Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells

Sci Rep. 2018 Nov 2;8(1):16283. doi: 10.1038/s41598-018-34536-y.


Monitoring virus assembly at the nanoscale in host cells remains a major challenge. Human immunodeficiency virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100 nm in diameter. Interestingly, HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble the immature virus. Here, we monitored VLP formation at the plasma membrane of host CD4+ T cells using a newly developed workflow allowing the analysis of long duration recordings of single-molecule Gag protein localisation and movement. Comparison of Gag assembling platforms in CD4+ T cells expressing wild type or assembly-defective Gag mutant proteins showed that VLP formation lasts roughly 15 minutes with an assembly time of 5 minutes. Trapping energy maps, built from membrane associated Gag protein movements, showed that one third of the assembling energy is due to direct Gag capsid-capsid interaction while the remaining two thirds require the nucleocapsid-RNA interactions. Finally, we show that the viral RNA genome does not increase the attraction of Gag at the membrane towards the assembling site but rather acts as a spatiotemporal coordinator of the membrane assembly process.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD4-Positive T-Lymphocytes / cytology
  • CD4-Positive T-Lymphocytes / metabolism*
  • CD4-Positive T-Lymphocytes / virology
  • Cell Membrane / metabolism*
  • HIV-1 / physiology*
  • Humans
  • Intravital Microscopy / methods
  • Jurkat Cells
  • Microscopy, Fluorescence / methods
  • Mutagenesis, Site-Directed
  • Mutation
  • RNA, Viral / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Single Molecule Imaging / methods
  • Transfection
  • Virus Assembly*
  • gag Gene Products, Human Immunodeficiency Virus / genetics
  • gag Gene Products, Human Immunodeficiency Virus / metabolism*


  • RNA, Viral
  • Recombinant Proteins
  • gag Gene Products, Human Immunodeficiency Virus