directly impair peptide binding (2
). A stretch of the N-HR including the GIV sequence has been shown to be important for T20 binding to gp41, and this region is frequently mutated in resistant isolates from T20-treated patients (1
). A recent clinical study reported that T20-resistant viruses are less fit (23
). Residues outside the N-HR, including gp120 sequences, however, have also been shown to modulate sensitivity to T20 (10
). Given the mutability of Env, it is likely that several pathways for resistance can emerge, and a pathway may involve several factors that contribute to resistance.
Aside from providing insights into mechanisms of resistance, our escape mutant virus also identified residue 577 as having a role in both activation of Env-mediated fusion and stability of the 6HB. Thus, residue 577 helps to regulate more than one conformation of Env, including the fusogenic 6HB conformation and the native or fusion-intermediate conformations that influence Env activation. This dual effect appears to be analogous to the conformational switch region that has been recently described for the fusion protein (F) of a paramyxovirus (34
), in which a region near the C-HR regulates both activation of F-mediated fusion and 6HB stability. Residues in the N-HR region of gp41 have previously been reported to be important for contacts with gp120 (6
), further supporting the idea that this residue may be involved in a conformational switch. Notably, residue 577 lies in a hydrophobic pocket region of the N-HR, which has been proposed to be an important region for stabilizing the coiled coil (13
) and a good target for developing inhibitors (7
). The demonstration that residue 577 tolerates substitutions (40
), and indeed mutates under selection as shown here, has implications for drugs targeting this pocket.
In summary, our studies identify new viral determinants that confer cross-resistance to N- and C-type peptide fusion inhibitors and change susceptibility to inhibitors mimicking CD4. The N mutation, which altered sensitivity to sCD4, also shows that this region of N-HR can play a dual role in both receptor activation and 6HB stability. The combined N and C mutations confer more resistance against several inhibitors than the individual mutations (Table ) and point to an indirect mechanism of resistance, where coupled changes in the N- and C-HRs may regulate several aspects of Env-mediated fusion to provide escape from a dominant-negative inhibitor. The importance of residues 577 and 648 in the development of resistance to peptide fusion inhibitors and the fusion process was not predicted from the 6HB high-resolution structures. Our findings underscore the complexity and plasticity of Env-mediated fusion and some of the challenges in designing fusion inhibitors.