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

Abstract

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.

Statistical Tests for Force Inference in Heterogeneous Environments

“…In the experiments, the HIV-1 Gag precursor was genetically modified to contain a photoactivable fluorescent tag mEOS2 protein. It allowed to record VLP assembly in human CD4 T cells using single-particle tracking photoactivated localization microscopy (sptPALM) (Manley et al 2008;Floderer et al 2018). Several VLPs can assemble in parallel in the same observation region.…”

“…4F bear evidence of a force with rather high Bayes factors (log 10 (K) ≥ 1). In such a complex system, the high K values in these individual domains may stem from local membrane activity, failed capsid assembly (Floderer et al 2018) or be false detection. In the rest of the region, the Bayes factor is | log 10 (K)| <1 meaning neither of the models is favored at the chosen level of statistical significance.…”

Reconstructing wells from high density regions extracted from super-resolution single particle trajectories

“…3A1-A2, A3-A4). Note that when the covariance matrix is recovered, the coefficients λ x and λ y can be recovered using equation (25) from the diffusion coefficient D and the semi-axes a, b…”

“…Although the interpretation of high density regions as potential wells for AMPA receptors was first anticipated in [24] and discovered in [3], the nature of these wells and others in general, remains unclear [13]. Potential wells were found for membrane proteins such CaV [11], GAG [12,25] and recently for G-protein [26]. Potential wells could be generated by protein clusters, or by membrane cusp of vesicle fusion points, by membrane-membrane con-tact at location of organelle interactions [27].…”

“…The role of wells could vary: they retain receptors for hundreds of milliseconds to seconds at specific locations in order to increase the probability of an optimal signal transduction, such as during synaptic transmission. Transient wells allow to trap proteins to create aggregates as proposed for capsid assembly [12,25]: once the energy of the well decreases, molecules are not interacting with the well. Another possible role is that wells could regulate the fluxes of receptors in micro-compartments such as dendritic spines [16].…”