HIV-1 Env exhibits a high degree of antigenic diversity and appears to have limited sites of vulnerability to neutralizing antibodies. However, the region of gp120 that contacts its primary receptor, CD4, is functionally conserved and is vulnerable to neutralizing antibodies. Some HIV-1-positive sera contain broadly reactive neutralizing antibodies directed to the CD4bs, and there is one well-characterized neutralizing monoclonal antibody, b12, that targets the CD4bs. Hence, the CD4bs has become a focus of vaccine design efforts to elicit neutralizing antibodies that would be effective against most circulating strains of HIV-1 (16
). In this regard, the atomic-level structure of the core of gp120 bound to monoclonal antibody b12 provides detailed architectural information that can form the basis of immunogens design efforts. One potential limitation to the success of these efforts is the natural occurrence of b12-resistant viruses. If the atomic-level structure of the b12 contact surface is to be used for vaccine design, we have to understand the structural basis of b12 resistance to prospectively design immunogens that have the capacity to generate neutralizing antibodies to both b12-sensitive and b12-resistant viruses. The analysis described here reveals specific antigenic and phenotypic alternations that can confer b12 resistance in a set of naturally occurring clade B and C viruses.
Prior to the elucidation of the atomic-level structure of b12 bound to the gp120 core and the knowledge of b12 contact sites, numerous studies described amino acid alterations that account for b12 resistance in some viral isolates. Sodroski and colleagues studied the emergence of b12 resistance for two strains of chimeric simian-human immunodeficiency virus, SHIV-HXBc2 and SHIV-89.6P, after passage in monkeys. They documented specific amino acid changes in regions V2 (SHIV-89.6P) or V1, V2, V3, and gp41 (SHIV-HXBc2) that were associated with b12 resistance. These mutations did not influence binding to monomeric gp120 but did affect binding to cell surface-expressed Env, suggesting that b12 resistance was the result of alterations in Env conformation that restricted the access of b12 to its epitope (13
). Pantophlet and colleagues performed a comprehensive alanine mutagenesis of the JR-CSF isolate and described numerous alterations that affected b12 binding to gp120 or the b12 neutralization of the resulting Env pseudoviruses. In some cases, these mutations resulted in decreased b12 binding to gp120 and were in positions that we now know are specific b12 contact sites. In other cases, mutations conferred b12 resistance without affecting b12-gp120 binding (37
). Similarly, Parren and colleagues and Moore and colleagues studied primary viral isolates and found that the lack of binding to the relevant gp120 could account for only some of the observed resistance (17
). Additional cell culture passage and mutagenesis studies have described amino acid alterations that can increase or decrease viral neutralization sensitivity to b12 (3
). In summary, these studies revealed that b12 resistance could result either from poor epitope recognition on gp120 or from steric constraints that limit antibody access to its cognate epitope.
Our study provides a novel analysis of the mechanism of b12 resistance among relevant circulating strains of HIV-1 clades B and C. Guided by the known HXBc2 gp120 contact sites for b12 and the precise Env sequence of each virus studied, we used sequence analysis, molecular modeling, and site-directed mutagenesis to analyze the structural basis of b12 resistance. To assess the total level of amino acid variation for b12 contact sites on gp120, we mapped HIV-1 sequence variations for all group M as well as clade B and clade C viruses onto the surface structure of gp120. The majority of b12 contacts were highly conserved, but there was substantial variation at several sites within the CD4-binding loop that forms the dominant binding surface for b12. Monoclonal antibody b12 interacts with all 10 continuous amino acids that make up the CD4-binding loop (positions 364 to 373), while CD4 interacts with only 6 core residues. Interestingly, the highest variation occurred among the four b12 contact residues that were not sites of CD4 contact. These were amino acid positions 364, 369, 372, and 373 (Fig. ). We next used the HXBc2 structural data to computationally model each site of amino acid variation observed among the b12-resistant viral strains. This analysis predicted that amino acid variants at three specific sites would result in clashes that interfere with b12 binding. Each of these variants (S364H, P369L/T/Q, and T373M) was within the CD4-binding loop, and each residue was identified by our sequence analysis to be subject to high levels of variation. In addition, computational modeling showed that each of these three b12 contact sites were just adjacent to the known CD4 contact surface. Hence, among the b12-resistant viruses, the contact sites predicted to interfere with b12 binding were concentrated within the CD4-binding loop just proximal to the CD4 contact surface and were sites of significant overall sequence variation. A somewhat similar mechanism of viral escape has been proposed for human rhinovirus 14, which is a nonenveloped picornavirus. The canyon hypothesis suggested that conserved residues within the floor of the viral capsid bind to the cellular receptor ICAM-1. The small dimensions of the canyon limit accessibility to antibodies, and hence neutralizing antibody contacts were at residues adjacent to the canyon that could vary and permit escape from neutralization (44
To further assess the impact of amino acid variants on b12 binding and neutralization activity, we performed site-directed mutagenesis on b12-resistant viruses to revert specific contact residues back to the wild-type (HXBc2) sequence. As predicted by computational modeling, alterations at residues 364, 369, and 373 could decrease b12 binding and render a virus neutralization resistant. As an example, the clade B viruses 6101.10 and TRO.11 became sensitive to b12 when amino acids at positions 369 and 373 were reverted to the wild-type (HXBc2) sequence. Similarly, the single residue 364H in viruses 7165.18 and ZM109F.PB4 accounted for both poor b12-gp120 binding and a lack of the neutralization of these viruses. Because the 364H sequence is relatively common in clade C, 364H may be an important mechanism contributing to b12 resistance in clade C viruses. It is interesting that some broadly neutralizing clade C sera contain neutralizing antibodies directed to the CD4bs (4
), and that these clade C sera can neutralize viruses with the 364H alteration. This suggests the existence of anti-CD4bs antibodies that have an epitope sequence different from that of b12.
While computational modeling did predict the importance of residues 364, 369, and 373 for b12 recognition, our modeling of potential b12 clashes was not always predictive of poor b12 binding and neutralization, particularly among clade C viruses. Although a proline at residue 369 was critical for high-affinity b12 binding and the neutralization of clade B viruses, a P369L alteration was present in all 18 clade C reference viruses regardless of b12 sensitivity. This suggests that computational modeling based on the clade B HXBc2 sequence is less predictive of the b12 interaction with the CD4bs of clade C viruses. Likewise, the T373M mutation among clade B viruses did contribute to neutralization resistance, but this single- amino-acid mutation also was present on several neutralization-sensitive viruses. It is important to note that our computational modeling makes some simplifying assumptions related to protein dynamics. To reduce the complexity of computation, we assumed a rigid backbone of the HXBc2 template and allowed only side-chain movements. In reality, certain degrees of flexibility, including backbone variation, likely are present on gp120, and the antigen-combining site of an antibody often adapts to changes on the contact surface of its ligand. This induced fit may allow b12 to tolerate some amino acid alterations, such as the single P369L or T373M alteration, with only small changes in binding affinity. Finally, the molecular modeling based on the atomic-level structure of the core of gp120 does not take into consideration the interaction of variable loops, the structure of gp41, or the interaction of the three gp120 surface unit molecules (quaternary factors) on the overall structural stability and conformation of the viral spike.
The prior data on the role of Env conformation in restricting b12 access (3
), and our own data demonstrating clear examples of b12 resistance despite a wild-type (HXBc2) sequence at known b12 contact sites and despite strong binding to monomeric gp120, prompted further investigation to address the potential impact of Env conformation on b12 neutralization. We therefore introduced several previously described mutations that alter the quaternary conformation of HIV-1 Env but lie outside the b12-binding surface of gp120. In one example, the clade B virus PVO.4 was fully resistant to b12 despite having a fully wild-type (HXBc2) sequence at all known b12 contact sites and despite a high relative binding capacity of b12 to the PVO.4 gp120 monomer. The removal of a single glycosylation site, position 301 at the base of the V3 loop (19
), improved b12 neutralization potency from an IC50
of >50 μg/ml to 2.0 μg/ml. Several similar examples of this effect for clade B and C viruses, including the effect of mutations in gp41 (6
), are shown in Table . In some cases, the conformational restriction to b12 access was quite dramatic. Virus CAAN5342.A2 was b12 resistant and remained resistant despite reversions to a wild-type (HXBc2) sequence that increased b12-gp120 binding by 200-fold (the EC50
decreased from 28 to 0.14 nM). However, the wild-type CAAN5342.A2 was rendered highly sensitive to b12 neutralization by one or two mutations in gp41 that had no effect on b12 binding. This suggests a strong interplay between the relative b12 affinity for its target surface on gp120 and the level of steric restriction by the native trimer. If the trimer is rendered highly flexible or open, even modest levels of binding affinity will result in a high level of virus neutralization. In almost every case, increased access by b12 was accompanied by an increase in the neutralization activity of sCD4. Without atomic-level structural data of the native trimer, we can only speculate on the interaction of the gp120 and gp41 molecules that make up each viral spike. One possibility is that the quaternary packing of the V2 and V3 regions of each protomer are affected by the removal of glycans at the base of the V2 or V3 loop. The result is a more flexible native configuration that no longer restricts access to the CD4-binding region of gp120 (Fig. ).
We can attempt to explain the mechanism of b12 resistance among the entire set of b12-resistant viruses studied. However, we caution that mutations altering the overall quaternary packing of the viral spike are likely to create Env conformations that are unusually flexible or open and not commonly found among naturally occurring viral isolates. With this caveat in mind, we reviewed the impact of sequence and conformational alterations on all 14 b12-resistant viruses. Of six resistant clade B viruses with mutations at b12 contact sites, reversion to the wild-type (HXBc2) sequence resulted in three viruses that became b12 sensitive (viruses 7165.18, 6101.10, and TRO.11). The gp120 of the remaining three viruses (BG1168.1, CAAN5342.A2, and TRJO4551.58) each demonstrated increased gp120-b12 binding but remained neutralization resistant. Each of these three viruses could be rendered neutralization sensitive by conformation-altering mutations. One clade B virus, PVO.4, contained wild-type (HXBc2) sequence at all b12 contact sites and became sensitive to b12 upon specific gp120 glycan removal distal from the actual contact surface. We also made b12 contact site mutations in five of seven resistant clade C viruses, but only one (ZM109F.PB4) became b12 sensitive. In addition, the reverted clade C viruses did not demonstrate an increase in b12-gp120 binding affinity. As noted previously, this suggests a limitation in modeling, based on the HXBc2 sequence, to predict b12 clashes for clade C viruses. As observed for clade B viruses, all but one of the resistant clade C viruses (CAP244.2.00.D3) could be rendered b12 neutralization sensitive by mutations that altered the Env conformation. Therefore, we can explain the precise structural basis for neutralization resistance for some viruses tested, particularly clade B viruses, and we can demonstrate the interaction of antigenic variation and conformational restriction on viral resistance to the anti-CD4bs monoclonal antibody b12.
In summary, HIV-1 is vulnerable to neutralizing antibodies that target the site of attachment to its primary receptor, CD4. Our data provide a novel mechanistic explanation for how resistance to the anti-CD4bs monoclonal antibody b12 is achieved among a relevant sampling of circulating strains of HIV-1. Using the known HXBc2 gp120 contact sites for b12 and the precise Env sequence of each virus studied, we were able to precisely define the structural context of key amino acid variants that diminish b12 binding and neutralization. These mutations were clustered within the CD4-binding loop of gp120, which forms the dominant binding surface to b12. Notably, amino acid variants conferring resistance resided just proximal to the known contacts of CD4, suggesting a preferential tolerance for variation surrounding the CD4 contact sites. In addition to alterations at b12 contact sites, resistance was influenced by the conformation of the native envelope trimer that restricts access to CD4-binding regions of gp120. To design vaccine immunogens that elicit anti-CD4bs antibodies similar to b12, researchers need to consider the antigenic variation within contact sites on gp120 and the restricted access to the CD4bs imposed by the native configuration of the trimeric viral Env spike.