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Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag

Yu-Fen Chang1, Shiu-Mei Wang, Kuo-Jung Huang

  • 1Institute of Public Health, National Yang-Ming University School of Medicine, Taipei, Taiwan.

Journal of Molecular Biology
|May 29, 2007
PubMed

Insights

Mutations in HIV-1

Area of Science:

  • Virology
  • Structural Biology
  • Molecular Biology

Background:

  • The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is essential for viral assembly.
  • Gag multimerization and membrane binding are critical steps in the HIV-1 replication cycle.

Purpose of the Study:

  • To investigate the role of the major homology region (MHR) and adjacent C-terminal regions in HIV-1 Gag assembly.
  • To analyze the impact of specific mutations on Gag multimerization, membrane binding, and virus-like particle (VLP) production.

Main Methods:

  • Site-directed mutagenesis to introduce alanine substitutions at key residues (K158, F168, E175) within the HIV-1 MHR and C-terminal regions.
  • Analysis of VLP assembly and production.
  • Assessment of Gag membrane-binding capacity using velocity sedimentation and detergent-resistant membrane assays.
  • Evaluation of Gag multimerization and the effect of MA domain removal.

Main Results:

  • Mutations at K158, F168, and E175 significantly impaired VLP production and Gag membrane binding.
  • Assembly-defective mutants showed increased Gag multimerization, particularly upon removal of the MA domain.
  • K158A mutant demonstrated enhanced VLP production after MA removal, binding to detergent-resistant membranes.
  • F168A and E175A mutants, despite improved multimerization, failed to significantly increase VLP production without MA, suggesting defects in raft-like membrane association.

Conclusions:

  • The HIV-1 MHR and adjacent downstream regions are crucial for Gag multimerization and tight packing during VLP assembly.
  • Enhanced Gag multimerization promotes Gag membrane binding and facilitates efficient VLP formation.
  • Specific interactions with raft-like membrane domains are essential for optimal Gag assembly and VLP production.

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