Multiplex 3D Nanoscopy Resolves Nanoarchitecture of Human Immunodeficiency Virus

Area of Science:

• Biophysics
• Structural Biology
• Virology

Background:

• Understanding the nanoscale organization of viral and host proteins is crucial for deciphering virion assembly and infectivity.
• High-resolution imaging techniques are needed to visualize protein distribution within individual virus particles.

Purpose of the Study:

• To develop a robust framework for multiplexed optical 3D super-resolution microscopy of human immunodeficiency virus type 1 (HIV-1) particles.
• To achieve isotropic localization precision below 10 nm for five target proteins using minimal fluorescence photon flux (MINFLUX) nanoscopy and DNA point accumulation for imaging in nanoscale topography (DNA-PAINT).

Main Methods:

• Utilized MINFLUX nanoscopy and DNA-PAINT for multicolor 3D super-resolution imaging.
• Assessed linkage errors using the HIV-1 matrix layer as a reference structure.
• Developed the matFLUX software for accurate visualization and quantitation of imaging data.

Main Results:

• Achieved isotropic localization precision below 10 nm for five target proteins in HIV-1 particles.
• Mapped the spatial organization of tetraspanin proteins CD9 and CD81 in single virus-like particles.
• Demonstrated the framework's ability to visualize and quantify protein components in 3D.

Conclusions:

• The presented framework enables high-resolution spatial mapping of protein components within individual virus particles.
• This approach is generally applicable to studying nanoscale architectures in 3D.
• The findings contribute to a deeper understanding of viral structure and assembly mechanisms.