Article Figures & Data
Figures
- 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.
