Evidence for a stable interaction of gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1 particles

Abstract

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55(Gag) in infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55(Gag) in HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55(Gag) in HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55(Gag) at the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55(Gag) and the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55(Gag) with gp41 may regulate the function of the envelope proteins during HIV-1 maturation.

FIG. 1

Isolation of immature HIV-1 core structures. Immature HIV-1 particles were harvested from 293T cells transiently transfected with the R9.PR⁻ proviral plasmid and were filtered to remove cellular debris. Concentrated virions were sedimented through a layer of 1% Triton X-100 into a linear 30 to 70% sucrose gradient. Fractions (1 ml) were collected from the top of the gradient and assayed for CA concentration (p24) by ELISA and for density by refractometry. (A) Virions subjected to ultracentrifugation on a control gradient lacking detergent. (B) Detergent-treated virions.

FIG. 2

Immunoblot and ELISA analysis of immature HIV-1 virions and cores. (A) The peak Pr55^Gag-containing fractions of virions (lanes 1 to 3) and cores (lanes 4 to 6) were diluted with STE buffer, and particles were pelleted by ultracentrifugation. Pellets were dissolved in 1× SDS loading dye, subjected to electrophoresis on a 4 to 20% acrylamide gel, transferred to PVDF membrane, and probed with the indicated antisera. Protein bands were revealed by chemiluminescent detection after probing with the appropriate HRP-conjugated secondary antiserum. For comparative purposes, twofold serial dilutions of the peak cores fraction (lanes 7 to 9) were analyzed on the same gel. Molecular mass markers are shown in kilodaltons on the left of each panel. (B and C) Quantitation of gp120 associated with immature HIV-1 virions and cores by ELISA. Gradient fractions of HIV-1 immature virions and cores were analyzed for the presence of gp120 and Pr55^Gag by ELISA. The gp120 concentration for each sample was determined using a standard curve of recombinant HIV-1_LAV gp120. (B) Virions subjected to ultracentrifugation on a control gradient lacking detergent. (C) Detergent-treated virions.

FIG. 3

Immunoblot analysis of immature HIV-1 cores and virions lacking the cytoplasmic tail of gp41. The peak Pr55^Gag-containing fraction of cores (C, lanes 1 and 3) and virions (V, lanes 2 and 4), containing either a full-length form of gp41 (WT) or a truncated form of gp41 lacking the cytoplasmic tail (Tr712), were diluted with STE buffer and pelleted by ultracentrifugation. Pellets were dissolved in 1× SDS loading buffer, subjected to electrophoresis on a 4 to 20% acrylamide gel, and transferred to PVDF membrane, and the blot was probed with the indicated antisera. Protein bands were revealed by chemiluminescent detection after incubation of the blot with appropriate HRP-conjugated secondary antisera. Molecular mass markers are shown in kilodaltons on the left of each panel.

FIG. 4

Immunoblot and ELISA analysis of HIV-1 virions and cores inhibited for cleavage at the MA-CA junction of Pr55^Gag. (A) The peak MA-CA-containing fractions of virions (lanes 1 to 3) and cores (lanes 4 to 6) were analyzed by immunoblotting as described in the legend to Fig. 2. (B and C) The gp120 concentrations of the gradient fractions were determined as described in the legend to Fig. 2. (B) Analysis of virions. (C) Analysis of cores.

FIG. 5

Association of HIV-1 Env proteins on immature HIV-1 cores independently of the presence of lipid rafts. Immature HIV-1 cores were isolated from a PLAP-encoding virus by incubation for 1 h in detergent at either 4 or 37°C, followed by sedimentation centrifugation. Fractions were examined for the presence of PLAP and HIV-1 Env proteins. (A and B) Levels of PLAP were determined by incubation of gradient fractions with an AP substrate and quantitated on a luminometer as relative units (RU) of luminescence. A CA-specific ELISA was used to determine the amount of virus in each gradient fraction. (C) Immunoblot analysis of the peak Pr55^Gag-containing gradient fractions from immature cores treated with detergent at either 4 or 37°C (lanes 1 and 4, respectively) was performed as described in the legend to Fig. 2. Threefold serial dilutions of the peak Pr55^Gag-containing fractions were also analyzed (lanes 2 and 3 and lanes 5 and 6).

FIG. 6

Analysis of phospholipids present immature HIV-1 virions and cores. Immature HIV-1 particles were harvested from ³²P-labeled 293T cells transiently transfected with the R9.PR⁻ proviral plasmid. The virions were concentrated by ultracentrifugation and then treated with 1% Triton X-100 for 1 h at 37°C. The remaining core structures were separated from free proteins and lipids by pelleting them through a 20% sucrose cushion. Lipids from virions (V) and cores (C) were extracted by chloroform-methanol (2:1) extraction, analyzed by thin-layer chromatography, and visualized by autoradiography. Lipids were extracted from similar quantities of immature virions (lane 1, 30 μg of p24) and immature cores (lane 3, 26 μg of p24) and analyzed. A 1:50 dilution of the intact virions (lane 2) was analyzed for quantitative comparison. Phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC) standards were analyzed in parallel.