Detection of a trimeric human immunodeficiency virus type 1 Gag intermediate is dependent on sequences in the matrix protein, p17

Abstract

Previous studies have shown that single amino acid changes in the amino-terminal matrix (MA) domain, p17, of the human immunodeficiency virus type 1 Gag precursor Pr55, can abrogate virion particle assembly. In the three-dimensional structure of MA such mutations lie in a single helix spanning residues 54 to 68, suggesting a key role for this helix in the assembly process. The fundamental nature of this involvement, however, remains poorly understood. In the present study, the essential features of the MA helix required for virus assembly have been investigated through the analysis of a further 15 site-directed mutants. With previous mutants that failed to assemble, residues mapped as critical for assembly were all located on the hydrophobic face of the helix and had a key role in stabilizing the trimeric interface. This implies a role for the MA trimer in virus assembly. We support this interpretation by showing that purified MA is trimeric in solution and that mutations that prevent virus assembly also prevent trimerization. Trimerization in solution was also a property of a larger MA-capsid (CA) Gag molecule, while under the same conditions CA only was a monomer. These data suggest that Gag trimerization driven by the MA domain is an intermediate stage in normal virion assembly and that it relies, in turn, on an MA conformation dependent on the hydrophobic core of the molecule.

FIG. 1

Thin sections of Sf9 cells expressing wild-type Pr55 (top panel) or Pr55 bearing the Tyr79Ala change in the MA domain (bottom panel). Compared to the wild type, Tyr79Ala produced deformed, only partly budded particles and an accumulation of Gag antigen at the membrane typical of other virus assembly mutants previously described (reference and references therein).

FIG. 2

Sedimentation profiles of purified Gag antigens on glycerol gradients. Purified soluble Gag antigens were sedimented through 15 to 30% glycerol gradients made in 20 mM Tris (pH 7.4)–100 mM NaCl–1 mM dithiothreitol–0.5 mM EDTA at 48,000 rpm in an SW50 rotor and 4°C for 40 h (for analysis of MA and CA) or for 27 h (for analysis of MA-CA). Molecular mass markers in the gradients were provided by high (analysis of MA-CA)- or low (analysis of MA and CA)-molecular-mass calibration kits (Pharmacia), are shown in the upper section of each panel in all cases, and are marked SM (sedimentation markers). The high-molecular-mass range consisted of the following: catalase, 4 × 58 kDa = 232 kDa; lactate dehydrogenase, 4 × 36 kDa = 140 kDa; and serum albumin, 67 kDa. The low-molecular-mass range consisted of the following: phosphorylase b, 94 kDa; serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa; and a-lactalbumin, 14.4 kDa. Gel markers (M) are prestained molecular mass markers (Bio-Rad). Gradients were fractionated from the bottom, and Gag antigen was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The middle and bottom sections of each panel show an analysis of antigen prepared under low- and high-salt conditions, respectively. (A) MA protein; (B) MA-CA protein; (C) CA protein.

FIG. 3

Sedimentation analysis of assembly-defective and -competent Gag mutant MAs. The MA domain of several Gag mutants was rescued by PCR for expression as a purified MA domain by using pGEX expression as described previously (28). The antigen was prepared under conditions of low salt throughout and analyzed by glycerol velocity gradients under the same conditions described in the legend to Fig. 2. The panels show the analysis of MA antigen prepared from (top to bottom) Arg58Ala, Gln59Ala, Gln63Ala, Pro66Ala, Gln59Ala/Pro66Ala, Cys57Ser, Leu64Ala, and Leu61Ala/Leu68Ala mutants. Prestained markers (Bio-Rad) are visible in the leftmost lane of each gel. A trace of trimer (less than 5% by gel scan) was present in panels containing Cys57Ser and Leu64Ala, but none was visible in Leu61Ala/Leu68Ala mutants.