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. 2000 Jun;74(11):5091-100.
doi: 10.1128/jvi.74.11.5091-5100.2000.

Variable-loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits

R W Sanders  1 , L Schiffner, A Master, F Kajumo, Y Guo, T Dragic, J P Moore, J M Binley
Affiliations

Variable-loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits

R W Sanders et al. J Virol. 2000 Jun.

Abstract

We have described an oligomeric gp140 envelope glycoprotein from human immunodeficiency virus type 1 that is stabilized by an intermolecular disulfide bond between gp120 and the gp41 ectodomain, termed SOS gp140 (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627-643, 2000). In this protein, the protease cleavage site between gp120 and gp41 is fully utilized. Here we report the characterization of gp140 variants that have deletions in the first, second, and/or third variable loop (V1, V2, and V3 loops). The SOS disulfide bond formed efficiently in gp140s containing a single loop deletion or a combination deletion of the V1 and V2 loops. However, deletion of all three variable loops prevented formation of the SOS disulfide bond. Some variable-loop-deleted gp140s were not fully processed to their gp120 and gp41 constituents even when the furin protease was cotransfected. The exposure of the gp120-gp41 cleavage site is probably affected in these proteins, even though the disabling change is in a region of gp120 distal from the cleavage site. Antigenic characterization of the variable-loop-deleted SOS gp140 proteins revealed that deletion of the variable loops uncovers cryptic, conserved neutralization epitopes near the coreceptor-binding site on gp120. These modified, disulfide-stabilized glycoproteins might be useful as immunogens.

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Figures

FIG. 1
FIG. 1
Schematic representation of the V1, V2, and V3 regions of JR-FL gp120 and the deletions made in the various mutants. The structures and residue numbering scheme are based on the representation of JR-FL gp120 in reference , which is in turn based on the gp120 secondary structure described by Leonard et al. (27).
FIG. 2
FIG. 2
Analysis of cleavage and intermolecular disulfide bond formation in full-length and variable-loop-deleted gp140 proteins. Envelope glycoproteins secreted from transfected 293T cells were immunoprecipitated with MAb 2G12. Furin was cotransfected only with the SOS gp140 proteins. The WT gp140 proteins were produced in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of cotransfected furin. The SOS gp140 proteins were produced in the presence of cotransfected furin (lanes 5 and 6). In each lane, the upper band represents a gp140 protein containing gp120 plus the gp41 ectodomain. These bands from WT proteins comprise uncleaved gp140 species in which the gp120-gp41ECTO cleavage site is unprocessed. In the case of SOS gp140 proteins, the upper bands represent disulfide-stabilized, proteolytically cleaved gp140s. In all cases, the lower bands are gp120 proteins, with or without deletions in their variable loops. The precipitated proteins were treated with DTT or not treated, as indicated. The full-length proteins are analyzed in panel A, and different variable-loop-deleted proteins are analyzed in panels B through H.
FIG. 3
FIG. 3
Antigenic structure analysis of variable-loop-deleted gp140 proteins. Envelope glycoproteins from (A) full-length, (B) ΔV3, and (C) ΔV1V2* WT and SOS gp140 proteins were precipitated with MAb 23A to the gp120 C5 region or with MAb 2F5, 25C2, or 7B2 to the gp41 ectodomain, as indicated. The WT gp140 proteins were expressed in the absence of cotransfected furin, yielding gp120 and uncleaved gp140 (odd-numbered lanes). Furin was cotransfected with the SOS gp140 proteins (even-numbered lanes).
FIG. 4
FIG. 4
Exposure of CD4BS and CD4i epitopes on variable-loop-deleted SOS gp140 proteins. Envelope glycoproteins expressed from the full-length SOS gp140 protein and the ΔV2 SOS, ΔV3 SOS, and ΔV1V2* SOS gp140 proteins in the presence of cotransfected furin were immunoprecipitated with (A) the CD4-IgG2 molecule or (B) MAb 17b to a CD4i epitope in the presence and absence of sCD4.
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