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

Abstract

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.

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Abbreviations

CA:

Capsid domain

dSTORM:

Direct stochastic optical reconstruction microscopy

EMCCD:

Electron-multiplying charge device

HIV-1:

Human immunodeficiency virus type 1

MA:

Matrix domain

MEA:

Mercaptoethylamine

NC:

Nucleocapsid domains

ROI:

Region of interest

Ryr:

Ryanodine receptors

TIRF:

Total internal reflection fluorescence

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Acknowledgments

This work was supported by the Systems Biology Initiative (FORSYS) of the German Ministry of Research and Education (BMBF), Project VIROQUANT and Grant No. 0315262.

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Correspondence to Mike Heilemann.

Additional information

S. Malkusch and W. Muranyi contributed equally.

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Malkusch, S., Muranyi, W., Müller, B. et al. Single-molecule coordinate-based analysis of the morphology of HIV-1 assembly sites with near-molecular spatial resolution. Histochem Cell Biol 139, 173–179 (2013). https://doi.org/10.1007/s00418-012-1014-4

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Keywords

  • Super-resolution microscopy
  • Single-molecule fluorescence microscopy
  • Cluster analysis
  • Cellular structures
  • Human immunodeficiency virus
  • Gag