Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity

Abstract

We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR(+)) or inactive (PR(-)) viral PR. We observed that PR(-) virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.

FIG. 1.

PR⁻ virions display a fusion defect in a cell-cell fusion-from-without assay. env-defective HIV-1 NL4-3 (NL4-3/KFS) (19, 22) and its PR⁻ counterpart were cotransfected into 293T cells with pUC19, with vectors expressing the WT NL4-3 Env (pIIINL4env) (39) or the CT truncation mutant CTdel-144 (pNL4envCTdel-144) (39). Virus-containing supernatants were harvested 2 days posttransfection, and virions were concentrated (10 to 20×) by centrifugation (20,000 × g for 2 h). The concentrated pseudovirions were added to Jurkat cells. After a 20-h cultivation, the number of syncytia (whose diameters were more than four times those of unfused Jurkat cells) was scored. The number of syncytia was expressed as a ratio (%) of those obtained with PR⁻ versus PR⁺ virions. The average number of syncytia induced by PR⁺ virions bearing WT and CTdel-144 Env was 171 and 116, respectively. Data are means of the results of four independent experiments. Error bars indicate standard deviations.

FIG. 2.

PR⁺ and PR⁻ virions incorporate comparable levels of gp41. Virion lysates, prepared from concentrated virus stocks as described in the legend of Fig. 1, were transferred to polyvinylidene difluoride membranes and immunoblotted with the anti-gp41 MAb T32 (upper panel) and AIDS patient serum (lower panel) to detect p24 (capsid) and/or Pr55^Gag. To confirm equal loading of virion-associated material, blots were reprobed with an anti-Vpr antibody (data not shown). Quantitative Western blotting was performed with a Fluor-S MAX MultiImager (Bio-Rad, Hercules, Calif.). T32 was obtained from P. Earl (14), and AIDS patient serum was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. The data shown are representative of the results of at least five independent experiments.

FIG. 3.

PR⁺ and PR⁻ virions display comparable levels of CD4-dependent cell surface binding. Molt-4 clone 8 cells (5 × 10⁵) (30) were incubated with concentrated HIV-1 pseudovirions (prepared as described in the legend of Fig. 1) in the presence of anti-CD4 MAb (Beckman, 13B8.2) or control IgG1. Virus-bound cells were stained with a rat anti-gp120 MAb (W#10) followed by the addition of phycoerythrin-conjugated goat anti-rat IgG. The amount of virus bound was determined by flow cytometry. CD4-independent binding determined by measuring the amount of virus bound in the presence of the anti-CD4 MAb was subtracted from the total binding. Data are means ± standard deviations of the results of four independent experiments.

FIG.4.

PR⁺ and PR⁻ virions display comparable levels of 6HB formation. Concentrated HIV-1 virions (prepared as described in the legend of Fig. 1, except that in this case NL4-3 [1] and CTdel-144 [39] and their PR⁻ counterparts were used) were incubated with an anti-6HB antiserum (rabbit serum no. 948) or preimmune serum (pre) in the presence or absence of 1 μg of sCD4 (Immuno Diagnostics) at 37°C for 2 h. Virions were then pelleted by centrifugation (20,000 × g for 2 h) prior to lysis and immunoprecipitation with protein G Sepharose beads. The immunoprecipitated material was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to quantitative Western blotting with the anti-gp41 MAb T32. An amount of viral lysate equivalent to input virus was run as a positive control. Gels were run for WT Env (A) and CTdel-144 Env (B). The increase in anti-6HB reactivity (n-fold) induced by sCD4 in the PR⁺ (solid bar) and PR⁻ (open bar) context (C) is shown. Also shown is the percentage of anti-6HB-reactive gp41, calculated by dividing the amount of 6HB-reactive gp41 following sCD4 addition by input gp41 in PR⁺ (solid bar) and PR⁻ (open bar) virions (D). Rabbit serum no. 948 was obtained from C. Weiss (12, 23). Data are means ± standard deviations of the results of at least three independent experiments.