Multiplex 3D Nanoscopy Resolves Nanoarchitecture of Human Immunodeficiency Virus

Resolving the nanoscale organization of viral and host proteins is important to understanding virion assembly and infectivity. Here, we present a robust framework for multiplexed optical 3D super-resolution microscopy of human immunodeficiency virus type 1 (HIV-1) particles using minimal fluorescence photon flux (MINFLUX) nanoscopy and DNA point accumulation for imaging in nanoscale topography (DNA-PAINT), achieving isotropic localization precision below 10 nm for five target proteins. First, we assessed linkage errors introduced by different labeling strategies by employing the HIV-1 matrix layer as a reference structure. We then extended the approach to display five viral and host proteins and mapped the spatial organization of tetraspanin proteins CD9 and CD81 in single virus-like particles. For accurate visualization and quantitation of multicolor 3D MINFLUX imaging data, we developed the analysis workflow and software matFLUX. The approach presented here enables high-resolution spatial mapping of protein components within individual virus particles and is generally applicable to the study of nanoscale architectures in 3D.