HIV-1 and HIV-2/SIV share ancestry and many properties, including the ability to infect CD4+
T lymphocytes, monocytes/macrophages, and microglia. Low-resolution cryoelectron tomograms of HIV-1 and SIV virions indicate that the envelope glycoprotein trimers of these viruses exhibit a similar overall architecture (45
). A recent study has shown that the HIV-1 gp120 core has a propensity to assume the CD4-bound conformation spontaneously once the V1/V2 and V3 variable loops are removed (40
). Thus, in the unliganded envelope glycoprotein complex, gp120 must retain a stable association with the trimer but must also be restrained from sampling downstream conformations, such as the CD4-bound state. Some of the HIV-1 gp120 elements (N/C termini, inner domain β-sandwich and layer 2, and V3 [in primary strains]) that contribute to the stable association of gp120 with the envelope glycoprotein trimer have been characterized (21
). Here we have provided evidence that the SIVmac239 gp120 N/C termini and inner domain β-sandwich and layer 2 also contribute to gp120-trimer association. Thus, the global architecture of the envelope glycoprotein structures that govern gp120-trimer association is likely shared by HIV-1 and SIVmac239. Some differences exist in the trimer association phenotypes associated with changes in particular residues within these elements of HIV-1 and SIV gp120, suggesting that there may be subtle differences in the specific structural relationships between the envelope glycoproteins of these virus lineages.
Our study highlights two structural elements that differ between the HIV-1 and SIV gp120 exterior envelope glycoproteins, i.e., layer 1 of the inner domain and the Phe 43 cavity-lining residue 375. We investigate the function of these elements in the SIVmac239 envelope glycoproteins, and we show that these elements interdependently determine phenotypes related to CD4 binding. Relative to layer 1 of HIV-1 gp120, the SIVmac239 gp120 layer 1 plays a more prominent role in maintaining gp120-trimer association and is minimally involved in promoting CD4 binding. HIV-2/SIVsmm gp120 glycoproteins, like those of most monkey SIVs, typically have a tryptophan residue at position 375 (39
). In HIV-1, substitution of tryptophan for serine 375, which is well conserved in the major group of HIV-1, results in spontaneous sampling of a conformation closer to the CD4-bound state (76
). The S375W HIV-1 mutant binds CD4 more efficiently and exhibits increased sensitivity to sCD4-induced shedding of gp120 and neutralization (21
). These observations suggested the hypothesis that relative to HIV-1 gp120, the HIV-2/SIVsmm gp120 glycoproteins, by virtue of the Phe 43 cavity-filling Trp 375, might naturally exhibit a greater propensity to sample the CD4-bound conformation. We found that a W375S change in the SIVmac239 envelope glycoproteins decreased CD4 binding and eliminated the enhancing effect of sCD4 on infection of CD4+
target cells (3
). This sCD4-induced activation of SIVmac239 infection was also dependent on the integrity of gp120 layer 1. Moreover, some additional decrease in CD4 binding was associated with SIV gp120 layer 1 mutants in the context of the W375S, but not the wild-type, SIVmac239 envelope glycoproteins. The cooperative influence of both residue 375 and layer 1 in determining CD4 binding and its consequences is supported by the converse studies of the HIV-1 envelope glycoproteins. The H66A and W69L layer 1 changes decreased the CD4-binding affinity of HIV-1 gp120 but had no effect on CD4 binding of an S375W HIV-1 mutant (21
). Moreover, changes in these HIV-1 layer 1 residues, particularly in combination with the S375W change, allowed the formation of a longer-lived activated intermediate upon sCD4 binding, closer to the sCD4-induced phenotype observed for SIV (21
). Thus, the gp120 inner domain layer 1 and residue 375 exert reciprocal influences on the affinity of CD4 binding and the stability of the CD4-activated state.
These observations suggest that some differences between the SIVmac239 and HIV-1 gp120 subunits in the assembled envelope glycoprotein trimer exist, as summarized in . By virtue of the Phe 43 cavity-filling Trp 375, SIVmac239 gp120 has a strong propensity to assume the CD4-bound conformation. Since premature movement into the CD4-bound conformation can have negative consequences (gp120 shedding, exposure of neutralization epitopes, and rapidly decaying intermediate states), this propensity in SIVmac239 gp120 is compensated by the strong contribution of the inner domain layer 1 to the association of gp120 with the trimer. With Trp 375 filling the Phe 43 cavity, layer 1-layer 2 interactions make minimal contributions to CD4 binding. In contrast, in HIV-1 gp120, which lacks a Phe 43 cavity-filling residue at position 375, the propensity to assume the CD4-bound conformation is lower than that in SIVmac239 gp120. The association of HIV-1 gp120 with the unliganded trimer is adequately maintained by the N/C termini and the inner domain β-sandwich and layer 2. In HIV-1, therefore, the major function of layer 1 is to promote high-affinity CD4 binding by solidifying the interaction with layer 2 and decreasing the off rate of CD4.
Fig 9 Summary of functional differences between HIV-1 and SIVmac239 gp120 glycoproteins. One of the three gp120 subunits of the SIVmac239 and HIV-1 envelope glycoprotein trimer is depicted. In the orientation shown, the trimer axis runs vertically on the left (more ...)
The SIVmac239 envelope glycoproteins were chosen for this study because they exhibit a level of CD4 dependence comparable to that of the previously studied HIV-1YU2
envelope glycoproteins (5
). However, SIVmac239 is more CD4 dependent, more neutralization resistant, and less able to infect monkey macrophages than most other members of the SIVsmm clade. How generalizable are the results that we obtained with the SIVmac239 envelope glycoproteins? First, despite the similar levels of CD4 dependence of the SIVmac239 and HIV-1YU2
envelope glycoproteins, the phenotypes of the gp120 inner domain layer 1 mutants differed. Thus, layer 1 function in other members of the SIVsmm/HIV-2 clade, which are less dependent on CD4 than either SIVmac239 or HIV-1YU2
), is also expected to differ from that of HIV-1. Second, the SIVmac239 gp120 residues identified in this study as important for gp120-trimer association and CD4 binding are well conserved in all SIVsmm and HIV-2 strains. Therefore, all SIVsmm and HIV-2 gp120 envelope glycoproteins likely share common structural and functional inner domain elements. However, although the Phe 43 cavity-filling tryptophan 375 residue is found in the gp120 glycoproteins of HIV-2 and most monkey SIVs, differences in the inner domain layer 1 and layer 2 sequences occur among the distinct SIV lineages associated with different African monkey species. Additional studies will be required to determine the functional implications of these layer 1/layer 2 differences in SIV lineages other than the HIV-2/SIVsmm clade.
What was the sequence of events during PIV evolution that led to the observed changes in the gp120 inner domain and Phe 43 cavity? Based on our current understanding of PIV evolution (66
) and the patterns observed in , we can suggest a model. The ancestral pool of indigenous SIVs likely had short inner domain layer 1 structures and a Phe 43 cavity filled with a tryptophan residue. At some point in time and for reasons that are unknown, the SIVgsn/SIVmus/SIVmon gp120 glycoproteins evolved longer disulfide loops in layer 1 without changing the Phe 43 cavity-filling tryptophan residue. The longer layer 1 sequences in the gp120 glycoproteins of SIVgsn/SIVmus/SIVmon are not likely to result in an extension of the α0 helix, in contrast to the situation in HIV-1 gp120 (54
). This assertion is supported by the presence of Pro 65 and Gly 66 residues, both unfavorable for stable helix formation, in layer 1 of the SIVgsn/SIVmus/SIVmon gp120 inner domains. Nonetheless, some advantage resulted in fixation of the longer layer 1 in the gp120 glycoproteins of these SIVs. SIVgsn, SIVmus, and SIVmon infect three closely related Cercopithecus
species (C. nictitans
, C. cephus
,, and C. mona
, respectively), which are hunted by chimpanzees in central Africa. Chimpanzees are thought to have become infected with one of these SIVs, which, through a recombination event with a coinfecting SIVrcm, supplied the env
gene to the SIVcpz precursor of HIV-1 (4
). With the evolution of SIVcpz, His 66 makes its first appearance in the elongated layer 1, allowing stacking interactions to occur with the preexisting Pro 212 in layer 2. Thus, by the time SIVcpz variants became indigenous in African chimpanzees, the ability of layer 1-layer 2 interactions to bolster CD4 binding was established. This may have relaxed the evolutionary pressure to maintain a tryptophan 375 residue in the Phe 43 cavity, as evidenced by the preference in SIVcpzPtt, SIVgor, and HIV-1 strains for residues other than tryptophan at position 375. Subsequent adaptation in humans further influenced the identity of residue 375. The less successful group N and group O HIV-1 strains retain the large residues at position 375 found in their respective chimpanzee and gorilla SIV ancestors. The extremely successful group M HIV-1 evolved an empty Phe 43 cavity. Thus, a series of sequential structural changes in the gp120 inner domain and Phe 43 cavity apparently provided the circumstances conducive to the evolution of a group M HIV-1 envelope glycoprotein well suited for transmission and spread in humans.
The different arrangements of the Phe 43 cavity and inner domain of HIV-1 and SIVsmm gp120 glycoproteins helps to explain why SIVsmm envelope glycoproteins are generally less dependent on CD4 for CCR5 interaction, are able to infect cells that express low levels of CD4, and can maintain a long-lived sCD4-activated state (17
). What biological necessities shaped the divergent evolution of these HIV-1 and SIV gp120 elements? The very low level of CD4 expression on the surface of tissue macrophages of monkeys (5
) may provide selective pressure on the SIV envelope glycoproteins to achieve and maintain the CD4-bound conformation after engaging CD4 at low stoichiometry. Indeed, the nearly undetectable levels of CD4 expression on monkey macrophages have been shown to represent the major factor limiting their infection by T cell-line-tropic SIVsmm strains and, notably, by macrophage-tropic HV-1 strains (5
). The ability of the latter viruses to infect primary human macrophages suggests that human macrophages targeted for HIV-1 infection must have higher levels of surface CD4 available for envelope glycoprotein interaction than primary macrophages from monkeys. Freed of the necessity to maintain the ability to infect cells with extremely low levels of CD4 expression, HIV-1 may have taken advantage of the benefits of avoiding the CD4-bound conformation. Transmitted/founder HIV-1 typically retains the ability to infect T lymphocytes but not monocytes/macrophages (53
). Thus, HIV-1 tropism for cells with low CD4 levels, like monocytes/macrophages and microglia cells, may need to be acquired as the virus evolves in particular tissue compartments, such as the central nervous system (16
). An understanding of PIV adaptation to species-specific environments may assist efforts to intervene in virus transmission, spread within the infected host, and pathogenesis.