Cryoelectron Tomography of HIV-1 Envelope Spikes: Further Evidence for Tripod-Like Legs

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Abstract
A detailed understanding of the morphology of the HIV-1 envelope (Env) spike is key to understanding viral pathogenesis and for informed vaccine design. We have previously presented a cryoelectron microscopic tomogram (cryoET) of the Env spikes on SIV virions. Several structural features were noted in the gp120 head and gp41 stalk regions. Perhaps most notable was the presence of three splayed legs projecting obliquely from the base of the spike head toward the viral membrane. Subsequently, a second 3D image of SIV spikes, also obtained by cryoET, was published by another group which featured a compact vertical stalk. We now report the cryoET analysis of HIV-1 virion-associated Env spikes using enhanced analytical cryoET procedures. More than 2,000 Env spike volumes were initially selected, aligned, and sorted into structural classes using algorithms that compensate for the “missing wedge” and do not impose any symmetry. The results show varying morphologies between structural classes: some classes showed trimers in the head domains; nearly all showed two or three legs, though unambiguous three-fold symmetry was not observed either in the heads or the legs. Subsequently, clearer evidence of trimeric head domains and three splayed legs emerged when head and leg volumes were independently aligned and classified. These data show that HIV-1, like SIV, also displays the tripod-like leg configuration, and, unexpectedly, shows considerable gp41 leg flexibility/heteromorphology. The tripod-like model for gp41 is consistent with, and helps explain, many of the unique biophysical and immunological features of this region. The envelope (Env) spikes on the surface of HIV-1 and SIV virions facilitate target cell tropism, binding, and entry, and serve as the sole targets of humoral (antibody-mediated) immunity. X-ray crystallography has previously revealed the atomic structures of key core domains and peptides of the gp120 and gp41 Env spike subunits, but the manner by which these components are arranged in the Env spike is still speculative. Cryoelectron tomography (cryoET) affords a view of the entire Env spike in the context of the intact virion. We have previously published a cryoET model of the SIV Env spike which showed a unique tripod-like leg configuration for the solvent-exposed (external) gp41 stalk region. This model is consistent with, and helps explain, many of the unique biophysical and immunological features of this region. Subsequently another group using similar technology and virions reported a spike model displaying a compact gp41 stalk inconsistent with our splayed-leg spike model. In this report, we apply enhanced analytical cryoET procedures to show that HIV-1 also displays the tripod-like leg configuration, and shows considerable gp41 leg flexibility/heteromorphology. These results have implications for the design of effective vaccines targeting this region and may provide new insights into Env spike function.

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