Influences on Trimerization and Aggregation of Soluble, Cleaved HIV-1 SOSIP Envelope Glycoprotein

Abstract

dWe describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies. One of the most substantial obstacles to the development of an effective vaccine to prevent infection by human immunodeficiency virus type 1 (HIV-1) is our collective inability to design immunogens that are able to induce broadly active neutralizing antibodies (bNAbs) at sufficient titers (1-4). However, NAbs do invariably target the envelope (Env) glycoprotein complexes that are present as spikes on the surface of virions, but often in a strainspecific manner. The Env spikes mediate virus-cell attachment and fusion, processes that are prevented by NAb occupancy. Hence one rational vaccine design strategy for bNAb induction involves the use of recombinant versions of Env spikes as immunogens.The Env spike is a trimer, each subunit containing a gp120 surface glycoprotein linked noncovalently to a gp41 transmembrane glycoprotein. The three gp120/gp41 protomers are also noncovalently associated, predominantly via their gp41 components but with additional contributions from gp120-gp120 interactions near the spike apex (5-8). The native Env spike must undergo a complex series of conformational changes, triggered by receptor interactions, to fulfill its fusion functions. Accordingly, the intersubunit bonds are fairly weak, and the spikes can spontaneously decay or otherwise lose function over time (9-11). Recombinant Env proteins are often expressed in soluble form, as gp120 or as gp140, which lacks the transmembrane and cytoplasmic domains of gp41 (12-18). However, trimers of gp140 are labile: unless stabilizing mutati...

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

“…S1). This density is far larger than can be accounted for by the additional 17 amino acids, but resembles a micelle that associated with similar KNH1144.681 trimers when a small amount of detergent was added to prevent aggregate formation (23,24). Although there was some aggregation of the MPER-containing BG505 trimers, the extent was much less than with the KNH1144 versions.…”

“…MPER Deletion Does Not Explain the Rarity of Native-Like gp140 UNC Trimers. The above experiments used gp140s from which the MPER was deleted to increase trimer homogeneity (23,24). To assess whether the absence of the MPER influenced trimer configuration, we made SOSIP.R6-MPER, SOSIP.SEKS-MPER, and WT.SEKS-MPER constructs that are identical to the corresponding variants described above except for truncation at residue 681, not residue 664.…”

“…Variants bearing a D7324 epitope tag at the gp41 ECTO C terminus were purified in the same way, and designated by appending "-D7324" to the gp140 descriptor (e.g., SOSIP.R6-D7324, WT.SEKS-D7324). MPER-containing SOSIP.R6-MPER, WT.SEKS-MPER, and SOSIP.SEKS-MPER constructs were based on the SOSIP.681 gene (23,24). See also SI Materials and Methods.…”

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

“…3A). The deletion of the membrane-proximal external region (MPER) and the occlusion of other hydrophobic areas of gp41 ECTO by the overlying gp120 subunits accounts for why SOSIP.664 proteins aggregate only minimally (1,8,45,56). In contrast, the SEC profiles of the Ni-NTA-purified gp140 UNC -Fd-His proteins revealed a quantitatively dominant single peak that contained high-molecularweight (MW) protein aggregates, followed by trimers, with a small amount (1 to 3% of the total) of later-eluting gp140 monomers also present (Fig.…”

“…The gp140 UNC -Fd-His, gp140-FL20, gp140-FL20-IP, and gp140-FL20-SOSIP proteins were isolated from culture supernatants via their His tags, using nickel-nitrilotriacetic acid (Ni-NTA) affinity columns and elution with 250 mM imidazole, essentially as recommended by the manufacturer (Life Technologies Inc.) (2,42). The SOSIP.664-His proteins were purified from culture supernatants using affinity columns containing either Galanthus nivalis agglutinin (GNA)-lectin or one of the 2G12, PGT145, or PGT151 bNAbs, and then similarly fractionated by size exclusion chromatography (SEC) (18,45). The affinity columns were made using a CNBr-activated Sepharose 4B resin (GE Healthcare) as previously described (18).…”