Hunting Down HIV-1 Gag Proteins at the Plasma Membrane of Human T Lymphocytes

Newly formed HIV-1 particles are one of the possible targets for an efficient cure against AIDS. It is therefore important to understand at a single molecule level how assembly is occurring in living CD4 T lymphocytes, the main host cells for HIV-1 replication. HIV-1 Gag has been shown to be the most important component for virus assembly as it is sufficient to produce newly formed particles, called virus-like particles (VLPs). Assembly of retroviral Gag proteins is mainly occurring at the inner leaflet of the infected cell plasma membrane. ¹ Interestingly, Gag can be tagged internally by a fluorescent protein without impairing its capacity to assemble into a VLP. ^2,3 Therefore, we are currently deciphering VLP assembly at the plasma membrane of human T lymphocytes by monitoring the dynamic organization of Gag-i(mEOS2) protein, at the nanometric level, with the help of single-protein tracking photo-activable localization microscopy (spt-PALM) coupled to total internal reflection fluorescence (TIRF) microscopy.

This super-resolution microscopy method ⁴ produces high-resolution images (Fig. 1A) where assembly platforms made of Gag-i(mEOS2) are distinguishable as areas enriched in Gag proteins. Using that type of image, we can monitor the mean number of assembling sites occurring after 18 to 24 h of viral protein expression. On average we observed between 100 and 250 assembly platforms per cell. Since we are performing time lapse experiments, we can also determine the mean time of existence of these assembly platforms inside the cell. We find it in between 6 and 15 min, which corresponds to what Ivanchenko et al. ² and Jouvenet et al. ³ found in human adherent HeLa cells expressing GFP-tagged Gag. Interestingly, as an advantage of the method, we can identify and characterize the final step of the process by observing some VLPs released in the extracellular medium. These VLPs are ∼100–140 nm in diameter and do not exhibit any dynamic or evolution in time, which is a signature of an achieved particle.

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FIG. 1.

(A) A living Jurkat T cells-expressing HIV-1 Gag-i(mEOS2) 24 h post-transfection is shown. The PALM/TIRF image of Gag-i(mEOS2)-expressing Jurkat T cells corresponds to the sum of all Gag-i(mEOS2) localizations, the color scale corresponds to the number of localizations per pixel. The resolution is 50 nm. The total number of localizations is 17.8 × 10⁶. The color scale bar indicates Gag localization density: single molecule Gag (close to “zero” value, blue color), small assembly complexes (close to “15” value, magenta color), and assembled particles (“30” value, white color). Scale bar is 5 μm. (B) The sum of reconstructed Gag-i(mEOS2) trajectories for 40 s in a Jurkat cell (1490 trajectories represented) is shown. The total number of trajectories for the time of acquisition is 1.1 × 10⁶. One Gag molecule trajectory is represented by one color. Scale bar is 5 μm. PALM, photo-activable localization microscopy; TIRF, total internal reflection fluorescence. Color images available online at www.liebertpub.com/aid

Spt-PALM ⁴ also allows to study the time-resolved evolution of the assembly, molecule after molecule at the nanoscale level (∼50 nm), that is, with a resolution higher than the virus assembly platform size of 110–140 nm in diameter. The total number of localizations for the entire acquisition time is in the order of 10⁷ per cell, leading to ∼10⁵ to 10⁶ analyzable trajectories. By tuning acquisition frequency, we can select mainly slow diffusing proteins (D<2 μm²·s⁻¹), that is, membrane-associated proteins. Thus, by reconstructing images of the different Gag trajectories (Fig. 1B), we can identify Gag proteins getting trapped into the assembly sites as well as others freely diffusing at the cell plasma membrane of Jurkat T cells (transposable to primary CD4 T lymphocytes). We are currently analyzing these trajectories using newly developed tools and big data facilities to give access to new molecular details about HIV-1 Gag assembly dynamic in its natural host cells and to improve our knowledge about the late stages of HIV-1 replication in CD4 T lymphocytes.