The results presented in this study show that a single-amino-acid mutation, D179N, in the V2 domain of gp120 can convert a highly neutralization-resistant virus to a neutralization-sensitive virus. The fact that the D179N mutation increased sensitivity to neutralization by MAbs and antiviral drugs, targeting both gp120 and gp41, suggests that the D179N mutation induces a conformation change that affects accessibility of multiple neutralizing epitopes, rather than affecting the contact residues of a single neutralizing antibody binding site. These results suggest a far greater level of interaction between these two subunits, with respect to neutralization sensitivity, than was previously appreciated. The fact that D179 is conserved in HIV-1, SIV, and HIV-2 suggests that D at position 179 may have been preserved throughout evolution in order to preserve resistance to neutralization by antibodies targeting epitopes in both gp120 and gp41.
Our results are consistent with previous studies that have identified the V2 domain of gp120 as the “global regulator of neutralization sensitivity” (51
). Because the V2 domain can be deleted entirely in some viruses while preserving virus viability (10
), it seems unlikely that the V2 domain provides a contact surface required for infectivity or virus fusion. Rather, it appears to provide an epitope-“masking” function that is thought to conceal important neutralizing epitopes from neutralizing antibodies until the envelope protein undergoes a conformational change triggered by CD4 binding (35
). This hypothesis is supported by studies showing increased binding of antibodies to neutralizing epitopes in the V3 and C4 domains by envelope proteins lacking the V2 domain (10
). In this regard, the single-amino-acid substitution of N for D at position 179 appears to confer the same phenotype as that observed when the entire V2 domain is deleted from the SF162 virus (5
). Further data supporting the role of the V2 domain in regulating neutralization sensitivity is provided by studies showing that sensitivity and resistance to neutralization can be transferred by moving the V2 domain from a neutralization-sensitive virus (e.g., SF162) onto a neutralization-resistant virus (e.g., JR-FL) backbone. Conversely, the neutralization-sensitive SF162 virus can be converted to a neutralization-resistant virus by exchange of the V2 domain with that of JR-FL (52
Although conformational masking by the V2 domain appears to explain most of the data relating to the ability of the V2 domain to modulate neutralization sensitivity and resistance (10
), the molecular interactions determining how the mask is “raised and lowered” have not been characterized. Our results suggest that D179 mediates a key interaction required for maintenance of the neutralization-resistant, “masked” state. Replacement of D with N at position 179 seems to open up the structure of the gp160 trimer and makes the virus more sensitive to neutralization by exposing epitopes in both gp120 and gp41. Aspartic acid at position 179 appears to be unique, since it appears in all but two of more than 5,918 virus sequences in the 3 datasets examined and since all of the other mutations created in vitro
at this position resulted in either noninfectious viruses or viruses with increased neutralization sensitivity. The lack of representation of viruses with mutations at position 179 in other data sets might reflect the fact that all other variants are noninfectious or are so sensitive to neutralization that they are rapidly eliminated from circulation once envelope-specific antibody responses have developed. The fact that transfer of the D179N mutation to five unrelated viruses (YU2, JRCSF, QH0692.42, TRO-11, and 108048) all resulted in noninfectious viruses is consistent with the importance of D179 in preserving the functional structure of the envelope protein and suggests that compensatory mutations are required in other parts of the molecule to preserve infectivity when D179 is replaced with N. In this regard, the need for compensatory mutations may be similar to that observed with V2 domain deletions where deletion of the V2 domain in the SF162 strain results in infectious viruses, whereas deletion of the V2 domain in other strains (e.g., HXB2) requires compensatory mutations to maintain virus infectivity (57
). This possibility is supported by the V1/V2 domain replacement experiment (Table ), where it was found that replacement of the entire V1/V2 domain could increase sensitivity to neutralization by HIV-1-positive sera, while preserving infectivity. With respect to mutations at position 179, the amino acid substitutions that destroyed infectivity may have stabilized the masking function to such an extent as to prevent the conformational changes required for infectivity following receptor binding.
Our data are also consistent with the hypothesis that the V2 masking function is dependent on quaternary interactions between the gp160 subunits that associate to form the trimeric envelope structure that mediates virus infectivity and fusion (13
). Based on structural studies involving cryoelectron tomography and X-ray data fitting, the V1 and V2 domains appear to be located at the apex of an intermolecular contact region within the envelope glycoprotein trimer (41
). According to this model, the native trimer is held together by strong contacts at the gp41 base and the V1/V2 regions, with little or no contact elsewhere. Upon CD4 binding, the monomers rotate with respect to the core of the trimer to “open” the center of the trimer, exposing CCR5 binding sites, shifting gp41 up toward the cell membrane to form the six-helix bundle, and exposing the fusion peptide at the target cell membrane (see Fig. S2 in the supplemental material). When viewed in the context of these observations, our data are consistent with the possibility that D179 provides interactions required to maintain the unligated trimeric structure. Accordingly, mutations at position 179 may weaken the quaternary, intersubunit interactions, thereby providing increased access of antibodies to parts of the molecule, such as the V3 domain, the CD4 binding site, and the MPER, that are normally located in the interior of the molecule and exposed only after CD4 binding. Further investigations using conformation-dependent antibodies to the V2 domain, such as the newly described PG9 and PG16 antibodies (68
), might provide additional support for this model; studies using these antibodies as well as cryoelectron tomography are planned to further investigate this mutation.
The results reported herein confirm and extend our previous studies, in which swarm analysis has proved useful in identifying single-amino-acid substitutions that appear to trigger conformational changes that expose or conceal epitopes recognized by bNAbs. Envelopes with exposed neutralizing epitopes may represent a source of immunogens potentially more effective in eliciting bNAbs than those previously tested. Envelope proteins with deleted V2 domains have been tested as candidate HIV-1 vaccine antigens and were shown to elicit higher titers of neutralizing antibodies than wild-type proteins (5
). Studies are in progress to determine whether immunization with the D179N mutant described in these studies exhibits broader neutralizing activity, as seen with the V2-deleted envelope antigens.