Nonstructural protein 4 of human norovirus self-assembles into various membrane-bridging multimers

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Positive-sense single-stranded RNA ((+)RNA) viruses form replication compartments within host cells.
• Noroviruses, a genus of (+)RNA viruses, cause significant human gastroenteritis, with GII.4 strains being predominant.
• Nonstructural protein 4 (NS4) is crucial for norovirus replication-associated membrane rearrangements, but its precise function remains unclear.

Purpose of the Study:

• To produce, purify, and structurally and functionally characterize the GII.4 norovirus NS4 protein.
• To investigate the membrane-interacting properties and self-assembly behavior of GII.4 NS4.
• To determine if GII.4 NS4 can recapitulate key membrane events of norovirus replication in vitro.

Main Methods:

• Protein production and purification of GII.4 NS4.
• AlphaFold modeling integrated with experimental data for structural analysis.
• Liposome-based assays to study membrane binding and fusion.
• Cryo-electron microscopy (Cryo-EM), Nuclear Magnetic Resonance (NMR), and membrane flotation assays to analyze NS4 assemblies.

Main Results:

• GII.4 NS4 self-assembles into distinct structures.
• NS4 exhibits membrane-bridging capabilities, promoting liposome aggregation.
• At least two distinct NS4 assemblies were identified that bridge membranes.
• NS4 alone can induce membrane apposition, a key feature of norovirus replication.

Conclusions:

• The GII.4 norovirus NS4 protein possesses intrinsic membrane-binding and -rearranging properties.
• NS4 self-assembly drives membrane apposition, essential for forming viral replication compartments.
• These findings provide in vitro evidence for NS4's role in generating norovirus replication organelles.