Using ASLV-A as a model for class I envelope function, residues that impact sensitivity to an HR2 peptide inhibitor were identified in two distinct regions of TM, the N-terminal heptad repeat (HR1), and the amino terminus of the subunit (Fig. ). The diversity of the thirteen distinct envelope variants described here, as well as the ease with which they were selected, suggests that they represent only a fraction of the possible sensitivity determinants. These mutations increase the R99 IC50 from approximately threefold to more than 1,500-fold (Table ). The mutations presented two distinct phenotypes, those that displayed expanded tropism and a reduced envelope activation threshold, and those that did not. The former class was only observed with HR1 mutations, although not all HR1 mutations were of this type.
One mechanism whereby substitutions within HR1 could influence R99 sensitivity is by changing HR2 peptide-binding affinity. However, proposing a mechanism of peptide escape for the other mutations (residues 2 to 12 of TM) is less straightforward. A disulfide bond between cysteines at positions 9 and 45 of TM (10
) would be expected to bring the amino terminus of TM in proximity to HR1, the target of R99, and thus mutations around position 9 might influence R99-binding affinity through steric hindrance. Indeed, four of the five substitutions in this region are of residues with larger side chains than the wild-type envelope (Table ). However, this is less apparent for D11N, the change with the strongest resistance to R99. D11E, which preserves the charge at this region while increasing the bulk, has only a modest fourfold effect on peptide sensitivity. This raises the possibility, then, that the negative charge of D11 interacts with other regions of TM within one or more envelope conformations, the disruption of which significantly affects R99 sensitivity.
HIV-1 escape mutants to the HR2 inhibitor enfuvirtide map to residues 36 to 45 of gp41 within the target-binding site for the inhibitor (20
). A three-residue motif, GIV (amino acids 36 to 38) within HR1, is a particularly significant region for resistance mutations. Interestingly, all of the ASLV resistant mutations within HR1 also lie within the N-terminal region and none were found in the HR1 C terminus. It is possible that destabilizing mutations at the HR1 N terminus, near the fusion peptide end of the coiled coil, are tolerated because coiled-coil formation initiates at the C terminus and mutations that affect the initiation region are not readily selected. Thus, C terminal mutants could theoretically provide resistance, but they are not seen because function of the TM protein is compromised.
Determinants of baseline sensitivity to peptide inhibitors, however, can also be located in gp120 domains that govern coreceptor specificity (12
) as well as a region of gp41 C-terminal to HR1 (21
). As the TM subunit of lentiviruses has an N-terminal fusion peptide (15
), there is no obvious HIV counterpart to the ASLV segment between the SU/TM cleavage site and its fusion peptide. The results presented here implicate this intervening segment, which is also found in filoviral glycoproteins (16
), as a functional component of envelope activation, in addition to identifying an important new region governing HR2 peptide sensitivity. The study of these mutants might provide insight into the role of the TM amino terminus and its relationship to the internal fusion peptide in ASLV and filoviruses. One possibility is that this region acts similarly to the capping structure of influenza HA and helps to bring the apposed membranes together during fusion. It will be interesting to determine if the peptide resistant mutations within the ASLV intervening segment affect the rate of membrane fusion.
An expanded tropism phenotype as was seen in this analysis of ASLV has not been previously reported for any HR2 peptide escape mutant. Determinants of HIV-1 coreceptor specificity within gp120 have been reported to modulate enfuvirtide sensitivity (12
), with envelopes that bind coreceptor more efficiently being less sensitive to the inhibitor (45
), although this observation has been questioned (20
). Thus envelopes with greater coreceptor affinity might be triggered more efficiently, providing less of a target for enfuvirtide inhibition. Whether this increased coreceptor affinity also translates into increased use of alternative coreceptors and expanded tropism of enfuvirtide-insensitive variants has not been studied. Similarly, CD4 independence, and thus an altered glycoprotein activation threshold, of an HIV-2 isolate has been shown to involve either one of two changes within gp41 (48
); interestingly, one of these two changes is within the GIV motif that is associated with enfuvirtide resistance, and the other change flanks this motif. The impact of these changes on sensitivity to an HR2-based peptide has not been investigated.
The native structure of influenza hemagglutinin (HA) has been described as metastable (7
), where the native form of the glycoprotein on the virion is blocked from a more energetically favorable structure by a thermodynamic barrier. Conformational changes in HA, which can be triggered by acid, heat, or urea, convert the envelope to its lower-energy, fusion-active state. It has been proposed that receptor binding could perform a similar destabilizing role for other viral envelopes (7
). As mentioned above, HIV envelopes that exhibit CD4 independence or greater coreceptor affinity demonstrate altered interactions between envelope and receptor(s), and these changes appear to be linked to enfuvirtide sensitivity. Similarly, our analysis of ASLV mutants suggests a model for peptide resistance that, for a subset of the mutants, involves altered triggering of the envelope glycoproteins. Of the thirteen ASLV-A mutants described here, four displayed detectable infectivity on 293T cells. This represents one-half (four out of eight) of the alterations within HR1, illustrating that this phenotype is not uncommon. The activation threshold for the expanded tropism mutant envelopes appears to be lower, as reflected by their in vitro requirements for receptor-mediated triggering (Fig. ), revealing a potential mechanism by which alternative receptors might trigger these mutants. Such a mechanism would not require a change in the binding affinity for the putative alternative receptor(s). A single amino acid substitution in the SU subunit of the ASLV-B envelope also confers a similar expanded tropism, and it has been suggested that the mechanism for this phenotype involves not receptor affinity but rather the fusogenic capability of the envelope (43
The TM substitutions that conferred expanded tropism were distinguishable by their slightly increased sensitivity to neutralization as well (Fig. ). Some enfuvirtide resistance mutations of HIV have also been noted to be more sensitive to neutralization (46
). This change in sensitivity may reflect a destabilized native conformation, in which access to neutralizing epitopes is enhanced; alternatively, it may indicate a modification of the conformational changes in envelope such that a distinct set of epitopes is displayed, or that the epitopes are exposed for a longer period of time.
The experiments described here characterize envelope function with single-round infection assays, so it is possible that some or all of the mutations might not be well tolerated in the absence of R99 selection. For HIV-1, viral fitness is decreased for enfuvirtide-selected mutants (30
). However, all of the ASLV-A mutant envelopes reported here mediated infection of avian QT6 cells with wild-type efficiency (Fig. and data not shown). In addition, production of replication competent ASLV encoding the L62A envelope variant that displays peptide resistance and expanded tropism did not result in rapid reversion to wild-type sequence (data not shown). Furthermore, the T2I substitution has been found in natural ASLV isolates (28
), and the D11E change has been observed in an endogenous, subtype J-like sequence (11
). The functional impact of these natural variants has not been explored.
Peptide inhibitors analogous to enfuvirtide and R99 have been described or proposed for a wide variety of human pathogens that encode class I membrane fusion proteins, including severe acute respiratory syndrome (SARS) coronavirus (4
), Ebola virus (56
), Nipah and Hendra viruses (6
), HTLV (41
), influenza virus, respiratory syncytial virus, parainfluenza virus, and measles virus (27
). The demonstration of the reduced activation threshold of some HR2 peptide-resistant variants may have important consequences for the effective use of this class of inhibitor in antiviral therapy. Although expanded viral host range would be detrimental in vivo, our results also suggest that immune system pressure might provide a counterbalance by selecting against such variant envelopes due to their increased sensitivity to neutralization. These studies thus highlight the requirement for more analysis of variant viruses that are resistant to peptide entry inhibitors.