Live single molecule microscopy of HIV-1 assembly in host T cells reveals a spatio-temporal effect of the viral genome.

Monitoring virus assembly dynamic at the nanoscale level 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 100nm in diameter. HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble immature virus. Here, we monitored Gag assembly in host CD4 T lymphocytes using single molecule dynamics microscopy and energy mapping. A workflow allowing long time recordings of single Gag molecule localization, diffusion and effective energy maps was developed for robust quantitative analysis of HIV assembly and budding. Comparison of numerous cell plasma membrane assembling platforms in cells expressing wild type or assembly-defective Gag proteins showed that VLP formation last 15 minutes, with an assembly time of 5 minutes, and that the nucleocapsid domain is mandatory. Importantly, it reveals that the viral genome coordinates spatio-temporally HIV-1 assembly.