Single-molecule coordinate-based analysis of the morphology of HIV-1 assembly sites with near-molecular spatial resolution.

We apply single-molecule super-resolution microscopy and coordinate-based cluster analysis to extract information on the distribution and on the morphology and size of clusters of the human immunodeficiency virus (HIV-1) Gag polyprotein in fixed cells. Three different patterns of Gag distribution could be distinguished. A major type of assembly observed was in accordance with previous electron microscopy analyses revealing ~140 nm-sized HIV-1 buds at the plasma membrane of virus-producing cells. The distribution of Gag molecules in the 2D projection at these sites was consistent with a semi-spherical 3D assembly. We compared different methods of cluster analysis and demonstrated that we can reliably distinguish different distribution patterns of the Gag polyprotein. These methods were applied to extract information on the properties of the different Gag clusters.