Stimulus-responsive assembly of nonviral nucleocapsids

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• Controlled assembly of protein shells around genetic material is crucial for viral replication.
• Existing methods for in vitro nucleocapsid formation have limitations in cargo encapsulation and assembly control.

Purpose of the Study:

• To develop a strategy for regulating the co-assembly of nonviral proteins and nucleic acids into ordered nucleocapsids in vitro.
• To enable controlled, RNA-templated formation of engineered protein cages.

Main Methods:

• Engineered an NC-4 protein cage by fusing maltose binding protein to its subunits to block spontaneous assembly.
• Utilized selective proteolysis to remove the steric block and initiate RNA-templated capsid formation.
• Employed transmission and cryo-electron microscopy to analyze the structure of the assembled nucleocapsids.

Main Results:

• Successfully blocked spontaneous capsid assembly, allowing isolation of soluble protein monomers.
• Achieved RNA-templated nucleocapsid formation upon enzymatic trigger, producing structures identical to in vivo assemblies.
• Demonstrated that the method allows for broader RNA encapsulation by the NC-4 cage.

Conclusions:

• Enzymatically triggered protein cage formation offers a novel method for controlled nucleocapsid assembly.
• This approach provides new opportunities to study the co-assembly of viral/nonviral capsids and their genetic cargo.
• The technique has potential applications in studying diverse viral and nonviral assembly processes.