In this study we have demonstrated by several different techniques that polyanions interact with the V3 loop and the newly characterized conserved coreceptor binding surface on gp120 in a manner dependent on viral origin and coreceptor usage. Thus, the monomeric X4 and R5X4 gp120 molecules tested here bind polyanions strongly whereas R5 gp120 binds polyanions relatively weakly (gp120Bal) or not at all (gp120JRFL). Most of this variation comes from changes in the charge and structure of the V3 loop, but variability in the conserved coreceptor binding surface, which displays some interisolate differences in electrostatic potential (Fig. and results not shown), may also influence polyanion binding. The data that we have obtained from kinetic and MAb inhibition studies of gp120-heparin binding strongly suggest a phenomenon based on an initial high-affinity association via the V3 loop followed by weaker binding via a second site, most probably the conserved coreceptor binding region. Although we do not have unequivocal evidence of a direct interaction between polyanions and the conserved coreceptor binding surface, the data presented here provide a strong case for this. Thus, (i) polyanions potently inhibit the attachment of MAbs specific for the conserved coreceptor binding surface and regions of the V3 loop but, with the exception of one V2 loop-specific MAb, have little or no effect on MAbs to other exposed regions of gp120; (ii) CD4i-specific and V3 loop-specific MAbs inhibit the heparin-gp120 interaction in an additive manner; (iii) heparin binds mutants of gp120 from which the NH2 and COOH termini and the variable loops V1, V2, and V3 have been deleted, and the CD4i MAb 48d interferes with the binding of heparin to this mutant; and (iv) weak binding of heparin to gp120HXBc2ΔV3, gp120JRFLΔV3, and gp120HXBc2Δ82ΔC5ΔV1V2V3 can be observed under the appropriate conditions.
The molecular model of the electrostatic potential on the coreceptor-binding face of gp120 shows that the basic nature of the combined V3 loop-conserved coreceptor binding surface is dominated by the V3 loop charge. Although the models presented here and in reference 45
are of low resolution, it is clear that relatively long-chain sulfated polysaccharides such as heparin would be capable of spanning the V3 loop and the conserved coreceptor binding surface, which exhibit considerable spatial overlap. Moreover, several molecules of gp120 could bind a single heparin chain. V3 loop charge and conformation (12
), in association with the variable loops V1 and V2 (11
), determine coreceptor usage and viral tropism in vitro. It seems likely, based on our current understanding of HIV-1 attachment to receptor-bearing cells, that polyanion binding to gp120 parallels, at least to some extent, the interaction between gp120 and its coreceptors. Binding of gp120 to the appropriate coreceptor is thought to take place initially via the V3 loop, an interaction that determines coreceptor-gp120 specificity (12
), followed by exposure and binding of the conserved coreceptor binding region (64
). The importance of electrostatic interactions in the gp120-coreceptor association is underscored by a series of studies that demonstrate a reduction in gp120-CCR5 binding and coreceptor function in virus infectivity and fusion assays following substitution of acidic or potentially sulfated amino acids in the amino terminus of CCR5 (21
). However, interactions other than those mediated by the chemokine receptor NH2
terminus are also required for virus fusion to be fully activated, such as those based around the second extracellular loops of CCR5 (20
) and CXCR4 (5
There is no direct evidence for HIV-1 adaptation to HSPG use in vivo; the evolution of the V3 loop toward a more basic structure appears to come from adaptation to CXCR4 usage (70
). However, adaptation of HIV-1 to replicate in T-cell lines in vitro may be driven, at least in part, by increased interaction between the V3 loop and cell surface HSPG, since human T-lymphotropic leukemia virus type 1-immortalized T-cell lines such as H9, MT-2, and c8166 that have been used routinely to isolate, propogate, and phenotype viruses from infected individuals express large amounts of surface HSPG (57
). Indeed, this may help to explain why viruses passaged only in primary cells such as activated CD4+
T cells and macrophages, which express only small amounts of HSPG (40
), do not have V3 loops with net positive charges greater than +5 or +6 whereas viruses passaged in immortalized cell lines that express high levels of HSPG frequently have very basic V3 loops with net positive charges of +8 and +9 (28
). In the present study, analysis of X4 gp120 was carried out with TCLA virus-derived clones. Clearly, further studies of PI X4 virus-derived sgp120 will have to be done as this becomes available. However, the finding that the R5X4 PI gp120 89.6 binds heparin implies that PI X4 virus sgp120 will have a similar phenotype.
Although HIV-1 may use HSPG as a low-affinity attachment receptor, allowing the virus to scan the cell surface for specific entry receptors (34
), the biological implications of gp120 binding to HSPG are unclear, since the principal target cells for HIV-1 in vivo, CD4+
T cells and macrophages, have little HSPG on their surfaces. Nevertheless, even low levels of HSPG influence the efficiency of viral attachment and therefore entry and so may have important consequences for infectivity and cellular tropism in vivo. A selective ability of X4 and R5X4 viruses to attach to these cells via HSPG may give these viruses an advantage over R5 viruses that are potentially less able to do so. Other cell types that carry large amounts of HSPG are endothelial and epithelial cells (50
), and certain tissues, such as the liver, are very rich in HSPG expression (66
). These cell and tissue types are considered to be nonpermissive for HIV infection, but one potential consequence of HSPG expression may be the preferential trapping of X4 and R5X4 viruses onto their surfaces, leading to selective exhaustion of these viral phenotypes in vivo (83
). Such a phenomenon may help to explain the preferential transmission and early dominance of R5 viruses over R5X4 and X4 viruses in vivo.
Our observation that polyanions bind recombinant, monomeric sgp120 was reproduced with oligomeric, gp41-associated forms of X4 gp120 expressed on HIV-1-infected cells, suggesting that the monomeric form may represent to some extent the functional heterotrimeric form of gp120. However, it is very likely that the trimeric form will have a greater avidity for membrane HSPG complexes. Experimental evidence supporting this comes from a study by Roderiquez et al. (65
), who demonstrated that whereas no binding of monomeric TCLA X4 gp120 to cell surface HSPG was detectable, oligomeric TCLA X4 gp140 derived from the same isolate bound specifically. Thus, the 220 nM affinity constant observed for monomeric sgp120HXBc2
binding to immobilized heparin may be of high avidity when multiple interactions of this type take place on the surface of a virion. This idea is consistent with the predicted occupation of four or five molecules of gp120 by one heparin chain under our experimental conditions. Moreover, although we observed only a weak association between soluble or immobilized polyanions and monomeric R5 sgp120, the interaction with oligomeric gp120, either soluble or virion associated, could be stronger, as predicted by the electrostatic properties of the gp120 trimer. Future studies will need to address the kinetics of binding of oligomeric gp140 or whole virus particles to immobilized heparin or membrane HSPG.
The use of polyanionic compounds such as DexS as anti-HIV therapeutic agents systemically has not had obvious success in clinical trials, since there has been no obvious benefit to the patients (1
), adsorption has been considered poor (47
), and some toxicity has been observed (26
). However, other studies are more optimistic with regard to the use of polyanionic compounds as anti-HIV and antiviral therapeutic agents either systemically (30
) or locally (74
). One reason for the failure of these molecules in vivo may be their weak neutralization activity for R5 viruses (Fig. ), the viral phenotype associated with HIV-1 transmission and early infection. Another is that HIV variants resistant to DexS inhibition are rapidly generated in vitro, suggesting that similar events would occur in vivo (22
). However, the finding that the basic and highly conserved coreceptor binding region interacts with polyanions and the recent availability of the gp120 core crystal structure may allow for novel strategies in the rational development of small-molecule inhibitors of X4 and perhaps R5 HIV-1 infection based on electrostatic interactions with gp120.