Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers

Abstract

The mature envelope glycoprotein (Env) spike on the surfaces of human immunodeficiency virus type 1 (HIV-1)-infected cells and virions is derived from proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1 Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ∼10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, cross-linked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared by negative-stain electron microscopy to sample multiple conformations. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from those of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding-site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies. The entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shapes of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against the virus. Here, we evaluate the effects of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes but expose some Env elements differently than the natural Env.

Conformational Differences between Functional Human Immunodeficiency Virus Envelope Glycoprotein Trimers and Stabilized Soluble Trimers

“…The similarity of these high-resolution Env trimer structures has led to the widely held assumption that these structures represent the pretriggered state 1 conformation. However, differences in the antigenicity, glycosylation, and global architecture of sgp140 SOSIP.664 trimers and functional membrane Envs have been observed (39)(40)(41)(42)(43). Moreover, mutagenesis studies have tested the predictions of the sgp140 SOSIP.664 and EnvΔCT structural models with respect to trimer subunit interactions and failed to obtain the expected Env phenotypes (44)(45)(46).…”

Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function

“…Despite its relatively poor processing, we found that the EnvΔ712 DS mutant could mediate a moderate level of cell-cell fusion in a reducing environment, and this allowed us to investigate the conformation of the functional EnvΔ712 DS glycoprotein. Although these studies were performed with the cytoplasmic tail-deleted EnvΔ712, the ectodomain conformations of the EnvΔ712 and full-length Env from many HIV-1 strains, including HIV-1 JR-FL , are similar (42,101,132). Consistent with this similarity, the unmodified full-length Env and cytoplasmic tail-deleted EnvΔ712 exhibited nearly identical antigenic profiles ( Fig.…”

“…The metastability of Env creates immense challenges for structural studies and for the creation of vaccine immunogens. The pretriggered state 1 Env readily transitions into lower-energy conformational states upon removal from its native membrane environment (39)(40)(41)(42). Soluble Env trimers lacking the transmembrane region and cytoplasmic tail are labile and easily dissociate into monomers and dimers (39,(43)(44)(45)(46).…”

“…Such evidence has spurred consideration of membrane Envs from cells or viruslike particles (VLPs) as immunogens that are potentially more representative of the functional, state 1 Env. Proteolytically mature Envs better resist the binding of weakly neutralizing antibodies (42,(78)(79)(80)(81)(82)(83) and therefore may have advantages as immunogens; however, the noncovalent nature of the gp120-gp41 association can create lability in Env trimer preparations. Moreover, binding to CD4 in humans or monkeys can drive Env immunogens into state 2 or state 3 conformations (14,(84)(85)(86), potentially diverting antibody responses away from state 1 Env.…”

“…The antigenicity of HIV-1 JR-FL Env, EnvΔ712, and the corresponding SOS and DS mutants expressed on the surface of HOS cells was examined. The HIV-1 Env precursor is processed efficiently in HOS cells (78), allowing a better correlation between antibody binding to the cell surface Env and virus neutralization (42,89,101). We used a cell-based enzyme-linked immunosorbent assay (ELISA) to measure the recognition of cell surface Envs by a panel of broadly neutralizing antibodies as well as poorly neutralizing antibodies, in the absence and presence of 1 mM DTT.…”