Enhancing the Proteolytic Maturation of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins

Abstract

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.The Env glycoprotein complex mediates receptor binding and membrane fusion during human immunodeficiency virus type 1 (HIV-1) infection of susceptible cells (66). It is synthesized as a polypeptide precursor (gp160) that oligomerizes to form a heavily glycosylated trimer (20,24). At a late stage of synthesis, most probably in the trans-Golgi network (TGN), gp160 is cleaved by furin (17,18,(55)(56)(57)(58) or other, related subtilisin-like proteases (17,18,28,38,58,90) into the surface (SU; gp120) and transmembrane (TM; gp41) subunits (34,43,(55)(56)(57)(58)82). Cleavage occurs at a motif at the gp120-gp41 juncture that contains a basic amino acid tetrad, R-X-(R/K)-R (where X is any amino acid). The gp120 and gp41 proteins then remain noncovalently associated, forming the functional, native gp120 3 -gp41 3 complex (20,24,66).During fusion, the gp120 protein interacts with the virus receptor and coreceptor on target cells. This triggers conformational changes that lead to the insertion of a hydrophobic fusion peptide, located at the N terminus of gp41, into the target cell membrane (66). Cleavage of gp160 is essential for fusion, since uncleaved gp160 is fusion incompetent (9,33,39,48). Generally, only cleaved Env is incorporated into virions (22), although uncleaved Env can be virion associated (39,48). By analogy with other enveloped viruses such as influenza A virus (5,...

Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers

“…In the current study, we used monomeric gp120s from the subtype B strain JR-FL. The KNH1144 SOSIP.R6 gp140 protein contains the trimer-stabilizing A501C, T605C, and I559P substitutions and a hexa-arginine (R6) motif at the C terminus of gp120 that increases the efficiency of gp120-gp41 proteolytic cleavage (44,45,53,54). The sequence was also modified to contain epitopes for mAbs 2F5, 4E10, and 2G12 (47).…”

“…The gp140 was modified to reconstitute the 2G12 epitope (S295N substitution), and the 2F5 and 4E10 epitopes in the MPER (A662E, G664D, S668N, and N671T substitutions) (47). The gp140 also contains the A501C and T605C substitutions to create the SOS disulfide bond between gp120 and gp41 (44), the I559P mutation to promote trimerization (45), and a hexa-arginine cleavage site (R6) to enhance cleavage efficiency (54). The type of N-glycan indicated to be present at each glycosylation site (oligomannose, complex or undefined) is based on glycan characterization of IIIB gp120 (83).…”

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

“…The resulting cleaved trimers are designated SOSIP gp140s (14). Additional modifications have improved their stability, homogeneity, and antigenicity (15)(16)(17). Our current design, based on the BG505 subtype A env gene, yields SOSIP.664 trimers that mimic native, virion-associated Env spikes antigenically and when viewed by negative-stain electron microscopy (EM) (17)(18)(19).…”

“…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).…”