The entry of HIV into cells can be blocked by ENF, which binds to HR1 and prevents subsequent HR2 interactions that are needed for membrane fusion (10
). In addition to being the first in the new class of antiretroviral drugs collectively referred to as entry inhibitors, ENF is unusual in that it targets a structural intermediate of the viral entry process (29
). The ENF binding site is not exposed in the native Env trimer but rather becomes accessible as a consequence of receptor binding (16
). The transient exposure of the ENF binding site during the entry process makes possible a variety of mechanisms by which HIV could potentially acquire resistance to this novel drug. With ENF being administered to a growing number of patients, the mechanisms by which HIV can become resistant to ENF and the implications of ENF resistance for sensitivity to other classes of entry inhibitors, neutralizing antibodies, and viral fitness assume greater importance.
There are at least two general mechanisms by which changes in Env could affect ENF sensitivity. Perhaps the most obvious is the classic mechanism in which changes in the drug binding site confer resistance and a selective growth advantage in the face of therapy. While limited work has been done thus far, it is known that selection for highly drug-resistant viruses in vitro can be associated with single amino or double acid changes in the ENF binding site in HR1 (37
). Importantly, some of these changes have also been observed in patients who have experienced viral rebound while on ENF therapy (17
), either alone or in combination with other antiretroviral drugs, and it is these mutations that we examined in this study. While changes in HR1 can directly affect ENF binding (10
), they are also likely to impact binding of the viral HR2 region. If so, reduced affinity between HR1 and HR2 might be expected to negatively impact membrane fusion activity. Two of the HR1 changes that we examined in this study have recently been shown to reduce virus fitness in direct competition with wt virus when grown on cell lines in vitro (25
). In this study, we find that changes in HR1, without compensatory mutations in HR2, make fusion less efficient and delay fusion kinetics, perhaps accounting for their negative impact on virus fitness in vitro.
A second general mechanism by which sensitivity to ENF can be altered is through changes in membrane fusion kinetics. Since ENF targets a structural intermediate of the membrane fusion process, altering the time during which the ENF binding site is exposed would logically be expected to modulate virus sensitivity to this entry inhibitor. All other things being equal, we have found that alterations in fusion kinetics can have a significant impact on ENF sensitivity (34
). Fusion kinetics, in turn, can be increased by higher coreceptor expression levels and by higher-affinity interactions between Env and coreceptor (34
). Both of these serve to accelerate what appears to be the rate-limiting step in HIV entry—coreceptor binding—and minimize the period of time during which the ENF-binding site in HR1 is exposed. Our work here identifies changes in the HR1-HR2 binding site as being yet another factor that can affect the rate of HIV Env-induced membrane fusion to a significant degree.
An important finding in our study is that resistance to ENF resulting from changes in HR1 has little or no effect on virus sensitivity to a range of other entry inhibitors, including the fusion inhibitor T-1249, a peptide more potent than ENF that binds to a site on HR1 that overlaps that of ENF and which can block ENF-resistant viruses in vitro and in vivo (15
). The fact that Env proteins containing ENF resistance mutations remain sensitive to different classes of entry inhibitors is encouraging, since there is a good theoretical basis for employing entry inhibitors in combination (34
). For example, coreceptor inhibitors reduce the rate of membrane fusion by reducing coreceptor availability, which in turn results in prolonged exposure of the ENF-binding site. The interplay between receptor binding and the exposure (from CD4 binding) and ultimate loss (after coreceptor binding) of the ENF binding site likely accounts for the ability of entry inhibitors to synergistically inhibit HIV infection in vitro (45
). Since we have found that resistance to ENF need not impact sensitivity to other entry inhibitors, the rationale for combination entry inhibitor therapy is strengthened.
Our results also help explain why mutations in HR1 that increase ENF resistance may be selected against, in vitro and in vivo, in the absence of ENF. The HR1 mutations examined here decreased membrane fusion activity as well as viral infectivity by various amounts depending on the mutation and whether cell lines or human PBMCs were used. Further, the HR1 mutations delayed membrane fusion kinetics by an appreciable amount. It is not clear if the slower fusion kinetics resulting from the HR1 mutations were the cause or the result of reduced membrane fusion activity. In the context of virus, slower membrane fusion kinetics could increase the probability that virus will be internalized and delivered to lysosomes prior to membrane fusion, resulting in less efficient virus infection (41
). Alternatively, the change in free energy associated with the formation of the six-helix bundle is thought to provide the motive force needed to elicit membrane fusion (26
), and a reduction in the strength of HR1-HR2 interactions could increase the likelihood that receptor-induced conformational changes fail to elicit membrane fusion. Given the effects of HR1 mutations on virus-membrane fusion and virus fitness (25
), it will be important to determine if prolonged ENF therapy results not just in drug resistance but also in the selection of compensatory mutations that restore virus fitness. Interestingly, a virus dependent upon ENF for entry has recently been described (3
Another, significant consequence of the delayed fusion kinetics resulting from HR1 mutations was that HIV became more sensitive to neutralization by some broadly cross-reactive MAbs as well as antibodies in the sera of some HIV-infected individuals. The Env protein of HIV is well adapted to function in the face of a robust and ever-changing humoral immune response (7
). Conserved neutralizing epitopes may be inaccessible, sterically constrained, or shielded by carbohydrate and variable regions of the gp120 subunit (7
). We found that the HR1 mutations enhanced sensitivity of virus to 2F5 and 4E10, two broadly cross-reactive neutralizing antibodies that bind to the base of the gp41 ectodomain, without obviously affecting their binding to native Env trimers. Thus, we favor the idea that the epitopes to which these antibodies bind either are more exposed as a consequence of receptor binding or become more accessible as a result of receptor-induced conformational changes (4
). By lowering the rate of these conformational changes, antibody binding may be enhanced. Thus, in addition to spatial constraints that limit antibody binding to the native Env trimer, there are likely to be kinetic constraints as well. This clearly does not apply to all antibodies, as the sensitivity of virus to neutralization by 2G12, a MAb that binds to a surface-accessible epitope on gp120, was unaffected by the HR1 mutations. In contrast, MAbs to the coreceptor binding site that normally neutralize HIV weakly or not at all, due to the inaccessibility of their epitopes in the native trimer, exhibited enhanced neutralizing activity when certain mutations that reduced infectivity and delayed fusion kinetics were introduced into HR1. This was especially apparent with the G36D mutation, which fused at lower CD4 concentrations but exhibited the slowest fusion kinetics and may thus expose CD4i
epitopes for the longest time. Thus, Env proteins that remain in a CD4-triggered state for a longer period of time due to reduced levels of coreceptor, reduced Env affinity for coreceptor, or mutations in the HR1-HR2 binding region that slow fusion kinetics may present epitopes that are present on structural intermediates of the fusion process or that are sterically constrained in the native trimer for a longer period of time, enhancing antibody binding and virus neutralization as a result.
T-1249, like ENF, targets a structural intermediate of the fusion process. Therefore, T-1249 might be expected to exhibit more potent inhibitory activity against mutant viruses that fuse more slowly, although fusion kinetics has less of an impact on T-1249 than on ENF sensitivity, which may be due to the enhanced potency of T-1249 (35
). On the other hand, ENF-resistant mutations in the HR1 binding site might be expected to confer some degree of cross-resistance to T-1249. Therefore, it is possible that the minimal impact of ENF resistance mutations on T-1249 sensitivity might reflect an interplay between increased exposure of the T-1249 binding site and reduced T-1249 binding efficiency due to some cross-resistance to ENF resistance mutations.
Resistance to antiretroviral drugs is typically associated with a reduction in virus fitness, though compensatory mutations may restore virus fitness to wt levels (27
). Our work shows that resistance to ENF, and perhaps to other classes of entry inhibitors, may affect virus fitness in a way that is not readily apparent by standard in vitro replication assays. By prolonging the kinetic window during which structural intermediates of the membrane fusion process exist, novel epitopes or epitopes that are not readily accessible in the native Env trimer may become better exposed. Can this be exploited during antiretroviral therapy so as to limit the emergence of drug-resistant viruses? Some of the viruses studied here were more sensitive to neutralization by antibodies present in the sera of some but not all HIV-infected individuals tested. It is not known how commonly neutralizing antibodies such as 2F5, 4E10, and 17b are elicited in HIV-infected individuals. However, if immunization strategies can be identified that elicit such antibodies, then a mutually beneficial relationship between the humoral immune response and entry inhibitor therapy may arise. By slowing virus entry either by reducing available receptor levels or by selecting for mutations that prolong the entry process, entry inhibitors may make virus more sensitive to neutralization and may even increase the possibility that neutralizing antibodies may be elicited. The humoral immune response, in turn, may provide additional selective pressure against the emergence of virus strains that are resistant to ENF and perhaps other types of entry inhibitors as well. However, HIV can mutate to escape neutralizing antibody pressure as well as antiretroviral therapy, but escape may come at a cost to viral fitness.