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

Ping Zhu,Hanspeter Winkler,Elena Chertova,Kenneth A Taylor,Kenneth H Roux,Michael Farzan

doi:10.1371/journal.ppat.1000203

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.

Highlights

HIV-1 and the closely related SIV envelope (Env) spikes are composed of a trimer of heterodimers [1,2,3,4,5,6]
The base of the Env spike is comprised of three gp41 subunits, each of which possesses, from N-terminal to C-terminal, a fusion peptide, N-terminal heptad repeat, disulfide loop, C-terminal heptad repeat, membrane proximal external region (MPER), transmembrane domain, and cytoplasmic tail (CT)
Electron microscopy (EM) is an important adjunct to atomic structural studies and has the potential to allow the placement of the atomic structures of gp120 and gp41 core fragments and peptides, as well as the unresolved flexible components, into the global structural context of the Env spikes in situ [12]

Summary

Introduction

HIV-1 and the closely related SIV envelope (Env) spikes are composed of a trimer of heterodimers [1,2,3,4,5,6]. The atomic structure of the unliganded SIV core has recently been described [10] For both atomic structures, some of the more flexible elements, including V loops, N and C-terminal peptides and much of the glycan shield, were either deleted from the crystallization construct or were not resolvable due to flexibility [8,9,10]. Electron microscopy (EM) is an important adjunct to atomic structural studies and has the potential to allow the placement of the atomic structures of gp120 and gp core fragments and peptides, as well as the unresolved flexible components, into the global structural context of the Env spikes in situ [12]. By negative stain electron tomography, clear evidence for 3-fold symmetry was observed for a mutant form of SIV exhibiting Env with a truncated cytoplasmic tail [6]. Because of the potential for morphological artifacts resulting from the use of the negative staining EM technique, including the attachment to a carbon substrate, pH changes, and drying, definitive analyses of the spike architecture could not be performed by this method [6]

Author Summary

Findings

Materials and Methods