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Opposing effects of human immunodeficiency virus type 1 matrix mutations support a myristyl switch model of gag
1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. Heinrich_Gottlinger@DFCI.harvard.edu
Abstract:
Targeting of the human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55(gag) to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain. N-myristylation of MA is essential for the stable association of Pr55(gag) with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation. Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion. Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA. The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group. Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane.
Insights
Human immunodeficiency virus type 1 (HIV-1) matrix (MA) domain targeting to membranes is crucial for virus assembly. Mutations affecting MA
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- The N-terminal matrix (MA) domain of human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55(gag) directs it to the plasma membrane for virus assembly.
- N-myristylation of MA is essential for stable Pr55(gag) membrane association and efficient virus production.
Purpose of the Study:
- To investigate the role of the N-terminal region of the MA domain in Pr55(gag) targeting and HIV-1 assembly.
- To elucidate the mechanism by which N-terminal modifications affect myristyl group accessibility and membrane insertion.
Main Methods:
- Site-directed mutagenesis of the MA domain near the N terminus and in the globular core.
- Subcellular fractionation to assess Pr55(gag) membrane binding.
- Analysis of viral particle assembly and yield.
Main Results:
- Single amino acid substitutions near the MA N terminus impaired Gag membrane binding and virus assembly, even with intact myristylation.
- These defects were suppressed by second-site mutations in the MA globular core, which enhanced membrane binding and viral particle production.
- Compensatory mutations increased the exposure of the myristyl group, suggesting improved membrane insertion.
Conclusions:
- The MA domain's N terminus and globular core regulate myristyl group exposure for controlled insertion into the plasma membrane.
- The MA globular core sequesters the myristyl group to prevent aberrant binding to intracellular membranes.
- Controlled N-terminal exposure of the myristyl group is critical for efficient HIV-1 assembly at the plasma membrane.