Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET

Abstract

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic 1 – 8 . Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3) 8 – 10 . It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers) 5 , 11 – 18 . Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1 JR-FL strain in complex with the antibody PGT151 19 . Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pretriggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies—and thus of interest for the design of immunogens—remains unknown.

Cryo-EM structure of full-length HIV-1 Env bound with the Fab of antibody PG16

“…We infer that the antigenic differences at the trimer apex between SOSIP forms of soluble, recombinant gp140s and full-length, processed gp160 are due primarily to the extent to which they visit somewhat more open conformations, rather than to a difference in the most stable, "ground state" conformation. This conclusion acquires importance in view of on-going debate about whether the SOSIP structures indeed represent the conformation of native Env on infectious virions [21,22].…”

“…Introduction of the SOSIP modifications into BG505 Env on virions also appears to broaden the distribution of occupancies, with a shift toward higher FRET states [21], but those measurements do not by themselves distinguish particular regions of the molecule.…”

“…Moreover, the acceptor fluorophore is attached through a coenzyme A "handle" with a potential contour length of over 20 Å. The smFRET data have been analyzed into three "states", with low (state 1), intermediate (state 3) and high (state 2) FRET signals, respectively [6,21,39]. Munro et al [6] reported that when Env on virion was bound with PG16 (and certain other broadly neutralizing antibodies), the low-FRET, state 1 predominated, but the same group reported recently that soluble SOSIP.664 instead gave a much higher FRET signal than did Env on virions and suggested that the SOSIP.664 structures determined by cryo-EM and x-ray crystallography do not represent the predominant conformation on the virion [21].…”

“…Envs that yield stable, homogeneous, trimeric, recombinant ectodomain (gp140) fall into the latter group [7,17,18]. Studies using fluorescence resonance energy transfer (FRET) or DEER spectroscopy have probed the ranges of relevant conformational ensembles [6,21,22].…”

HIV-1 Envelope and MPER antibody structures in lipid assemblies

“…Low expression levels and poor long-term stability of native Env together with structural flexibility and shedding of gp120 subunit (Hammonds et al, 2003), have led structural biologists to stabilize the trimer with mutations and/or antibodies so as to achieve higher resolution details. Structural intermediates have been captured after receptor binding and in complex with antibodies, which illustrate the intricately coordinated structural transitions of Env (Lu et al, 2019;Ozorowski et al, 2017;Tran et al, 2012). This flexibility is compounded when parts below the ectodomain are included, so structures of MPER, TMD and CTD have only been resolved in isolation using NMR (Chiliveri et al, 2018;Dev et al, 2016;Kwon et al, 2018a;Sun et al, 2008).…”