Stabilization of the Soluble, Cleaved, Trimeric Form of the Envelope Glycoprotein Complex of Human Immunodeficiency Virus Type 1

Abstract

The envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41 subunits) are associated by relatively weak, noncovalent interactions. The induction of neutralizing antibodies after vaccination with individual Env subunits has proven very difficult, probably because they are inadequate mimics of the native complex. Our hypothesis is that a stable form of the Env complex, perhaps with additional modifications to rationally alter its antigenic structure, may be a better immunogen than the individual subunits. A soluble form of Env, SOS gp140, can be made that has gp120 stably linked to the gp41 ectodomain by an intermolecular disulfide bond. This protein is fully cleaved at the proteolysis site between gp120 and gp41. However, the gp41-gp41 interactions in SOS gp140 are too weak to maintain the protein in a trimeric configuration. Consequently, purified SOS gp140 is a monomer Here we describe modifications of SOS gp140 that increase its trimer stability. A variant SOS gp140, designated SOSIP gp140, contains an isoleucine-to-proline substitution at position 559 in the N-terminal heptad repeat region of gp41. This protein is fully cleaved, has favorable antigenic properties, and is predominantly trimeric. SOSIP gp140 trimers are noncovalently associated and can be partially purified by gel filtration chromatography. These gp140 trimers are dissociated into monomers by anionic detergents or heat but are relatively resistant to nonionic detergents, high salt concentrations, or exposure to a mildly acidic pH. SOSIP gp140 should be a useful reagent for structural and immunogenicity studies.The envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 (HIV-1) mediates viral entry into CD4 ϩ cells. The sequential binding of the surface subunit gp120 to the CD4 receptor and a coreceptor, usually CCR5 or CXCR4, induces conformational changes in the Env complex. These alterations in protein structure eventually enable the insertion of the hydrophobic fusion peptide of the transmembrane subunit, gp41, into the cell membrane. Subsequently, the viral and cell membranes fuse, allowing the release of the viral core into the cytoplasm and the initiation of a new cycle of infection (for reviews, see references 17, 24, 25, 31, 61, and 99). gp120 and gp41 are synthesized as a gp160 precursor that is cleaved within the cell to yield the native, prefusion form of the Env complex (39,55,63). This is generally considered to be a trimeric structure, containing three gp120 and three gp41 moieties held together by noncovalent interactions (31,73,99). The native Env complex is unstable, because the noncovalent intersubunit interactions that hold gp120 to gp41 are weak, as are the intermolecular interactions between the gp41 moieties (31,73,99). This instability is probably essential for receptortri...

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

“…As noted, the IP.R6 and WT.R6 constructs formed trimers inefficiently (<5%). Thus, the only cleaved gp140 for which trimers predominated was SOSIP.R6, consistent with previous reports that SOS and I559P changes are both needed to stabilize cleaved trimers (13,14). However, the SOS bond alone (SOS.R6) was sufficient to allow some cleaved trimers to be expressed from BG505 env.…”

“…The critical changes are an appropriately positioned disulfide bond (referred to as "SOS") to link gp120 to gp41 ECTO covalently, and an Ile/Pro (IP) substitution at residue 559 to strengthen intergp41 ECTO interactions (13)(14)(15)(16)(17). The resulting cleaved trimers are designated SOSIP gp140s (14). Additional modifications have improved their stability, homogeneity, and antigenicity (15)(16)(17).…”

“…Our alternative approach is based on the premise that cleavage is a fundamental feature of Env structure and involves stabilizing fully cleaved gp140s. The critical changes are an appropriately positioned disulfide bond (referred to as "SOS") to link gp120 to gp41 ECTO covalently, and an Ile/Pro (IP) substitution at residue 559 to strengthen intergp41 ECTO interactions (13)(14)(15)(16)(17). The resulting cleaved trimers are designated SOSIP gp140s (14).…”

“…However, the resulting proteins, known as gp140s, are highly unstable and disintegrate into their gp120 and gp41-ectodomain (gp41 ECTO ) components, making them useless as immunogens. Two fundamentally different protein-engineering strategies have been used to create gp140s that can be produced and purified without falling apart (3,4,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The most common method involves eliminating the cleavage site between gp120 and gp41 ECTO , creating uncleaved gp140s (gp140 UNC ) where the two subunits remain covalently linked (7)(8)(9)(10)(11)(12).…”

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures

“…AY736812). JR-FL SOSIP gp140 constructs have been described previously (29). Codon-optimized SOSIP gp140 genes were subcloned into the PPI4 vector (11).…”