These studies highlight differences observed in quaternary structure upon sCD4-binding Env on HIV-1 BaL and on three SIV strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). HIV-1 BaL Env displays large conformational changes upon sCD4 binding, from a closed, native state to an open, CD4-bound state (28
) (). Env from all three strains of SIV displayed minor conformational changes upon sCD4 binding as compared to their respective native conformations (58
), which in SIVmneE11S and SIVmac239 are observed as a slight opening at the apex of trimeric Env. Despite similar minor conformational changes in Env with sCD4 binding, these SIV strains have varied pathogenicities, antigenicities, and neutralization sensitivities (8
Fig. 7. Top and schematic views of trimeric SIV envelope glycoproteins in native and sCD4-bound states. (A) Top views of the density maps (shown as a transparent blue isosurface) with fitted gp120 coordinates for SIVmne, sCD4-bound SIVmne, SIV CP-MAC, and sCD4-bound (more ...)
Early biochemical studies of the stoichiometry of CD4 binding to SIV Env suggested multimeric binding of sCD4 (17
), while more-recent investigations have concluded only one sCD4 molecule binds per gp120 trimer (9
). It has also been reported that the stoichiometry of binding to SIV Env is different from the binding to HIV-1, where three sCD4 molecules bind each gp120 trimer (9
). Because the structures we report are based on averaging thousands of individual trimers, we cannot determine the absolute sCD4 occupancy or exclude the possibility that a subpopulation of the Env complexes have only one bound sCD4. Nevertheless, the orientation and geometry of binding of sCD4 derived in our structural analysis show that three sCD4 molecules can be bound without steric hindrance to a single SIV trimer in the quaternary conformations displayed by all three SIV strains analyzed.
The architecture of the sCD4-bound state of SIV provides a structural basis to rationalize the striking differences between the sensitivities of SIV and most HIV-1 viruses to neutralization by sCD4 (10
). The finding that relatively small quaternary structural changes are induced in trimeric gp120 upon sCD4 binding is consistent with previous reports that sCD4 binding causes local changes in the exposure of V2 and V3 loops but without measurable gp120 dissociation (52
). The fact that this sCD4-bound state is stable in SIV, potentially capable of interacting with coreceptor molecules on the target cell membrane, also explains why cell surface CD4 is no longer required for SIV entry in the presence of exogenously added sCD4 (53
) and that SIV can remain infectious at higher concentrations of sCD4, under conditions when most HIV-1 viruses are neutralized (10
). Reports that sCD4 binding results in the formation of a transient intermediate in HIV-1 strains whose decay correlates with an irreversible loss of infectivity (27
) and that sCD4 concentrations too low to result in gp120 dissociation can enhance viral infection (1
) are both fully consistent with the proposal that the sCD4 complex with SIV Env may resemble a similar structural intermediate that occurs transiently in HIV-1.
An unresolved question from our present work is whether further conformational changes occur in SIV Env before viral entry into target cells. It could be that a large quaternary conformational change such as that seen with HIV-1 occurs upon contact with the cell and that we have not yet identified conditions to capture this Env intermediate in our experiments. It could also be that the more subtle changes observed for SIVmac239 and SIVmacE11S are sufficient to expose coreceptor binding sites and that further changes may not be necessary for viral entry, since both viruses are capable of infecting human cell lines that express human CD4 and an appropriate coreceptor (19
). Yet another possibility is that the large conformational change does occur transiently but that the equilibrium is shifted to favor this state in HIV-1 BaL but not in certain SIV strains, except for strains such as SIV CP-MAC, where the conformation is already in a fully open state.
Another possible origin of the difference in the extents of conformational change observed with sCD4 binding to HIV-1 BaL, as compared to the minor changes upon sCD4 addition to the SIV strains investigated here, is our use of human sCD4 instead of simian sCD4 for the binding studies. However, since these strains are fully capable of infecting cells expressing human CD4 and human CCR5, this is unlikely to explain differences we observed for SIV and HIV-1 with respect to the consequences of sCD4 binding. Although human versus rhesus CD4 sequences are highly conserved (92%), there are amino acid changes in the rhesus CD4 near residues which have been shown to be critical in binding gp120 (35
). It remains to be determined whether rhesus macaque CD4 residue mutations contribute to an alternate SIV Env conformation upon binding that is different than that observed with human sCD4.
While the detailed mechanistic implications of the structural differences between HIV-1 and SIV strains will require thorough analysis of a wider spectrum of viruses ranging from neutralization-resistant primary isolates to laboratory-adapted neutralization-sensitive strains, the present studies establish that sCD4 can bind and stabilize distinct conformations of trimeric Env and that cryo-electron tomography can be used to identify strain-dependent variations in the quaternary structures of unliganded and liganded Env. Continuing advances in computational strategies to separate distinct Env conformations from heterogeneous spike populations will provide further possibilities for separating conformational variants and improved prospects for understanding the structural diversity of trimeric Env as displayed on intact viruses.