Structural analysis of membrane-bound retrovirus capsid proteins

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• Retrovirus core (Gag) proteins are crucial for viral assembly and structure.
• Analyzing Gag protein interactions in vitro is challenging due to their complex assembly dynamics.
• Histidine-tagged (His-tagged) proteins offer a method for specific binding and analysis.

Purpose of the Study:

• To develop a novel system for analyzing His-tagged retrovirus Gag proteins in vitro.
• To investigate the in vitro assembly and structural organization of Moloney murine leukemia virus (M-MuLV) capsid (CA) protein.
• To elucidate the substructure of retroviral cores and potential mechanisms for protein exclusion during assembly.

Main Methods:

• Development of a lipid monolayer system using egg phosphatidylcholine (PC) and a novel nickel-chelating lipid (DHGN).
• Binding of His-tagged proteins to DHGN-containing monolayers.
• Analysis of membrane-bound arrays of N-terminal His-tagged M-MuLV CA protein (His-MoCA) using 2D crystallization and image analysis.

Main Results:

• DHGN effectively chelates nickel and specifically binds His-tagged proteins.
• His-MoCA proteins formed extensive two-dimensional (2D) protein crystals with high resolution (9.5 A).
• Image analysis revealed a cage-like network of His-MoCA arrays, with distinct hexamer rings and protein-free cage holes.

Conclusions:

• Gag hexamers likely form a fundamental substructure of the retrovirus core.
• The observed cage hole dimensions may dictate the exclusion of other proteins, such as envelope proteins, from the assembling virus particle.
• The 2D crystallization method provides a powerful tool for detailed analysis of retroviral Gag proteins and other His-tagged proteins.