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Detection of a trimeric human immunodeficiency virus type 1 Gag intermediate is dependent on sequences in the matrix
Y Morikawa1, W H Zhang, D J Hockley
1The Kitasato Institute, Minato-ku, Tokyo 108, Japan. ymorikawa@kitasato.or.jp
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.
Insights
Human immunodeficiency virus type 1 assembly relies on the matrix (MA) protein
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Single amino acid changes in the human immunodeficiency virus type 1 matrix (MA) domain can prevent virion assembly.
- Mutations affecting assembly cluster in a specific helix (residues 54-68) of the MA domain, suggesting its critical role.
Purpose of the Study:
- To investigate the essential features of the MA helix required for human immunodeficiency virus type 1 assembly.
- To elucidate the role of MA trimerization in the Gag polyprotein assembly process.
Main Methods:
- Site-directed mutagenesis of the MA domain of human immunodeficiency virus type 1 Gag precursor Pr55.
- Analysis of virion particle assembly in mutants.
- Biochemical analysis of purified MA and MA-capsid (CA) Gag molecules, including assessment of trimerization in solution.
Main Results:
- Mutations preventing virion assembly map to the hydrophobic face of the MA helix, indicating a role in stabilizing trimeric interfaces.
- Purified MA protein exists as a trimer in solution, and mutations that disrupt assembly also prevent MA trimerization.
- A larger MA-capsid (CA) Gag molecule also trimerizes in solution, whereas CA alone is monomeric.
Conclusions:
- Gag trimerization, driven by the MA domain, is a crucial intermediate step in human immunodeficiency virus type 1 virion assembly.
- MA trimerization is dependent on a specific conformation of the MA domain, influenced by its hydrophobic core.