The observations that coreceptor usage and CD4 affinity can be modulated by combinations of V3 charge, variant V1V2 regions, and V3 N-linked glycosylation led us to postulate that these alterations may also modulate the ability of HIV-1 to be neutralized by antibodies. In order to address this issue, we studied the profiles of neutralization of selected molecularly cloned viruses by well-characterized, envelope-directed, monoclonal antibodies (MAbs). We used three MAbs with defined epitopes (2F5, b12, and 2G12). Other MAbs were tested (15e, 17b, and 48d), but none was shown to possess neutralizing activity (data not shown). Since the +5 V3 panel of viruses (X, X.10, X.10ΔgV1, and X.10ΔgV3) covered the virus phenotypes we were interested in studying, we tested these viruses in neutralization assays with CD4+ lymphocytes isolated from an individual homozygous for the wild-type CCR5 allele, CCR5+/+, with the results shown in Fig. .
FIG. 4. Virus neutralization by MAbs 2F5, b12, and 2G12. The +5 V3 array of viruses (symbols: closed circles, X; open circles, X.10; open squares, X.10ΔgV1; closed squares, X.10ΔgV3) were tested for neutralization by MAbs 2F5 (A), b12 (more ...)
Different patterns of neutralization were observed for the three different MAbs. MAb 2F5, directed against an epitope in the gp41 region (39
), demonstrated no specific inhibitory profile, with all of the viruses showing similar neutralization profiles (Fig. ). For MAb b12, with its discontinuous epitope in the gp120 structure containing residues from the CD4BS of gp120 (24
), we found the neutralization profile of the X and X.10ΔgV3 viruses to be similar, with the X.10 and X.10ΔgV1 viruses clustering together (Fig. ). This distinctive pattern was the opposite of that observed for inhibition by sCD4 (Fig. ), where the “intermediate” dualtropic viruses were more resistant to neutralization than the more extreme R5 and X4 viruses. For MAb 2G12, directed against specific carbohydrate determinants (41
), the same virus clustering pattern was observed, but with the reverse profile (Fig. ). The observation of a reverse neutralization profile indicates that the results obtained are not due to general differences in the replication profiles of the viruses on CD4+
lymphocytes. With both b12 and 2G12 neutralization, we have identified an association between the V1V2 region and loss of N-linked glycosylation within the V3 region. Addition of the late-stage V1V2 region alters neutralization, which cannot be explained by the addition of the N-linked glycosylation event in the V1 region, with the effect being reversed by the loss of N-linked glycosylation within the V3 region.
Changes in HIV-1 tropism have previously been shown to have a tight association with HIV-1 neutralization profiles that can have a major influence on HIV-1 pathogenesis (35
). A number of recent reports have demonstrated that alterations of gp120 can be linked to HIV-1 immune escape from neutralizing-antibody responses (22
). The term “glycan shield” has been coined to describe the phenomenon in which alterations in N-linked glycosylation shift responses from appropriate sites on the gp120 envelope (55
). The gp120 conformation is highly dependent on the type of glycosylation events present and their distribution on the surface of the molecule (27
). Tight cooperation between gp120 structural elements, e.g., between the V1V2 and V3 loops, have previously been shown to mediate different structural states and functions (7
). We demonstrate here that alterations of the V1V2 and V3 regions can differentially modulate the response of the virus to different MAbs with variant specificities. Previous reports have stated that alterations of glycosylation events in the V2 region can modulate recognition by both anti-V3 and anti-CD4BS antibodies (33
). The opening of the gp120 envelope structure to reveal the CD4BS would have obvious implications for pathogenesis, with these viruses being open for antibody recognition and control.
The binding site for MAb 2G12 is known to have a conformation- and carbohydrate-dependent epitope on the gp120 surface, and this is the first description of an alteration in the V3 N-linked glycosylation altering neutralization by this antibody, likely through disruption of the structure of the gp120 envelope (41
). Whether similar antibodies directed against carbohydrate sites are commonly induced in vivo has yet to be established. The results described here indicate that the V3 N-linked glycosylation site can somewhat protect the 2G12 epitope from recognition and that there may be selective pressure for its presence when the immune system is intact. Since we have shown that loss of the V3 N-linked glycosylation event can alter relative coreceptor usage and favor stronger CXCR4 utilization, its removal may be preferentially selected when the immune system is weakened later in the disease course. This same effect may also help explain why the V3 N-linked glycosylation site is preserved in individuals with intact immune responses and why these viruses remain relative CCR5 users. It is of interest that the V1V2 and V3 changes we examined did not overlap any of the binding sites of the MAbs studied. A likely explanation is therefore conformational changes within the MAb binding sites (including CD4BS) induced by the V1V2 and V3 changes, similar to what has been previously shown (5
). Another explanation is that modified V1V2 and V3 can interact with residues that are close to these binding sites and that modify antibody recognition.
These results could indicate a short-term transitional state during the coreceptor shift process that renders the envelope open to neutralization by antibodies targeting the CD4BS. Xiang et al. have shown that an amino acid alteration within the gp120 molecule, S375W, can affect virus neutralization in a manner similar to what was observed for the X.10ΔgV1 viruses (56
). Moreover, the S375W mutants demonstrated a decreased affinity for the CCR5 receptor (56
). The replacement of serine 375 is a rare event among HIV-1 strains, while at the same time, S375H replacement is widespread among AE native recombinants, which possess a high rate of the syncytium-inducting phenotype, as well as the ΔgV3 N-linked glycosylation genotype. The similarity, in terms of phenotypic properties, of subtype D viruses also correlates with a high frequency of the ΔgV3 N-linked glycosylation mutation (HIV Sequence Compendium 2001, available at the hiv-web.lanl.gov website) (13
). These observations indicate that deglycosylation of the V3 region is a functional mechanism correlating with the X4 phenotype. These results should be considered with those indicating that the pattern of tropism is highly related to both neutralization susceptibility and CD4 binding (20
). These results suggest that a shift in HIV-1 coreceptor usage can result from compensatory alterations within gp120 selected through pressures such as CC/CXC chemokine and neutralizing-antibody responses.
In summary, the gp120 modifications that occur during disease progression and that coincide with the R5-to-X4 switch have consequences for in vivo viral fitness. This fitness will depend on an array of host responses, both innate and adaptive, that coincide with alterations in coreceptor usage and envelope structure.