High-throughput single-virion DNA-PAINT reveals structural diversity, cooperativity, and flexibility during selective...

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Influenza A virus, an RNA virus, possesses an eight-segment genome.
• Co-infections can lead to reassortant viruses, potentially causing pandemics.
• Selective genome packaging is crucial for influenza virus assembly.

Purpose of the Study:

• To investigate the role of segment pairings in selective influenza A virus genome packaging.
• To analyze packaging defects and inter-segment distances within individual virus particles.
• To understand the contributions of segment interactions to genome assembly.

Main Methods:

• Developed a multiplexed DNA-PAINT technique.
• Assessed segment stoichiometry in over 10,000 individual virus particles.
• Spatially resolved individual RNA segments within virus particles with ~10 nm precision.

Main Results:

• Influenza genome assembly occurs via multiple, redundant, and cooperative pathways.
• Preferentially interacting segment pairs guide the assembly process.
• Synergistic effects enhance genome assembly efficiency.
• Segment interaction strength influences the spatial configuration of gene segments in mature virions.

Conclusions:

• Influenza A virus genome packaging is a complex, multi-pathway process.
• Segment-segment interactions play a critical role in guiding and completing genome assembly.
• The developed method provides a template for quantifying sequence motif contributions to selective packaging.