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

Elise Boilley,Cyril Favard,Jean-Baptiste Masson,Sonia Georgeault,Charlotte Floderer,Maxime Dahan,Peggy Merida,Jean-Baptiste Sibarita,Philippe Roingeard,Mohamed El Beheiry,Delphine Muriaux

doi:10.1038/s41598-018-34536-y

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.

Highlights

Monitoring virus assembly at the nanoscale in host cells remains a major challenge
How are single viral proteins recruited to virus budding sites once at the cell plasma membrane? What are the relative contributions of viral protein-protein, or protein-RNA genome interactions to this membrane recruitment? A method to decipher the underlying dynamic molecular mechanisms of virus assemblies at cell membranes, molecule by molecule, is super-resolution microscopy applied to living cells
The plateau phase duration was about 6 min for all three Gag derivatives. These results suggest that: i) MACASP1 forms low-density assembling platforms that mainly do not reach the virus-like particles (VLPs) formation stage, ii) only WM and wild type (WT) Gag can form high-density assembly platforms that lead to VLP formation, iii) on average, the time needed to make a VLP seems not affected by the presence of a packageable viral RNA or by CA-CA interactions

Summary

Introduction

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. Recent progress in single-molecule localisation microscopy allows deciphering protein organisation and dynamics in a single cell at the nanoscale level[1,2,3] In this context, we studied HIV-1 assembly and budding at the plasma membrane of living host CD4+ T cells by tracking the viral membrane Gag proteins and its derivatives. By coupling live PALM, TIRF-microscopy, Bayesian inference and hidden Markov statistical analyses based on millions of Gag protein localisations and hundreds of buds, we were able to monitor HIV-1 Gag particle formation at the plasma membrane of host CD4+ T cells. By analysing the temporal correlation between changes in the density and the trapping energy of membrane Gag molecules during VLP assembly, we brought evidence that the cis-packageable viral genome, which encodes Rev/RRE dependant Gag, spatio-temporally coordinates the complete VLP assembly at the surface of the host CD4+ T cells

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Results

Discussion

Conclusion