Investigation of HIV-1 Gag binding with RNAs and lipids using Atomic Force Microscopy

Area of Science:

• Biophysics
• Structural Biology
• Molecular Virology

Background:

• Gag polyproteins are essential for retroviral particle assembly.
• Understanding Gag-RNA and Gag-lipid interactions is crucial for viral replication.
• Specific lipid interactions, like with phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), can influence Gag multimerization.

Purpose of the Study:

• To investigate the morphology and multimerization of Gag proteins.
• To examine the binding complexes of Gag with specific RNA (ΨRNA) and lipids (PI(4,5)P2).
• To elucidate the role of specific RNA and lipid interactions in Gag assembly.

Main Methods:

• Atomic Force Microscopy (AFM) was employed to visualize Gag and its complexes at high resolution (0.1Å vertical, 1nm lateral).
• Experiments were conducted in solution using positively or negatively charged mica substrates.
• Gel electrophoresis was used to confirm Gag multimerization states (monomers, dimers, tetramers).

Main Results:

• Gag proteins exist as monomers, dimers, and tetramers.
• Specific ΨRNA binding significantly increases Gag multimerization.
• Non-specific TARpolyA RNA did not enhance Gag multimerization.
• PI(4,5)P2 binding increases Gag multimerization, but less effectively than ΨRNA.
• Co-presence of ΨRNA and PI(4,5)P2 induces conformational changes and further enhances Gag multimerization.

Conclusions:

• ΨRNA and PI(4,5)P2 are key factors in promoting Gag multimerization and viral assembly.
• The combined effect of ΨRNA and PI(4,5)P2 leads to significant Gag conformational changes and enhanced multimerization.
• AFM provides critical insights into the structural dynamics of Gag-RNA-lipid complexes.