In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain

Abstract

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Deltap6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Deltap6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Deltap6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Deltap6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Deltap6 and nucleic acid are not sufficient for the formation of normal-sized particles.

FIG. 1

Schematic diagrams of proteins expressed in E. coli and purified proteins. (A) Schematic diagram of HIV-1 Gag and Gag Δp6 used for in vitro assembly. (B) Coomassie blue-stained SDS-polyacrylamide gel of purified proteins. Lanes: 1 and 2, 10 and 100 μg of purified HIV-1 Gag Δp6, respectively; 3, HIV-1 Gag purified in the same manner as Gag Δp6; 4 and 5, anti-p6 antibody affinity purification of HIV-1 Gag; 4, flow-through (FT) from the affinity column; 5, affinity-purified HIV-1 Gag (eluted with p6 peptide). Arrows indicate the protein band referred to in each lane. PC, purified on a phosphocellulose cation-exchange column.

FIG. 2

Negatively stained EM images of Gag Δp6 and Gag particles assembled in vitro. (A) Gag Δp6 particles assembled with yeast tRNA; (B) Gag Δp6 particles assembled with an in vitro transcript of HIV-1 packaging sequence (∼1 kb); (C) Gag particles assembled at 4°C with E. coli rRNA; (D) Gag Δp6 particles assembled with MS2 bacteriophage RNA (∼3.5 kb); arrows indicate tails protruding from particles. All panels were negatively stained with 2% uranyl acetate. Scale bars = 50 nm.

FIG. 3

Precipitation of HIV-1 Gag Δp6 with poly(dTG) oligonucleotides (Oligo) of different lengths. HIV-1 Gag Δp6 (100 μg at 5 mg/ml in a solution containing 20 mM Tris [pH 7.5], 0.5 M NaCl, 0.5% NP-40, and 10 mM DTT) was mixed with no oligonucleotide (lanes 1 to 3) or with a 5-base (lanes 4 to 6), 10-base (lanes 7 to 9), or 15-base (lanes 10 to 12) TG oligonucleotide or the 24-base arbitrary oligonucleotide referred to in the text (lanes 13 to 15). The reaction mixtures were then diluted fivefold in pH 8.0 buffer without NaCl (final conditions, 100 μg [at 1 mg/ml] of protein, pH 8.0, 0.1 M NaCl, 0.5% NP-40, 10 mM DTT, 4 μg of oligonucleotide) and incubated for 2 h at room temperature. The precipitates were pelleted by centrifugation in a microcentrifuge for 1 h. Equal portions of the total (T), pellet (P), and supernatant (S) fractions were analyzed by SDS-PAGE and Coomassie blue staining.

FIG. 4

Precipitation of HIV-1 Gag Δp6 with oligonucleotides of different lengths, sequences, and oligonucleotide/protein ratios. Assembly reactions with oligonucleotides of different lengths (10, 15, 20, 25, or 30 nt long) and at different oligonucleotide/protein ratios (0, 1, 2, 4, 8, or 16% [wt/wt]) were performed and the products analyzed as described in the legend to Fig. 3. The percentage of protein in each pellet fraction was quantitated by densitometry. (A) Poly(dA) oligonucleotides; (B) poly(dTG) oligonucleotides.

FIG. 5

Cross-linking of HIV-1 Gag Δp6 with oligonucleotides (Oligo) of different lengths. The total reaction mixtures from the experiment of Fig. 3 were cross-linked with increasing amounts (0, 0.5, 1, and 2 mM) of DMS prior to SDS-PAGE and Coomassie blue staining. Lanes: 1 to 4, no oligonucleotide (0); 5 to 8, 5-base poly(dTG); 9 to 12, 10-base poly(dTG); 14 to 17, 15-base poly(dTG); 18 to 21, arbitrary 24-base oligonucleotide referred to in the text. The sizes of the molecular mass markers (M; lane 13) are indicated on the left (in kilodaltons). The positions of the Gag Δp6 cross-linking products are indicated on the right.

FIG. 6

Precipitation of HIV-1 Gag Δp6 with RNA is reversible with RNase A or NaCl treatment. Lanes: 1 to 3, in vitro assembly reactions with HIV-1 Gag Δp6 and no oligonucleotide (0); 4 to 6, yeast tRNA, 4%; 7 to 9, E. coli rRNA, 4%; lanes 10 to 12, bacteriophage MS2, RNA 4%; 13 to 15, poly(dT-dG) 30 DNA, 4% oligonucleotide. T, total; P, pellet; S, supernatant. (A) Assembly reactions in 0.1 M NaCl; (B) assembly reactions from panel A to which NaCl was added to 0.5 M NaCl for 20 min prior to sedimentation; (C) assembly reactions from panel A to which RNase A was added (at 0.1 mg/ml) for 20 min prior to sedimentation.

FIG. 7

Cross-linking of NaCl-treated HIV-1 Gag Δp6–TG 15 assemblies. Assembly reactions using TG 15, at a oligodeoxynucleotide/protein ratio of 4% (wt/wt), were cross-linked with increasing amounts (0, 0.5, 1, and 2 mM) of DMS prior to SDS-PAGE and Coomassie blue staining. All reaction mixtures were assembled and cross-linked for the same amount of time. Lanes: 1 to 4, no oligonucleotide (Oligo) (0), in 0.5 M NaCl; 5 to 8, TG 15, in 0.1 M NaCl; 9 to 12, TG 15 assembly reaction (in 0.1 M NaCl) to which NaCl was added to 0.5 M for 20 min prior to cross-linking. The sizes of the molecular mass markers (lane 13) are indicated on the left (in kilodaltons). The positions of the Gag Δp6 cross-linking products are indicated on the right.