Redox-triggered infection by disulfide-shackled human immunodeficiency virus type 1 pseudovirions

Abstract

We previously described a human immunodeficiency virus type 1 (HIV-1) envelope mutant that introduces a disulfide bridge between the gp120 surface proteins and gp41 transmembrane proteins (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). Here we produced pseudovirions bearing the mutant envelope and a reporter gene to examine the mutant's infectious properties. These pseudovirions attach to cells expressing CD4 and coreceptor but infect only when triggered with reducing agent, implying that gp120-gp41 dissociation is necessary for infection. Further studies suggested that virus entry was arrested after CD4 and coreceptor engagement. By measuring the activities of various entry inhibitors against the arrested intermediate, we found that gp120-targeting inhibitors typically act prior to virus attachment, whereas gp41 inhibitors are able to act postattachment. Unexpectedly, a significant fraction of antibodies in HIV-1-positive sera neutralized virus postattachment, suggesting that downstream fusion events and structures figure prominently in the host immune response. Overall, this disulfide-shackled virus is a unique tool with potential utility in vaccine design, drug discovery, and elucidation of the HIV-1 entry process.

FIG. 1.

Analysis of the functional activity of SOS pseudovirions. Single-round infectivity experiments were performed using U87.CD4.CCR5 cells. (A) Infectivity of pseudovirions bearing gp160 WT (open circles), gp160ΔCT WT (closed circles), gp160ΔCT SOS (squares), and gp160ΔCT UNC (triangles). RLU, relative light units. (B) Effect of adding DTT to cultures after virus incubation with target cells. Symbols denote gp160ΔCT-Env bearing pseudoviruses as follows: circles, WT; diamonds, SOS; and triangles, UNC. Open symbols denote culture wells to which DTT was added, and closed symbols denote wells in which the medium was merely changed. (C) DTT treatment was titrated by adding graded amounts after virus incubation with cells. (D) SOS pseudovirions were exposed to DTT either before (stars) or after (circles) attachment to U87.CD4.CCR5 cells, both before and after (open triangles), or neither (closed squares). Similar results were obtained with Cf2.CD4.CCR5 and HOS.CD4.CCR5 cells (data not shown).

FIG. 2.

SOS pseudovirions engage both CD4 and CCR5 upon attachment. (A) The ability of the anti-CCR5 MAb PA14 to inhibit SOS-VLP infection before and after the attachment of virus to cells. Symbols are as follows: closed circles, cells pretreated with PA14, followed by washing; open circles, PA14 and virus added to cells concomitantly and coincubated thereafter; triangles, PA14 added after virus attachment to cells. (B) Ability of SOS pseudovirions to attach to CCR5-expressing Cf2.Th.synCCR5 cells in the presence (open circles) or absence (closed triangles) of sCD4. Triangles denote infection of positive control Cf2.Th.CD4.CCR5 cells.

FIG. 3.

Standard neutralization and postattachment neutralization using SOS pseudovirions. Various MAbs, CD4-IgG2, and the T-20 peptide were assayed for neutralization of gp160ΔCT SOS pseudovirions using the standard (open circles) or postattachment (closed triangles) assay format.

FIG. 4.

Reversibility of neutralization by MAb 2F5 and the T-20 peptide. (A) Comparison of IgG1b12, 2F5, and T-20 in standard (open circles) and preattachment (closed circles) neutralization assays. (B) The T-20 peptide and 2F5 MAb were compared in neutralization assays with modified wash steps. Symbols are as follows: open circles, standard assay; closed triangles, postattachment assay; closed circles, standard assay without a pre-DTT treatment wash step; open triangles, postattachment assay with an additional wash before the DTT incubation.

FIG. 5.

Serum neutralization in pre- and postattachment neutralization assays. Sera from several HIV-positive donors and HIVIG were assayed for neutralization of gp160ΔCT SOS pseudovirus using standard (open circles) and postattachment (closed triangles) neutralization assays.